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Suppressors of Cytokine Signaling-1 (SOCS-1) Mimetic and Antagonist Peptides: Potential as Therapeutic Agents for Treatm...

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PAGE 1

1 SUPPRESSORS OF CYTOKINE SIGNALING1 (SOCS-1) MIM ETIC AND ANTAGONIST PEPTIDES: POTENTIAL AS THERAP EUTIC AGENTS FOR TREATMENT OF IMMUNOLOGICAL DISEASES By LILIAN WANGECHI WAIBOCI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Lilian Wangechi Waiboci

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3 To the memory of my grandmother Mrs. Pris cilla Wangechi Matu a nd my grandfather Mr. Richard Mwaniki wa Nyangi

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4 ACKNOWLEDGMENTS I wish to thank my major supervisor Dr. Ho ward M. Johnson for funding this research, for helpful suggestions and discus sions, and for providing a conduc ive environment for learning. I also wish to thank the members of my gra duate committee Drs. Ayalew Mergia, Edward Hoffmann, Peter Kima, and Janet Yamamoto fo r their helpful suggestions, and time. I wish to thank members of the Johnson labor atory who helped with various aspects of this research. I wish to specially thank Drs. Chulbul Ahmed and Mustafa Mujtaba for teaching me many of the techniques used in this study. I wish to th ank Mr. Mohammed Haider for synthesizing all the peptides used in this study, Dr. Levy Omara-Opyene, Dr. Lawrence Flowers, James Martin, Ezra Noon-Song, Rea Dabelic, Linds ey Jager, and Lauren Thornton for helpful suggestions and assistance. I also wish to thank Janet Lyles and Mary A nn Soncrant for helping make sure that my studies and stay at the Microbi ology Department ran smoothly and the staff of the International Student Center for ensuring that my stay in the USA went smoothly. I wish to thank my parents Mr. Francis Waiboc i and Mrs. Esther Waiboci for all they have done for my education, for instilling the love for learning, and discipline in my siblings and I. I also wish to thank my siblings for their constant encouragement. Last, bu t not least, I wish to thank my family, my husband Dr. George Ka riuki for his love, understanding, and constant encouragement through out the rough tides and my son Vict or Kariuki for his love, understanding, and patience.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 Rationale...................................................................................................................... ...........14 Specific Objectives............................................................................................................ .....16 2 LITERATURE REVIEW.......................................................................................................18 Janus Kinases.................................................................................................................. ........18 Signal Transducers and Activators of Transcription (STAT) Proteins..................................19 Interferon Signaling Through the JAK/STAT Signaling Pathway.........................................21 Regulation of the JAK/STAT Signaling Pathway..................................................................23 Suppressors of Cytokine Signaling (SOCS)...........................................................................23 Physiological Role of SOCS-1 Protein...................................................................................25 SOCS-1 Mimetic Peptides......................................................................................................27 Inhibition of SOCS-1 Activity and Immunological Relevance..............................................28 3 MATERIALS AND METHODS...........................................................................................33 Cell Culture................................................................................................................... ..........33 Peptides....................................................................................................................... ............33 Binding Assays................................................................................................................. ......34 In vitro Kinase Assays............................................................................................................35 Immunoblot Analysis............................................................................................................ ..36 Macrophage Activity............................................................................................................ ..37 Tkip Cellular Targets.......................................................................................................... ....37 Antiviral Assays for SOCS-1 Antagonist Function................................................................39 Transfections of LNCaP Cells with SOCS-1 DNA................................................................40 GAS Promoter Activity..........................................................................................................41 Primer Design and PCR Amplifi cation of Murine SOCS-1 DNA.........................................42 Cloning SOCS-1 into pBlueBac4.5/V5-H is TOPO TA Expression Vector...........................42 Expression of SOCS-1 in Sf9 Cells........................................................................................43 Statistical Analysis........................................................................................................... .......44 4 RESULTS........................................................................................................................ .......46

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6 Tkip Family Members Bind to JAK2 Autophosphorylation Site...........................................46 Tkip Family Members Inhibit JAK2 Kinase Activity.....................................................47 Tkip Inhibits Superantigen-induced Proliferation of Mouse Splenocytes......................47 SOCS-1 Kinase Inhibitory Region (SOCS1 -KIR) Binds to JAK2 Autophosphorylation Site........................................................................................................................... ...........49 Tkip and SOCS1-KIR Bind to JAK2 Autophosphorylation Site....................................49 JAK2 Kinase Activity and STAT1 Activation.............................................................51 Tkip and SOCS1-KIR Inhibit IFN -induced Activation of Macrophages......................52 Tkip and SOCS1-KIR Inhibit Antigenspecific Lymphocyte Proliferation....................52 An Extended SH2 Sequence (SOCS1-ESS) Peptide does not Bind to pJAK2 (10011013).......................................................................................................................... ..53 Tkip Cellular Targets.......................................................................................................... ....54 Effect of Tkip and SOCS1-KIR on CD4+ T Cells...........................................................54 Effects of Tkip and SOCS1-KIR on CD8+ T Cells.........................................................55 Effect of Tkip and SOCS1-KIR on B Cells.....................................................................56 Effect of Tkip on Macrophages.......................................................................................56 SOCS-1 Antagonist Activity of pJAK2 (1001) Peptide...............................................57 Expression of SOCS-1 Protein...............................................................................................59 5 DISCUSSION..................................................................................................................... ....80 6 FUTURE WORK....................................................................................................................86 APPENDIX: Vector Map of the Transfer (Cloning) Vector.........................................................87 LIST OF REFERENCES............................................................................................................. ..88 BIOGRAPHICAL SKETCH.........................................................................................................94

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7 LIST OF TABLES Table page 2-1 A table showing the JAK kinases and STAT proteins utilized by some cytokines, growth factors, hormones, and oncogenes.........................................................................32 3-1 List of peptides used in this study. ....................................................................................45

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8 LIST OF FIGURES Figure page 2-1 A schematic representation of the domain structure of the JAK kinase family.................30 2-2 A schematic diagram showing the do main structure of SOCS-1 protein..........................31 4-1 JAK2 autophosphorylation site pept ides JAK2(1001-1013) and pJAK2(1001-1013) bind to SOCS-1 mimetic peptides......................................................................................61 4-2 Both soluble DRTkip and soluble Tk ip inhibit the binding of biotinylated JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip.................62 4-3 DRTkip and Tkip but not the control peptide, MuIFN (95-106), inhibit JAK2 autophosphorylation...........................................................................................................63 4-4 Tkip, but not DRTkip inhibits supera ntigen-induced splenoc yte proliferation.................64 4-5 JAK2 autophosphorylation site peptides bind to SOCS1-KIR. A) JAK2(1001-1013) peptide binds to both SOCS1-KIR and Tkip.....................................................................65 4-6 Both soluble SOCS1-KIR and soluble Tk ip inhibit the binding of biotinylated JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip or SOCS1-KIR...................................................................................................................... .66 4-7 Differences in the kinase inhibition patterns of SOCS1-KIR and Tkip in JAK2 autophosphorylation, STAT1 phosphorylation, and EG FR phosphorylation.................68 4-8 SOCS1-KIR and Tkip inhibit IFN -induced macrophage activation................................69 4-9 Both SOCS1-KIR and Tkip inhibit pr oliferation of murine splenocytes..........................70 4-10 Biotinylated pJAK2(1001-1013) binds to SOCS1-KIR but not to SOCS1-ESS...............71 4-11 Tkip and SOCS1-KIR i nhibit antigen-specific CD4+ T cell proliferation and CD4+ T cell-induced IFN production............................................................................................72 4-12 Tkip and SOCS1-KIR inhibit CD8+ T cell-induced IFN production...............................73 4-13 Tkip and SOCS1-KIR inhibit antigen -induced B cell proliferation and antibody production..................................................................................................................... .....74 4-14 Tkip inhibits LPS-i nduced macrophage activity................................................................75 4-15 pJAK2(1001-1013) peptide has SOCS -1 antagonist properties........................................76 4-16 SOCS-1 protein was expressed in ba culovirus infected Sf9 insect cells...........................78

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9 4-17 Recombinant baculovirus containing muSOCS-1 DNA....................................................79 A-1 A map of the pBlueBac4.5/V5-His vector.........................................................................87

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SUPPRESSORS OF CYTOKINE SIGNALING (SOCS-1) MIM ETIC AND ANTAGONIST PEPTIDES: POTENTIAL AS THERAP EUTIC AGENTS FOR TREATMENT OF IMMUNOLOGICAL DISEASES By Lilian Wangechi Waiboci May 2007 Chair: .Howard M. Johnson Major: Microbiology and Cell Science Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by interferon gamma (IFN ) by binding to the autophosphorylation si te of Janus kinase 2 (JAK2) and by targeting bound JAK2 to the proteosome for degr adation. Studies on a tyrosine kinase inhibitor peptide, Tkip, which is a SOCS-1 mimetic, ar e described. Tkip was synthesized from the physiological form of amino acids, L-amino ac ids. We synthesized two additional SOCS-1 mimetic peptides, DTkip and DRTkip, from D-ami no acids, which are potentially more resistant to degradation and therefore would likely be better therapeutic agents. DTkip and DRTkip bound to the unphosphorylated JAK2 autophosphorylati on site peptide, JAK2(1001-1013) and the tyrosine 1007 phosphorylated peptide, pJAK2(10011013). Further, DTkip and DRTkip inhibited JAK2 autophosphorylation and JAK2 phosphorylation of IFN receptor-1 (IFNGR-1). Tkip was also compared with the kinase in hibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN -induced biological activity. Tkip and a peptide corresponding to the KIR region of SOCS-1, 53DTHFRTFRSHSDYRRI (SOCS1-KIR), bound similarly to JAK2(10011013) and to pJAK2(1001-1013). Dose-response competitions suggested that Tkip and SOCS1-KI R similarly recognized the autophosphorylation

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11 site of JAK2. While Tkip inhibited J AK2 autophosphorylation as well as IFN -induced STAT1 phosphorylation, SOCS1-KIR, lik e SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1 activation. Both Tkip and SOCS1-KIR inhibited IFN activation of murine macrophages and antigen-specifi c splenocyte pr oliferation. The fact that SOCS1-KIR bound to pJAK2(10011013) suggested that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFN activity, blocked SOCS-1 induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN activation site (GAS) promoter activity, and e nhanced antigen-specific proliferation. Further, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can func tion as an antagonist of SOCS-1. In summary, Tkip and SOCS1-KIR recognized the autophosphorylation site of JAK2 similarly and pJAK2(1001-1013) peptide functione d as a SOCS-1 antagonist. Thus, we have developed peptides that function as SOCS-1 ag onists and antagonists, wh ich have potential for suppressing or enhancing the immune response.

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12 CHAPTER 1 INTRODUCTION Janus tyrosine kinases (JAKs) are an enzyme fa mily that mediates the biological effects of cytokines, hormones, and growth factors by ty rosine phosphorylation of signal transducers and activators of transcrip tion, STATs (Reviewed in Yamoaka et al. 2004; Parganas et al. 1998). The interferons (IFNs), including types I and type II, hormones such as growth hormone and angiotensin, and growth factor s such as thrombopoietin, ar e all among 50 related factors dependent on JAK tyrosine phosphorylation of appr opriate STAT transcription factors for their physiological functions (Subramaniam et al. 2001; Johnson et al. 2004). The immediate early signal transduc tion events associated with IFN and its receptor subunits involve the obligatory action of two tyrosine kinases, JAK1 and JAK2 (Reviewed in Kotenko and Peska 2000). The IFN receptor (IFNGR) system is a heterodimeric complex consisting of an -subunit (IFNGR-1) and a subunit (IFNGR-2), both of which are essential for the biological activity of IFN (Kotenko and Peska 2000). JAK1 is associated with the IFNGR-1 chain, whereas JAK2 is associated with the IFNGR-2 chain. The interaction of IFN primarily with the IFNGR-1 subunit, initiates a sequence of events that results in increased binding of JAK2 to IFNGR-1. This has important conseque nces for subsequent critical phosphorylation events. JAK2, in the process of binding to IF NGR-1, undergoes autophosphor ylation, and at the same time IFNGR-1 is phosphorylated. These events occur in concert with JAK1 function, resulting in recruitment and ty rosine phosphorylation of the IFN transcription factor, STAT1 (Reviewed in Kotenko and Peska 2000; Bromberg and Darnell 2000). A family of proteins called suppressors of cytokine signaling (SOCS) negatively regulates JAK/STAT signaling (Starr et al. 1997; Endo et al. 1997; Naka et al. 1997). SOCS proteins are also negative regulator s of signaling by other cytokines, growth factors, and hormones

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13 (Reviewed in Alexander et al. 2002; Larsen a nd Ropke 2002; Alexander and Hilton 2004). There are currently eight identified members of the SOCS family, SOCS-1 to SOCS-7 and cytokineinduced SH2 domain containing prot ein (CIS). SOCS-1 is of particular interest, because it is a negative regulator of the JAK kinases, as well as several cytokines, and hormone receptor systems including epidermal growth factor recep tor (EGFR) (Reviewed in Calo et al. 2003). Our laboratory recently designed and synthesized a tyrosine kina se inhibitor peptide, Tkip (WLVFFVIFYFFR) (Flowers et al. 2004). The characteristics of the Tkip 12-mer are summarized in Chapter 2. In this stu dy we show that Tkip inhibits IFN -induced macrophage activation and define Tkip cel lular targets in antigen-induced immune responses. We also describe a family of Tkip-related pept ides, DTkip (WLVFFVIFYFFR) and DRTkip (RFFYFIVFFVLW). DTkip and DRTkip, which like Tkip, bind to the JAK2 autophosphorylation site peptide, inhibit JAK2 autophosphoryla tion, and JAK2 phosphorylation of IFN receptor subunit, IFNGR-1. It has been suggested that the binding of SOCS-1 to JAK2 requires the SOCS-1 SH2domain and that the kinase i nhibitory region (KIR), while no t required for the binding, is essential for the inhibitory action of SOCS-1 (Yasukawa et al. 1999). We show here that a peptide corresponding to SOCS-1 KIR region, SO CS1-KIR, specifically binds to a peptide representing the JAK2 autophosphoryl ation site and inhibits STAT1 activation. Further, we show that SOCS1-KIR, as well as Tkip, inhibit IFN -induced macrophage act ivation. We also present data on a novel SOCS-1 antagonist pe ptide, pJAK2(1001-1013), which corresponds to the JAK2 autophosphorylation site. pJ AK2(1001-1013) enhances suboptimal IFN -induced antiviral activity, enhances IFN -activated sequences (GAS) pr omoter activity, and inhibits

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14 SOCS-1 suppression of STAT3 activation of LNCaP prostate cancer cells, thus functioning as a SOCS-1 antagonist peptide. Rationale SOCS proteins play an important role in the regulation of JAK/ST AT signaling. Disruption of the normal SOCS function may contribute to di sease onset, progression or death. It has been shown that deletion of SOCS-1-/or SOCS-3-/genes in mice results in death of the mice either as neonates (SOCS-1-/-) or embryos (SOCS-3-/-) (Naka et al. 1998; Starr et al. 1998). SOCS proteins regulate immune response by inhibiting JAK kina ses. Uregulated JAK kinases signaling may result in inflammation and cancers. Several cancers are characterized by constitutive activation of the JAK/STAT signaling pathway. SOCS proteins may play a role in inhibiting malignant transformation of cells by regulating JAK/STAT signaling, thereby preventi ng cancer onset and progression. This has been shown in a number of cancers including leuke mia (T cell acute lymphoblastic leukemia (ALL), Pre-B ALL, and atypical chroni c myelogenous leukemia (CML)), and hepatocellular carcinoma (Alexander and Hilton 2004). These examples raise the interesting possibility of the role that SOCS proteins and/or SOCS mimetics may pl ay in the management of these cancers. Dysregulation of JAK/STAT signaling also pl ays important roles in the pathogenesis of some inflammatory diseases including rheumatoid arthritis (Suzuki et al. 2001), inflammatory diseases of the gastrointestinal tract (SOCS2), (Suzuki et al. 2001; Lovato et al. 2003; Shouda et al. 2001) as well as inflammations of the central nervous system for example in experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis in humans (Maier et al. 2002). Hence, dysregulation of JAK/STAT signaling plays an important role in the pathology of some inflammatory diseases and therefore raises the possibility that SOCS proteins and/or SOCS

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15 mimetics that negatively regulate JAK/STAT si gnaling may be possible therapeutics for the control and treatment of th e inflammatory disease. There are several tyrosine inhibitors that are currently undergoing clinical trials for treatment of various cancers, especially cancers resistant to chemotherapy and radiation. Herceptin (Trastuzumab) is a humanized mono clonal antibody specific fo r a member of the epidermal growth factor receptor (EGFR) family called HER2, which binds to the extracellular binding domain of HER2/neu on tumor cells induci ng receptor internalization and inhibiting cell cycle progression (Reviewed in Shawver et al. 2 002). The discovery of Herceptin resulted in the treatment of aggressive forms of breast cance r, in which cancer cel ls overexpressed HER2. These cancers are usually less responsive to ch emotherapy (Shawver et al. 2002). A second drug, GleevacTM, is a small molecule inhibitor of the oncogenes BCR-Abl, Abl, PDGFR, and c-kit. GleevacTM blocks ATP binding to the kinase, there by preventing phosphorylati on events that are required for signal transduction. GleevacTM has been shown to increase the effectiveness of interferon therapy on ch ronic myelogenous leukemia patients (S hawver et al. 2002). Other small molecule tyrosine kinases targeted therapies incl ude Erbitux (EGFR) for treatment of colorectal cancer, Tarceva (EGFR) for treatment of pancreat ic cancer, and Iressa (EGFR) for treatment of non-small-cell lung cancer (Shawver et al. 2002; Vincentini et al. 2003). This provides additional evidence that tyrosine kinases targeted approach es may have potential as anti-cancer therapy. Hence, it is possible that the SOCS-1 mimetic peptides developed in our laboratory, which are tyrosine kinase inhibitors of STAT transcription factors such as the STAT3 oncogene, may have potential as anti-cancer and anti-inflammatory disease ag ents. The mimetics would likely augment the effect of endogenous SOCS-1. We w ill discuss these mimetic peptides and present data on their effect on the J AK/STAT signaling pathway, IFN -signaling, and cell proliferation.

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16 SOCS-1 regulates signaling by a vari ety of cytokines including IFN IFN and several interleukins. Put differently, SOCS-1 reduces the immune response mechanisms initiated by these cytokines. In an immune competent indivi dual, this is desirable because it prevents excessive signaling, which would likely re sult in inflammation. However, in an immunocompromised individual, there may be a need to enhance cytokine-induced immune response to infection. One way of enhancing imm une response to pathogen infection may be by inhibition of SOCS-1 activity. It has recently been shown that sile ncing of SOCS-1 in dendritic cells promoted cell activation, which led to enhanc ement of effective antigen-specific anti-tumor immunity (Shen et al. 2004). This implies that reagents that negatively regulate SOCS-1 may have potential in control and/or treatment of diseases arising fr om inadequate immune response. Hence, we designed a SOCS-1 inhibitor peptid e, pJAK2(1001-1013), that is derived from the JAK2 autophosphorylation site. I will present da ta showing that pJAK2(1001-1013) is a SOCS-1 antagonist. Specific Objectives In this dissertation Tkip and other SOCS-1 mimetic peptides, which I hypothesized, would function similar to Tkip and therefore similar to SOCS-1 are described. The first objective of this study therefore was to determine whether these peptides; DTkip and DRTkip have Tkip-related characteristics. Specifically, whether the peptid es bind to JAK2 autophosphorylation site, inhibit JAK2 autophosphorylation, and inhibit activated JAK2 activity. It has previously been stated that the SO CS-1 domains that bind directly to JAK2 autophosphorylation site ar e the SH2 domain and the extended SH2 region (ESS), and that the kinase inhibitory region (KIR) is not essential in the initial binding (Yasukawa et al. 1999). The second objective of this study wa s therefore to determine whet her a peptide corresponding to

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17 SOCS-1 KIR, SOCS1-KIR, binds directly to JAK2 autophosphorylation site, inhibits JAK2 autophosphorylation, and inhibits activated JAK2 activity. Since Tkip has been shown to be a SOCS-1 mimetic with possible therapeutic potential, the third objective of this study was to define Tk ip cellular targets. Specifically, to determine whether Tkip targeted B cells, CD4+ T cells, CD8+ T cells, and antigen presenting cells. This would provide insights on the dire ct effect of Tkip on specific cel ls of the immune system, which is important if Tkip proves be a potential therapeutic agent. The fourth objective of this study was to de fine a novel way of a ddressing inadequate cytokine-induced immune res ponse mechanism. Here, a nove l SOCS-1 antagonist peptide, pJAK2(1001-1013), is described and preliminary data showing that this pep tide reverses SOCS-1 inhibition is presented.

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18 CHAPTER 2 LITERATURE REVIEW Janus Kinases Janus tyrosine kinases (JAKs) are a small but indispensable enzyme family of nonreceptor tyrosine kinases that mediates the biological eff ects of cytokines, hormones, and growth factors by tyrosine phosphorylation of signal transduc ers and activators of transcription, STAT, (Reviewed in Ihle 1995; Parganas et al. 1998). Th e JAK family consists of four members (JAK1, JAK2, JAK3, and TYK2) that are differentially ac tivated in response to various cytokines (Ihle 1995). JAK proteins are approximately 120 to 13 0 kDa cytosolic proteins expressed in many types of tissue with the exception of JAK3 whos e expression is restricted to cells of the hematopoietic system (Ihle 1995, Reviewed in Thompson 2005). Members of the JAK protein family cont ain highly conserved structural domains designated JAK homology domains (JH). There are currently seven know n JH domains (JH1JH7) of which JH1 is the functi onal catalytic kinase. For JAK2, th is domain possesses a critical activation loop that beco mes phosphorylated in order to act ivate the kinase. The phosphorylation of tyrosine residue (Y1007) in the activation loop of JAK2 is essential for activation and downstream signaling events (Feng et al. 1997). It has recently been suggested that the phosphorylation of other tyrosines may also be necessary for JAK2 activation (Kurzer et al. 2004). Phosphorylation of Y1007 resu lts in a conformational cha nge in the activation loop, which allows substrate access to specific binding sites in the catalytic gr oove (Yasukawa et al. 1999). JH2 is a non-functional cataly tic kinase domain but it bear s sequence homology to typical tyrosine kinase domains, hence it is referred to as a pseudokinase domain. The functional role of the JH2 domain is unclear, however studies have suggested that it may have a kinase inhibitory

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19 function. Both the JH1 and JH2 domains are located near the carboxyl termi nus and comprise the major portion of the JAK molecule (Reviewed in Leonard 2001; Kisseleva et al. 2002). The JH3, JH4, and JH5 domains are poorly understood and require additional work to elucidate their functions. JH6 and JH7 amino terminal domains ha ve been implicated in the association between the JAK molecule and the specific cytokine r eceptor (Leonard 2001; Kisseleva et al. 2002). Figure 2-1 shows a diagrammatic representation of the domain st ructure of the JAK kinases. Comparisons of the JAK2 autophosphor ylation site amino acid sequences (LPQDKEYYKVKEP) revealed100% sequence ho mology among different mammalian species including of the human JAK2 ( Homo sapiens genebank accession number NM_004972) mouse ( Mus musculus AAH54807), rat ( Rattus norvegicus NP_113702), and pig ( Sus scrofa BAA21662) as determined using the basic local alignment se arch tool (BLAST search, http://www.ncbi.nlm.nih.gov/blast/). In addition, the amino acid sequence of the human JAK1 (IETDKEYYTVKDD, accession number NP_00 2218) was 100% homologous to the mouse JAK1 (NP_666257), as was hu man TYK2 (VPEGHEYYRVRED, accession number NP_003322) and mouse TYK2 (NP_061263). Human JAK3 (LPLD KDYYVVREP, NP_000206) and mouse JAK3 (LPLG KDYYVVREP, NP_034719), are ne arly identical with a one amino acid substitution, underlin ed. It is worth noting that in all four JAK kinases, the sequences are similar, but not id entical. The conservation in this region indicates the importance of the autophosphorylation site in JAK function. Signal Transducers and Activators of Transcription (STAT) Proteins STAT proteins are a family of cytoplasmic tran scription factors that pa rticipate in a variety of cellular events, including differentiation, proliferation, cell survival, apoptosis, and angiogenesis involving cytokine s, growth factors, oncogenes, and hormones. Some of the

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20 cytokines, growth factors, oncogenes, and hormo nes that utilize STAT proteins are shown in Table 2-1. There are currently seven known STAT family members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 (Darnell 1997 ). We are particularly interested in STAT1 and STAT3, both of which are negatively re gulated by SOCS-1. It is worth noting that other SOCS proteins may also nega tively regulate STAT1 and STAT3. The STAT proteins are comprised of six dom ains. These are oligomerization, coiled-coil, DNA binding, linker, SH2, and transc ription activation domains. Th e binding of STAT to the receptors occurs through interact ion of the SH2 to the receptor-doc king site. The critical tyrosine residues required for SH-phosphotyrosine inte raction are STAT1-Y701; STAT2-Y690; STAT3Y705; STAT4-Y693; STAT5-Y694, and STAT6-Y 641. These phosphotyrosines are located near the SH2 domain (Cal et al. 2003). The linker domain, which is alpha helical is the bridge between the DNA binding and the SH2 domains. The transcription activation domain, lo cated on the carboxyl terminus is involved in communication with transcription complexes. The domain has a conserved serine residue (except in STAT2 and STAT6) that when phosphor ylated regulates STAT transcription activity (Reviewed in Imada and Leonard 2000). The amino terminus region of STAT proteins is highly conserved and provides protein to protein interaction, such as dimer interaction of STAT molecules, which contributes to the stabil ity of STAT-DNA bindi ng, thereby increasing transcription activity (Imada and Leonard 2000). The coiled-coil domain may be involved in regulatory function and may be respons ible for nuclear export of STAT. Although some constitutively activated STATs have been observed in some human cancer cell lines and primary tumors, STAT proteins ar e generally activated by tyrosine phosphorylation

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21 (Cal et al. 2003). The STAT proteins become activated by a variety of receptors, such as cytokine, growth factor, and hormone receptors, wh ich may activate STATs directly or indirectly through JAK kinases. In JAK/STAT signaling, the bind ing of a cytokine to its receptor results in the associated JAK kinase becoming phosphorylat ed, and hence activated. The activated JAK kinase phophorylates the receptors cytoplasmic do main, at specific tyrosine residues, and opens docking sites for STAT proteins. The STAT prot eins docked on the recep tor, are phosphorylated, and thus become activated. Some models of JAK/STAT signaling indicate that the activated STATs dissociate from the receptor, dimerize, and translocate to the nucleus where they activate the transcription of specific genes (Bromberg and Darnell 2000). However, it has been shown that in the case of IFN signaling, IFN IFNGR-1, and the bound phospho-STAT1 dimer are translocated to the nucleus as a complex (Subramaniam et al. 2001, Ahmed et al. 2003, Ahmed and Johnson 2006), which implies that IFNGR-1 likely plays additional roles in signal transduction. Interferon Signaling Through the JAK/STAT Signaling Pathway There are two classes of interf eron, type I and type II, both of which utilize the JAK/STAT signaling pathway. Although type I and type II IF Ns signaling pathways are similar, distinct receptors, JAK kinases, and STAT proteins are utilized. IFN the type II IFN, signaling utilizes IFNGR, which is a heterodimeric co mplex comprised of two subunits, (IFNGR-1) and (IFNGR-2), both of which are essentia l for biological activity of IFN (Kotenko and Pestka 2000). IFNGR-1 is associated with JAK1, while the IFNGR-2 is associated with JAK2. Interaction of IFN primarily with IFNGR-1, causes the recep tor subunits to dimerize and brings the associated JAK1 and JAK2 into close proximity. The JAK kinases undergo

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22 autophosphorylation at specific tyrosine resi dues (1007 for JAK2) and become activated (Kotenko and Pestka 2000). The activated JAKs phosphorylate and activate the IFNGR subunits, which results in a cascade of events including the phosphorylation of STAT1 Two phosphorylated STAT1 monomers dimerize and are phosphorylated at specifi c serine residues to form an activated STAT1 transcription f actor. According to so me models of IFN signaling, the STAT1 dimers dissociate from the IFNGR1, translocate to the nucleus, bind to IFN activation sites (GAS), and induce expression of target ge nes (Bromberg and Darnell 2000). It has however been shown that IFNGR-1 accumulates in the nucleus and colocalizes with STAT1 in a time and dose dependent manner, implying that IFNGR-1 is likely also translocated into the nucleus (Subramanian et al. 2001, Ahmed et al. 2003, Ahmed and Johnson 2006). Thus, IFNGR-1 may play an active role in signal transduction even ts subsequent to binding of the receptor complex. Studies have indicated that IFNGR-2 likely is not translocated to th e nucleus (Ahmed and Johnson 2006). Type I interferons, IFN for example, utilize the in terferon alpha-receptor (IFNAR) for signal transduction. The IFNAR is comprised of two subunits, IFNAR-1 (associated with TYK2) and IFNAR-2 (associated with JAK1) (Kotenko and Pestka 2000). The signal transduction pathway is initiated when IFN binds to the receptor, which results in IFNAR-1 and IFNAR-2 forming a heterodimer with subsequent aut ophosphorylation and activa tion of both JAK1 and TYK2. The activated JAK kinases phosphorylat e IFNAR-2, providing docking sites for STAT2, which binds to the receptor and becomes phosph orylated at tyrosine 690. The phosphorylation favors the binding of STAT1 to the phosphoryl ated STAT2 (Durbin et al. 1996). STAT1 is phosphorylated (tyrosine 701) by the JAK kinases and the STAT2/STAT1 heterodimer is released from the receptor and translocates to the nucleus. The STAT2/STAT1 heterodimer

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23 associates with p48 nuclear factor to form th e IFN-stimulated gene factor (ISGF3) complex (Horvath et al. 1996), which stimulates activatio n of target genes within the IFN-stimulated response elements (IRSE) (Li et al. 1998). Regulation of the JAK/ STAT Signaling Pathway The JAK/STAT signaling pathway is caref ully regulated. Unregulated JAK/STAT signaling may result in excessive cytokine-induced immune res ponse, which would likely result in inflammation and be harmful to cells, and ultimately to the organism. Three classes of regulators of the JAK/STAT pathway are current ly known. These are the protein inhibitors of activated STATs (PIAS), tyrosine phosphata ses that include the Src-homology 2 (SH2)containing protein tyrosine phos phatases (SHPs), and the suppressors of cytokine signaling protein family (SOCS) (Reviewed in Kile et al. 2001; Kisseleva 2002; Larken and Rpke, 2002, Alexander and Hilton 2004). PIAS proteins regulate transcripti on through several mechanisms, including blocking the DNA-binding ac tivity of transcrip tion factors, recru iting transcriptional co-repressors and promoting pr otein sumoylation (Shuai 2006). SHPs regulate JAK/STAT pathway by dephosphorylating activated phosphotyr osine (Reviewed in Rico-Bautista et al. 2006). SOCS can block cytokine si gnaling by acting as (i) kinase inhibitors of JAK proteins (SOCS1 and SOCS3), (ii) binding competitors against STATs (SOCS3 and CIS) and (iii) by acting as ubiquitin ligases, thereby promoting the degradation of their partners (SOCS1, SOCS3, and CIS) (Rico-Bautista et al. 2006). I will discuss the SOCS proteins, in detail, as an example of negative regulators of JAK/STAT signaling, be cause SOCS proteins are essential for the regulation of JAK/STAT signali ng and other signaling pathways. Suppressors of Cytokine Signaling (SOCS) SOCS proteins are a family of cytoplasmi c proteins that negatively regulate signal transduction of cytokines, hormones, and growth factors that utilize the JAK/STAT signaling

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24 pathway (Naka et al. 1997, Starr et al. 1997, Endo et al. 1997). Loss or insufficient expression of SOCS proteins may result in diseases includ ing several immune disorders, inflammatory diseases, and cancers (Reviewed in Alexande r and Hilton 2004; Tan and Rabkin 2005). Mouse studies have shown that deletion of the SOCS-1 gene results in neonatal death (Naka et al. 1998; Starr et al. 1998), while deletion of the SOCS-3 gene results in embryoni c death (Croker et al. 2003). In the case of human disease, it has b een shown that a number of hematological malignancies are characterized by constitutive activation of JAK/STAT signaling pathway. These malignancies include T cell acute leuke mia and atypical chronic myelogenous leukemia (Alexander and Hilton 2004). Furt her, SOCS-1 is likely a tumo r suppressor since aberrant DNA methylation of SOCS-1 gene, resu lting in transcriptional silenc ing, has been observed in human hapatocellular carcinomas and hepa toblastomas. The restoration of SOCS-1 expression in cells in which SOCS-1 gene had been si lenced led to reduction in the transformed phenotype (Alexander and Hilton 2004), providing direct evidence that lack of SOCS-1 expression may have played a role in the development of the malignancies. In addition, constitutive activation of STAT3 and deregulation of SOCS3 expression have been obser ved in a variety of inflammatory diseases (Alexander and Hilton 2004). Thus SOCS proteins play a fundamental role in maintaining health. There are eight members of the SOCS famil y. These are the cytokine-induced SH2 domain containing protein (CIS), SOCS-1, SOCS-2, SO CS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7 (Reviewed in Alexander 2002; Larsen and Rpke, 2002; Alexander and Hilton, 2004). The SOCS proteins are also known as JAK-binding protein (JAB), STAT-induced STAT inhibitor (SSI), and cytokine-inducible SH2 containing (CIS) proteins (Larsen and Rpke, 2002). All SOCS proteins have three shared domains, wh ich are an N-terminal domain of varying length

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25 and sequences, a central SH2 doma in, which is essential for bi nding to the JH1 region of JAK kinases (Yasukawa et al. 1999), and a C-terminal SOCS box. The SOCS box couples substratespecific interactions of the SH2 domain to the ubiqutination machinery, resulting in proteosomal degradation of associated JAK (Alexander 2002). SOCS-1 and SOCS-3 also have the kinase inhibitory region, KIR, which is hypothesized to be involved in catalytic activity of SOCS-1, but not in the actual binding to JAK2 autophosphoryl ation site (Yasukawa et al. 1999, Giordanetto and Kroemer 2003). In this manuscript, data will be provided indicating th at the KIR region is likely involved in direct binding to JAK2 autophosphoryl ation site. Figure 2-2 contains a schematic representation of th e domain structure of SOCS-1. The physiological roles of f our SOCS family members (CIS, SOCS-1, SOCS-2, and SOCS-3) are well defined. I will br iefly discuss the physiological ro le of SOCS-1 as an example of the important role that SOCS protei ns play in maintaining homeostasis. Physiological Role of SOCS-1 Protein SOCS-1 plays a vital role in negative regulation of IFN signaling as shown by both in vivo studies and in vitro assays. SOCS-1 double knockout mice (SOCS-1-/-) die as neonates, displaying low body weight and liver damage including necrosis. Diseased livers are characterized by the presence of aggregates of granulocytes, eosinophils, and macrophages. The SOCS-1-/mice also exhibit monocytic invasion of th e pancreas, the lung, a nd the heart (Starr et al. 1998; Naka et al. 1998) and have a marked reduction in blood and spleen lymphocytes, as well as severe deficiencies in both mature Band T lymphocytes. In addition, mice thymuses are reduced in size and show increased numbers of a poptotic cells both in the spleen and the thymus when compared to normal mice. These symptoms indicate severe defici encies in the immune system (Naka et al. 1998; Starr et al. 1998) and show that SOCS -1 is indispensable for normal neonatal development.

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26 The pathologies observed in SOCS-1-/gene knock out mice were similar to those observed in wild-type mice administered excess IFN which led to the hypothesis that the disease observed in the SOCS-1-/gene knock out mice was likely due to excessive response to IFN Direct evidence that IFN was required for the development of the lethal disease observed in SOCS-1-/gene knock out mice was obtained when the mice were treated from birth with IFN neutralizing antibodies. Af ter three weeks of IFN treatment the anti-IFN treated mice remained healthy, while all the untreated mice had succumb ed to disease (Alexander et al. 1999). In addition, the SOCS-1-/-/IFN -/double knockout mice did not exhi bit the lethal phenotype observed in SOCS-1-/knockout mice (Alexander et al. 1999).Thus, SOCS-1 is a key physiological regulator of IFN signaling. It is worth noting that SOCS-1-/-/IFN -/double knockout mice eventually died 6 months after bi rth with inflammation and polycystic kidneys (Metcalf et al. 2002), which suggested that SOCS-1 regulation was not specific for IFN The prediction that SOCS-1 regulates IFN signaling was confirmed using biochemical studies. Upon injection of IFN into mice, STAT1 phosphorylation was evident in the livers of the SOCS-1+/+ mice within 15 min but declined after 2 h. However, in the SOCS-1-/gene knock out mice, phosphorylated STAT1 remained and was detectable 8 h after IFN administration (Brysha et al. 2001), i ndicating continued IFN signaling. These observations, taken together, indicated that SOCS-1 is a physio logical negative regulator of IFN signaling (Alexander and Hilton 2004) and that unregulated IFN activity contributed to the pathology observed in the SOCS-1-/gene knock out mice. Thus, SOCS-1 play s a fundamental role in regulating IFN signal transduction. It ha s also been shown that SOCS-1 plays a fundamental role in the regulation of IFN and IFN (Fenner et al. 2006) and other cytokines signaling through the JAK/STAT pathway.

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27 SOCS-1 Mimetic Peptides Our laboratory has designed a family of SOCS -1 mimetic peptides, which are being tested for SOCS-1-like activity. The first of these peptid es was tyrosine kinase inhibitor peptide (Tkip, WLVFFVIFYFFR), which was designed usi ng a complementary peptide approach for complementarity to the JAK2 autophosphorylation s ite (Flowers et al 2004). Tkip binds to the JAK2 autophosphorylation site a nd inhibits JAK2 autophosphor ylation and JAK2 mediated phosphorylation of the IFNGR-1 (Flowers et al. 2004). Tkip also inhibits the autophosphorylation of the epiderma l growth factor receptor (EGF R), consistent with the fact that EGFR is regulated by SOCS-1 and SOCS3. In contrast, Tkip doe s not bind or inhibit tyrosine phosphorylation of the vascular endoth elial growth factor receptor (VEGFR) or the substrate peptide of the protooncogene, c-Src (Flowers et al. 2004), both of which are not regulated by SOCS-1 suggesting specificity of Tkip-mediated inhibition. Although Tkip binds to unphosphorylated JAK2 autophosphorylation site peptide, JAK2(1001-1013), it binds significantly better to phosphorylated JAK2 autophosphorylation site peptide, pJAK2(10011013). It has been suggested that SOCS-1 rec ognizes and binds only to phosphorylated JAK2, therefore Tkip recognizes the JAK2 autophospho rylation site similar to SOCS-1, but not in precisely the same way. Consistent with inhibition of JAK2, Tkip also inhibits the ability of IFN to induce an antiviral state as well as upreg ulation of MHC class I molecules, and blocks the phosphorylation of both STAT1 and STAT3 (Flowers et al. 2004) Tkip also inhibits the proliferation of the prostate cancer cell lines DU145 and LNCaP, in a dose dependent manner (Flowers et al. 2005). In addition, Tkip has been shown to protect mice from EAE, an animal model for the human inflammatory disease, multipl e sclerosis (Mujtaba et al. 2005) via blockage activation of inflammatory cytokines. Hence, Tk ip appears to have both anti-inflammatory and anti-tumor properties.

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28 Tkip is synthesized from L-amino acids, whic h are the physiological form of amino acids in nature and therefore are poten tially readily degraded by proteases Hence, if Tkip were to be used as a therapeutic agent, there is potential that it may be readily degraded. This would likely limit Tkip usefulness as a therapeutic agent. To address this limitation, we synthesized other Tkip-related mimetic peptides. These we re DTkip (WLVFFVIFYFFR) and DRTkip (RFFYFIVFFVLW), which were synthesized fr om D-amino acids, which from a medicinal chemistry view, were likely to be more resistant to proteolytic digestion and therefore likely to be better therapeutics. DTkip has the same amin o acid sequence as Tki p, while in DRTkip the sequence is reversed, in other words DRTkip is a retro-inversion of Tkip. DTkip and DRTkip were tested for SOCS-1-like activity such as the ability to bind to JAK2, inhibit JAK2 autophosphorylation, and inhibit JAK2-mediated phosphorylation of the substrates of the JAK/STAT pathway. In addition, the peptides were also tested for eff ects on cell proliferation, IFN -induced antiviral activity and upregulation of MHC class I molecules. In addition some Tkip cellular targets were identified. I will pres ent data describing a family of SOCS-1 mimetic peptides and present a proof-of-concept fo r the therapeutic potential of Tkip. Inhibition of SOCS-1 Activity and Immunological Relevance As stated earlier, SOCS-1 is a negative re gulator of immune factors including IFNs, interleukins -3, -4, -6, -7, a nd tumor necrosis factor (TNF ) as well as a variety of hormones such as growth hormone (Tan and Rabki n 2005). This raises the possibility that one way of enhancing cytokine-mediated immune response to pathogen infection may be by inhibition of SOCS-1 activity. It has recently been shown that sile ncing of SOCS-1 in dendritic cells promoted cell activation, which led to enhan cement of effective antigen-specific anti-tumor immunity (Shen et al. 2004). This implies that reagents that regulate SOCS-1 may enhance immune responses and therefore ha ve potential in control and/or treatment of diseases arising

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29 from inadequate immune responses. A SOCS-1 inhibitor peptide, pJAK2(1001-1013), that is derived from the JAK2 autophos phorylation site was designed and synthesized. I will present data showing that pJAK2(1001-1013) is a SOCS-1 antagonist.

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30 C N JH3 65 aa JH7 65 aa JH5 65 aa JH6 125 aa JH4 150 aaJH1 215 aa Kinase domain, includes Y1007 JH2 245 aa Pseudo-kinase domain Receptor binding domain Figure 2-1. A schematic representation of the dom ain structure of the JAK kinase family. The JAK tyrosine kinase protein family contai ns four members: JAK1, JAK2, JAK3, and TYK2. Each JAK molecule contains se ven distinct regions : JH1-JH7. The JH1 domain is the catalytic domain, whic h includes the activation loop, in which autophosphorylation site (Y1007) is located. The JH2 domain is the pseudo-kinase domain and is believed to play a role in the autoregulatory ac tivities of JAK2. The JH6-JH7 domains mediate binding of JAK mo lecules to cytokine receptor proteins. Adapted with modifications from Imada and Leonard 2000.

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31 SOCS-1 N termin us KIR ESS SH2domain SOCSBox Figure 2-2. A schematic diagram showing th e domain structure of SOCS-1 protein. All SOCS proteins have an N-terminal region of va rying length and sequ ence, a central SH2 domain, and a C-terminal SOCS box. SOCS-1 and SOCS-3 have a kinase inhibitory domain (KIR), which lies between the Nterminal and the SH2-domain. In SOCS-1, the 12-amino acids N-terminal and conti guous to the SH2 domain form the extended SH2 (ESS) region, and the 12-amino acid resi dues N-terminal and contiguous with the ESS form the kinase inhibitory region (KIR). The SOCS1-KIR peptide is derived from the KIR, while the SOCS1-ESS pe ptide is derived from the ESS region.

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32 Table 2-1. A table showing the JAK kinases and STAT proteins utilized by some cytokines, growth factors, hormones, and oncogenes. Cytokines/hormones/oncogenes/ Growth factors JAK STAT Cytokines IFNJAK1 and JAK 2 STAT 1 IFN/ JAK1 and TYK-2 STAT 1, 2, 3, 4, 5A, 6 IL-2 JAK 1 and JAK 3 STAT 1, 3, 5A/5B IL-3 JAK 2 STAT 1, 3, 5A/B, 6 IL-4 JAK 1 and JAK 3 STAT 6 IL-6 JAK 1 STAT 3, 5A/B IL-10 JAK 1 and TYK-2 STAT 1, 3 IL-12 JAK 2 and TYK-2 STAT 1, 3, 4, 5 Growth factors/hormones EGF JAK2 STAT 1, 2, 3, 5 Growth hormone JAK2 STAT 1, 3, 5A/B Insulin JAK1 STAT3, STAT 5B Oncogenes V-abl JAK1 STAT 1, 3 and 5 V-src JAK1 and JAK2 STAT 3 Adapted with modification from Subramaniam et al. 2001

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33 CHAPTER 3 MATERIALS AND METHODS Cell Culture All the cell lines, except Sf9 insect cells, we re obtained from the American Type Culture Collection (Manassas VA). The human prostate cancer cells, LNCaP, and the murine macrophage cells, Raw 264.7, were maintained in RPMI (JRH Biosciences, Lenexa, KS) supplemented with 10% FBS (Hyclone, L ogan, CT), 100 U/mL penicillin, 100 U/mL streptomycin (complete media). Murine fibroblas t cells, L929, were maintained in DMEM (JRH Biosciences), supplemented with FBS, penici llin, streptomycin, non-essential amino acids, sodium bicarbonate, and sodium pyruvate. The muri ne monocyte cell line, U937, was maintained in RPMI complete media supplemented with 10 mM HEPES (Sigma-Aldrich, St. Louis, MO), and 1 mM sodium pyruvate (Sigma-Aldrich). All the cell types we re cultured at 37 C and 5 % carbon dioxide humidified incubato r. The Sf9 cells obtained fr om Invitrogen (Invitrogen Corporation, Carlsbald, CA ) were maintained at 27oC as adhesion cultures in complete TNM-FH media (Grace Insect Medium, Supplemented, c ontaining 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin) or as suspension cultur es in Sf-900 SFM media containing 5% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. Peptides The peptides used in this study are listed in Table 3-1, and were synthesized by Mr. Mohammed Haider, in our laboratory on an Applied Biosystems 9050 automated peptide synthesizer (Foster City, CA) using conventi onal fluorenylmethyoxycarbonyl (Fmoc) chemistry as previously described (Szente et al. 1996). A lipophilic group (p almitoyl-lysine) was added to the N-terminus of peptides, to facilitate entry in to cells, as a last step using a semi-automated protocol (Thiam et al. 1999). Peptides were characterized by mass spectrometry and where

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34 possible, HPLC purified. Peptides were dissolved in water or in DMSO (Sigma-Aldrich, St. Louis, MO). The control peptides used in this project did not s how significant biol ogical activity in the systems tested. Binding Assays Binding assays were performed as previously described (Flowers et al. 2004) with minor modifications. Tkip, SOCS1-KIR, and control peptide, MuIFN (95-106) at 3 g/well, were bound to 96-well plates, in binding buffer (0.1 M sodium carbonate and sodium bicarbonate, pH 9.6). The wells were washed three times in wash buffer (0.9% NaCl and 0.05% Tween-20 in PBS) and blocked in blocking buffer (2% gelati n and 0.05% Tween-20 in PBS) for 1 h at room temperature. Wells were then washed three times and incubated with various concentrations of biotinylated JAK2(1001-1013) or biotinylated pJAK2(1001-1013), in blocking buffer, for 1 h at room temperature. Following incubation, wells we re washed five times and bound biotinylated peptides were detected using HRP-conjugated neutravidin (Molecu lar Probes) and color detected using o -phenylenediamine in stable peroxidase buffer (Pierce, Rockford, IL). The chromogenic reaction was stopped by the addition of 2 M H2SO4 (50 L) to each well. Absorbance was measured at 490 nm using a 450-microplate read er (Bio-Rad Laboratories, Hercules, CA). Peptide competition assays were carried out as described above except following peptide immobilization, washing, and blocking, bio tinylated JAK2(1001-1013) or biotinylated pJAK2(1001-1013), which had been pre-incubated with various concentrations of soluble peptide competitors (Tkip, SOCS1-KIR or c ontrol peptide) was added. Detection of bound biotinylated peptide was conducted as describe d above. Data obtained fr om binding assays was plotted using Graph Pad Prism 4.0 software (Graph Pad Software, San Diego, CA).

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35 In vitro Kinase Assays Autophosphorylation activity of JAK2 was m easured in a reaction mixture containing GST-JAK2 kinase fusion protein (Cell Si gnaling Technology, Danvers, MA), ATP (20 M, Cell Signaling) and the appropriate peptide in kina se buffer (10 mM HEPES, pH 7.4, 50 mM sodium chloride, 0.1 mM sodium orthovanadate, 5 mM magnesium chloride, and 5 mM manganese chloride). It had previously been determined that soluble IFNGR-1 enhanced JAK2 activity (Flowers et al. 2004), therefore IFNGR-1 (2 g/ reaction) was adde d and the reaction mix incubated at 25 C for 30 min with intermittent agitation. The assays were carried out according to a JAK2 kinase protocol obtained from Cell Signaling (C ell Signaling Technology, Danvers, MA), but with modifications, derived in part from Flowers et al. ( 2004). The reactions were terminated by addition of appropriate volume of 6 X SDS-PAGE loading buffer (0.5 M Tris-HCl (pH 6.8), 36% glycerol, 10% SDS, 9.3% DTT, 0.012% bromophenol blue), and heating at 95 C for 5 min. The proteins were separated on a 12% SDS-polyacrylamide gel (Bio-Rad Laboratories), transferred onto ni trocellulose membrane (Amers ham Biosciences, Piscataway, NJ), and probed with anti-pJAK2 antibodies (Santa Cruz Biotec hnology, San Diego, CA). Membranes were then stripped and re-pr obed with anti-JAK2 antibody (Santa Cruz Biotechnology). Detection of proteins was accomp lished using ECL protein detection reagents (Amersham Biosciences). Autophosphorylation activity of epidermal grow th factor receptor (EGFR, Upstate) was measured in a 50 l reaction containing kinase buffer, 0.2 g EGFR (Upstate Biotechnology), 0.1 g EGF (Upstate Biotechnology), 20 M ATP (Cell Signaling), a nd the appropriate peptide (50 M) as described in Flowers et al. (2004). The reaction mix was incubated for 30 min at 250C, resolved by 12% SDS-PAGE, transferred onto nitrocellulose membrane, and

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36 immunoblotted with anti-pEGFR antibody. The membranes were then stripped and reprobed with anti-EGFR antibody. Detection of proteins was accomplished using ECL protein detection reagents (Amersham Biosciences). Immunoblot Analysis U937 murine fibroblast cells were plated on 6-well plates at a cell density of 1 x 106 cells/ well and after an overnight incubation at 37 C and 5% CO2, the cells were cultured in complete media containing varying concentrations of li pophilic Tkip, lipophilic SOCS1-KIR, or lipophilic control peptide for 18 h at 37 C in a 5% CO2 incubator. To activate the JAK/STAT signaling pathway, U937 cells were then incubated in the presence or absence of 1000 U/mL IFN (PBL Biochemical Laboratories, Piscataway, NJ) for an additional 30 min. The media was aspirated out and the cells washed twice with cold PBS to remove media and cell debris. Cell lysates were prepared by adding 250 L of cold lysis buffer (50 mM Tris-HCl (pH 7.4), 250 mM NaCl, 50 mM NaF, 5 mM EDTA, and 0.1% NP40) contai ning protease inhibitor cocktail (Amersham Bioscience) and phosphatase inhibi tors (Sigma-Aldrich, St. Louis, MO). Lysis was allowed to proceed for 1 h at 4 C (rocking) to ensure complete cell lysi s. Lysates were then centrifuged and supernatant transferred into fres h microcentrifuge tubes, protein concentrations determined, and protein lysates resolved by SDS-PAGE on a 12% polyacrylamide gel (Bio-Rad Laboratories). Proteins were transferred onto nitrocellulose membranes (Amersham Biosciences), placed in blocking buffer (5% nonfat dry milk and 0.1% Tw een-20 in TBS), and washed in 0.1% Tween20 in TBS. To detect phosphorylated STAT1 membranes were incubated with pY701-STAT1 antibody (1:400 dilution; Santa Cruz) in blocking buffer for 2 h at room temperature. After four washes, the membranes were incubated in HR P-conjugated goat anti-rabbit IgG secondary antibody (1:2000 dilution; Santa Cruz) in blocking buffer for 1 h at room temperature, washed

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37 four times, incubated for 1 min with ECL det ection reagents (Amersham Biosciences) and exposed to photographic film (Amersham Bi osciences) to visualize protein bands. Macrophage Activity Murine macrophage cells, Raw 264.7, were seeded on 24-well plates at a concentration of 3 x 105 cells/ well and allowed to adhere. Varying concentrations of the lipophilic peptides, Tkip, SOCS1-KIR or MuIFN (95-106), were then added to the wells and the cells incubated for 2 h at 37 C in a 5% CO2 incubator. Recombinant IFN in varying concentrations, was then added and the cells incubated for an additional 72 h at 37 C in a 5% CO2 incubator, after which supernatants were transferred into fresh tubes and assayed for nitrite levels as a measure of nitric oxide production using Griess reagent, according to manufactu rers instructions (Alexis Biochemicals, San Diego, CA). To test for s ynergy between Tkip and SOCS1-KIR, the cells were incubated in the presence of IFN and varying concentrations of peptides as described above and also in the presence of both lipophi lic Tkip and lipophilic SOCS1-KIR or lipophilic Tkip and lipophilic MuIFN (95-106). Supernatants were collected after 48 h and tested for nitric oxide production as described above. Tkip Cellular Targets SJL/J mice were immunized with bovine my elin basic protein (MBP) as previously described (Mujtaba et al. 2005). Briefly, 6 to 8 week old female SJL/J mice were immunized subcutaneously at two sites on the base of the tail, with MBP (300 g/mouse) in complete Freunds adjuvant. At the time of MBP immuni zation and 48 h later, pertusis toxin (400 ng/mouse) was administered (i.p ). This protocol was approved by IACUC at the University of Florida. The mice were observed daily for signs of EAE, and se verity of disease was graded using the following scale: 1) loss of tail t one; 2) hind limb weakness; 3) paraparesis; 4)

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38 paraplegia; and 5) moribund/death. The first physical signs of di sease were generally observed beginning on day 18 to 21 after MBP immunization. Spleens were extracted after disease onset and homogenized into a single cell suspension. Splenocytes (1 x 105 cells/well) were incubated with medium, MBP (50 g/mL), lipo-Tkip, lipo-SOCS1KIR or control peptide lipoMuIFNGR(253-287) for 48 h at 37 C in 5% CO2. To test the effect of the peptides on cell proliferation, the cultures were pulsed with [3H]-thymidine (1.0 Ci/well; Amersham Biosciences) for 18 h before harvesting onto filter paper discs using a cell harvester. Cell associated radioactivity was quan tified using a beta scintillation counter and data are reported as counts per minute (cpm). To test for the effect of Tkip on specific cellu lar targets, splenocytes obtained as described above were enriched for the desired cell type us ing negative isolation ki ts purchased from Dynal Biotech (Dynal Biotech, Oslo, Norway). The en richments were carried out according to the manufacturers inst ructions. Mouse B cells negativ e isolation kit, mouse CD4+ T cells negative isolation kit, or mouse CD8+ T cells negative isolation kit was used for B cells, CD4+ T cells, or CD8+ T cells, respectively. The use of the isolation kits results in cells enriched for the specific cell type, but to confirm the purity level, the cells may be stained w ith cell-type specific antibodies and FACS (fluorescence activated cell sorter) analysis carried out. This would have given a better indication of the actual purity level, and w ould provide an indication what contaminants (if any) were present. Enriched cells were incubated with varyi ng concentrations of appropriate lipophilic peptides in the presence or absence of antigen presenting cells (APCs), and in the presence or absence of antigen (MBP). The APCs were derive d from splenocytes obtained from nave SJL/J mice, which were incubated with MBP for 48 h, fixed with 2% paraformaldehyde for 30 min,

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39 and washed extensively to remove residual para formaldehyde. The cells were then transferred into mouse IFN -ELISPOT plates (Mabtech Inc, USA) and incubated for 48 h, after which the wells were washed, incubated with secondary antibody, washed, and spots developed according to manufacturers instructions. The plates were then blotted dry, spots counted, and the data plotted using Graph Pad Prism 4.0 software (Graph Pad Software, San Diego, CA). To test for the effect of Tkip on antibody production, B cells (5 x 105 cells/well) from MBP sensitized mice obtained two months after di sease remission were incubated with varying concentrations of lipophilic peptid es in the presence of MBP (50 g/mL) and APCs and incubated for 48 h. Culture supernatants were then harvested and tested for MBP-specific antibodies by enzyme-linked im munoabsorbent assay (ELISA). Antiviral Assays for SOCS-1 Antagonist Function Antiviral activity was determined using the standard viral cytopa thogenic effect assay described previously with minor modifications (Langford et al. 1981). Br iefly, human fibroblast WISH cells at 70% confluency, were incuba ted in media alone or 0.4 U/mL human IFN (PBL Biomedical Laboratories ) or both 0.4 U/mL human IFN and lipo-pJAK2(1001-1013) or lipo-JAK2(1001-1013) for 22 h in DMEM contai ning 2% FBS (maintenance media). Following incubation, WISH cells were washed once w ith maintenance media and infected with encephalomyocarditis virus (EMCV) (200 pfu/well) for 1 h at 37 C. The WISH cells were then washed once to remove unbound viral particles and incubated in fresh maintenance media for an additional 24 h at 37 C in a 5% CO2 incubator. Plates were subse quently blotted dry and stained with 0.1% crystal violet soluti on for 5 min. Unbound crystal violet was aspirated and the plates thoroughly rinsed with deionized water, blotted, and air-dried. Vi ral plaques were counted using

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40 a dissecting microscope and an tiviral activity was determined by comparing experimental treatment groups with the vi rus only control group. Transfections of LNCaP Cells with SOCS-1 DNA Transfections were carried out to introduce SOCS-1 DNA into mammalian cells and test the ability of pJAK2(1001-1013) peptide to reverse SOCS-1 inhibition of STAT3 phosphorylation. Human prostate cancer cells, LNCaP, were plated in a 6well plate and allowed to grow to 60% confluency. SOCS-1 plasmid DNA (1.6 g/well) (pEF-FLAG-I/mSOCS1), a gift from Dr. David Hilton (Walter and Eliza Hall Ins titute of Medical Research, Victoria, Australia) or an empty vector, was transfected into th e LNCaP cells using lipofectamine (Invitrogen Corporation, Carlsbad, CA) according to manufactu rers instructions, but with modifications. The cells were incubated for 4 h, after which the tr ansfection media was aspirated, fresh complete media (DMEM supplemented with 10% FBS and 100 U/mL streptomycin and 100 U/mL penicillin) added, and the cells incubated for an additional 72 h at 37 C. The complete media was then aspirated out, fresh media containing lipo-pJAK2(1001-1013) (20 M) or control peptide, lipo-MuIFN (95-125), added to the transfected cells and the JAK/STAT signaling pathway activated by adding IL-6 (50 ng/mL). Cells were incubated for 30 min prior to harvesting. Cell extracts were resolved by SDS-PAGE on a 12% polyacrylamide gel, transferred onto nitrocellulose membrane (Amersham Bioscien ces), and probed with phosphorylated (pY705) STAT3 antibody (Santa Cruz Biotechnology). The me mbranes were stripped and reprobed with unphosphorylated STAT3 antibody (Santa Cruz Bi otechnology). Detection of proteins was accomplished using ECL detection reagents (Amersham Biosciences). For immunoprecipitations, LNCaP cells growing on 60 mm plates and at 50% confluency were transfected with either SOCS-1 plasmid DNA (8 g/plate) or empty vector (8 g/plate) in

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41 lipofectamine and incubated for 4 h after whic h 5 mL complete media was added and cells allowed to grow for 72 h. The cells were harveste d in lysis buffer as described for western blot analysis, and the cell lysates ce ntrifuged at 10000 x g to remove cellular debris and nuclei. Supernatants were transferred into fresh tubes and incubated with 2 g/mL anti-Flag antibody (Sigma-Aldrich) for 2 h at 4C while rotating. Protein G PLUS-agarose beads (Santa Cruz Biotechnology) were added to th e supernatants and allowed to incubate for 2 h at 4C while rotating, followed by centrifugation to pellet prot ein G immune complexes. Supernatants were discarded and the immune complexes washed thre e times with lysis buffer and once with PBS. The immune complexes were then heated (95C/5 min) in 50 L of 1 X SDS sample buffer, resolved on a 12% polyacrylamide gel, transfer red onto nitrocellulose, and immunoblotted with anti-SOCS-1 antibody (Santa Cruz Biotechnology). Detection of proteins was accomplished using ECL detection reagents (Amersham Biosciences). GAS Promoter Activity A plasmid, pGAS-Luc, that cont ains the promoter for IFN -activated sequence (GAS) linked to firefly luciferase gene was obtained from Statagene (L a Jolla, CA). A constitutively expressed thymidine kinase promoter-driven Renilla luciferase gene (pRL-TK) (Promega Corporation, Madison, WI) was used as internal control in reporter ge ne transfections. WISH cells (1 x 105 cells/well) were seeded in a 12-well plate and incubated overnight at 37C, following which 3 g GAS promoter-driven firefly lucife rase expressing plasmid DNA and 10 ng pRL-TK were cotransfected into the WISH cells, using Fugene 6 (Roche Diagnostics Corporation, Indianapolis, IN). Two days later, the cell lysates were used to assay for firefly luciferase and Renilla luciferase, using a dua l luciferase assay kit (Promega Corporation).

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42 Luciferase activity, in relative luciferase units was calculated by dividing firefly luciferase activity by Renilla luciferase activity in each sample. Primer Design and PCR Amplific ation of Murine SOCS-1 DNA Primers were designed to amply full-length SO CS-1 from the mouse SOCS-1 intracellular expression vector, pEF-FLAG-I/mSOCS-1 previous ly described. Primers were designed for compatibility with the multiple cloning site (M CS) of pBlueBac4.5/V5-His TOPO TA vector (Invitrogen). The primers were purchased from Integrated DNA Technology Inc and the primer sequences are shown below. Forward primer 1 5AGG ATG GTA GCA CGC AAC CAG GT 3 Reverse primer 5GAT CTG GAA GGG GAA GGA AC 3 PCR reactions were carried out in 50 L reactions containing dNTPs (0.2 mM each), MgCl2 (1.5 mM), primers (0.2 M each), template DNA (1 g), platinum Taq DNA polymerase (1 unit), platinum Pfx polymerase (1 unit) 10 X PCR buffer (5 L) and DNAse free water. The thermocycler was programmed for 30 cycles at 94oC for 30 sec, 50oC for 30 sec, 72oC for 1 min, with initial denaturation at 94oC for 4 min and final extension at 72oC for 7 min. The PCR products were resolved on a 1% TAE agarose gel, stained with ethidium bromide, and photographed. Cloning SOCS-1 into pBlueBac4.5/V5His TOPO TA Expression Vector The PCR products of correct size (650 bp) were excised out of the gel, purified using Wizard PCR prep DNA purification system (P romega, Madison WI) and cloned into the pBlueBac4.5/V5-His TOPO TA expression vector according to the manufact urers instructions (Invitrogen). Following cloning, the recombinan t vector was used to transform TOP10

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43 chemically competent cells according to manuf acturers instructions (Invitrogen), and the transformed cells grown on LB-ampicilin nutrient agar plates overnight at 370C. Positive and negative control cloning reactions were carried out similar to t hose for SOCS-1 DNA, but in the absence of SOCS-1 DNA. Positive clones were id entified by PCR, and plasmid DNA isolated from the PCR positive clones. Restriction enzyme digestion and DNA sequencing were used to confirm the presence of SOCS-1 DNA. Expression of SOCS-1 in Sf9 Cells The recombinant pBlueBac4.5/His-V5 TOPO ve ctor carrying the murine SOCS-1 gene (pBlueBac4.5/muSOCS-1) was transfected into th e expression vector Bac-N-Blue (Invitrogen) according to the manufacturers inst ructions. Briefly, the Bac-N-Blue vector contains a triple-cut, linearized AcMNPV ( Autographa californica multiple nuclear polyhedrosis virus). The linearized virus lacks sequences essential for efficient propagati on, specifically sequences in the ORF1629. Hence, for successful propagation and is olation of viable virus, the essential ORF1629 sequences need to be supplied by a tr ansfer vector. The pBlueBac4.5/His-V5 TOPO vector contains these essential sequences. Th e pBlueBac/muSOCS-1 and Bac-N-Blue were cotransfected into Sf9 ( Spodoptera frugiperda ) insect cells growing at 50% confluency (Invitrogen) in the presence of cellfectin reagent (Invitrogen) in unsuppl emented Grace Insect Medium (Invitrogen). The transfection was carried out at room temperature and the transfection complexes incubated with the cells for 6 h following which complete TNM-FH medium was added and the cells allowed to grow for an addi tional 72 h. Half the volume of the culture media was harvested and used for plaque assay. The same volume of fresh TNM-FH media was added to the cells, and the cells allowed to grow for an additional 72 h at which point significant cell lysis was observed. The virus (P1 stock) was then collected by centrifugation and tested for the presence of murine SOCS-1 DNA by PCR using the Baculovirus primers.

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44 Forward primer 5TTTAC TGTTTTCGTAACAG TTTTG 3 Reverse primer 5 CAAC AACGCACAGAATCTAGC 3 In order to generate high titer recombinant virus, PCR positive clones were propagated further and used to infect fresh Sf9 cells grow ing in suspension culture. The cells were allowed to grow and harvested at differe nt time points to determine the best time for harvesting cells expressing SOCS-1 protein. It was determined th at 96 h infection provide d the highest SOCS-1 protein yield, and subsequently Sf9 cells were harvested 96 h post infection. Cell lysates were harvested using native conditions as described in the ProBond purification manua l (Invitrogen) and the expression of murine SOCS-1 confir med by immunoblot analys is using anti-SOCS-1 antibody (Santa Cruz). Statistical Analysis The data were not normally distributed therefor e nonparametric statistic al analyses tests, the Mann-Whitney and Wilcoxon tests were us ed. The Mann-Whitney signed rank sum test compares two groups and performs calculations on the rank of the values, rather than the actual data (Motulsky 1995). It is consider ed to be similar to the t-test except the data are not normally distributed. The Wilcoxon signed rank sum test co mpares two paired groups of nonparametric data (Motulsky 1995). It is similar to the paired t-test, but the da ta are not normally distributed. All calculations were performed using the Gr aphPad Prism statistical package (GraphPad Software Inc, San Diego, CA).

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45 Table 3-1 List of peptides used in this study. DT kip and DRTkip were synthesized from Damino acids. All the other peptides were synthesized from L-amino acids. The Y1007 in pJAK2(1001-1013), in italics, is phosphorylated. Peptide Sequences Tkip WLVFFVIFYFFR DTkip WLVFFVIFYFFR DRTkip RFFYFIVFFVLW JAK2(1001-1013) 1001LPQDKEYYKVKEP pJAK2(1001-1013) 1001LPQDKE Y YKVKEP MuIFN (95-106) 95AKFEVNNPQVQR MuIFN (95-125) 95AKFEVNNPQVQRQAFNELIRVVHQLLPESSL SOCS1-KIR 53DTHFRTFRSHSDYRRI SOCS1-ESS 68ITRASALLDACG MuIFNGR1(253-287) 253TKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYS MuIFN (95-106), MuIFN (95-125), and MuIFNR1(253-287) were used as control peptides. These peptides do not show signi ficant biological activity in th e assays for which they have been used as control peptides.

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46 CHAPTER 4 RESULTS Tkip Family Members Bind to JAK2 Autophosphorylation Site It has previously been shown that Tkip binds to the autophosphoryla tion site of JAK2 and inhibits JAK2 autophosphorylation and phosphorylat ion of IFNGR-1 (Flowers et al. 2004). Here I show that DTkip and DRTkip but not the control peptide, MuIFN (95-106), bound to the JAK2(1001-1013) peptide in a dose-dependent ma nner (Figure 4-1a). Thus, these SOCS-1 mimetic peptides bind JAK2 autophosphorylation site peptide in a dose-dependent manner. Current literature suggests th at phosphorylation of JAK2 ty rosine 1007 is important in SOCS-1 mediated JAK2 ubiquitin-proteosome-d ependent degradation (Ungureanu et al. 2002). Hence, I wanted to determine whether these peptides, like SOCS-1, bind to phosphorylated JAK2, pJAK2(1001-1013). Dose response solid-phase ELISA were carried out with biotinylated pJAK2(1001-1013) peptide in place of bioti nylated JAK2(1001-1013) peptide. DTkip and DRTkip bound with two to thr eefold greater affinity to pJ AK2(1001-1013) than to JAK2(10011013) as shown in Figure 4-1b. This is consistent with previous data showing that Tkip binds with a greater affinity to pJAK2(1001-1013) th an to JAK2(1001-1013) (Flowers et al. 2004). The binding observed was dose dependent, implying specificity in binding. Hence, the SOCS-1 mimetic peptides recognize JAK2 autophosphorylati on site similar to Tkip, implication of which is that they recognize JAK2 aut ophosphorylation similar to SOCS-1. In order to determine whether Tkip and DRTkip bind to the same site on JAK2 autophosphorylation site, competition for binding a ssays were carried out. Soluble Tkip and soluble DRTkip, but not th e control peptide (MuIFN 95) inhibited binding of biotinylated JAK2(1001-1013) to immobilized Tkip in a dose dependent manner (Figure 4-2a) Next, the competition for binding of Tkip with DRTkip to pJAK2(1001-1013) was determined. As shown

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47 in Figure 4-2b, Tkip and DRTkip inhibited the binding of pJAK2(1001-1013) to immobilized Tkip. Hence, Tkip and DRTkip likely bind to the same site on JAK2 autophosphorylation site, the implication of which is that DRTkip may have similar binding characteristics as Tkip to JAK2. The D amino acid isomer thus, binds similar to the L isomer, suggesting a stable form of Tkip for functional studies. Tkip Family Members Inhi bit JAK2 Kinase Activity SOCS-1 regulates JAK2 activity by interac ting with the autophosphorylation site and inhibiting JAK2 kinase activity. Therefore, DRTkip was tested for its ability to inhibit JAK2 autophosphorylation and JAK2 phosphorylation of substrate (IFNGR-1). DRTkip but not the control peptide, MuIFN (95-106) inhibited JAK2 autophos phorylation and JAK2 induced phosphorylation of IFNGR-1 (Figure 4-3). These results are similar to what has been previously shown with Tkip (Flowers et al. 2004). The da ta suggest that DRTkip may inhibit both JAK2 activation and JAK2-mediated phosphor ylation of IFNGR-1, the implic ation of which is that the SOCS-1 mimetic peptides, like SOCS -1, may potentially regulate IFN signaling. It is worth noting that like Tkip, DRTkip did not inhibit ty rosine phosphorylation of c-Src kinase (data not shown), which is consistent w ith the fact that c-Src kinase is not inhibited by SOCS-1. Tkip Inhibits Superantigen-induced Pr oliferation of Mouse Splenocytes The staphylococcus superantigens are potent T cell mitogens that ex ert their effects by forming complexes with MHC class II molecules on antigen presenting cells, and binding to the T cell receptor (TCR) via the V-element of the (TCR), resulti ng in activation of the T cells (Reviewed in Torres et al. 2001). Our laborator y has shown that superantigens such as staphylococcus entrotoxin A and B (SEA and SE B) can exacerbate immunological disease and induce relapses in the mouse model for multiple sclerosis, experimental allergic encephalomyelitis, EAE, (Torres et al. 2001). It ha s previously shown that Tkip inhibits antigen-

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48 induced proliferation of splenocytes (Mujtaba et al. 2005). Here the ability of Tkip and DRTkip to inhibit superantigen-induced proliferation of primary cells was tested. Mouse splenocytes were stimulated with SEB (500 ng/mL) in the pr esence of lipophilic Tki p, lipophilic DRTkip, or lipophilic control peptide, MuIFN (95-106) (a lipophilic group, lysy l-palmitate, is added to the N-terminal ends of the peptides to facilitate entry into the cells) and incubated for 72 h prior to pulsing with 3-[H]-thymidine. As shown in Figure 4-4a Tkip but not DRTkip inhibited SEB induced proliferation of the splenocytes. Next I te sted the ability of Tkip and DRTkip to inhibit SEA-induced splenocytes proliferation. Tki p, but not DRTkip inhibited SEA-induced splenocytes proliferation (Figure 4-4b). Next the ability of Tkip and DRTkip to inhibit STAT1 phosphorylation in murine fibroblast cells (U937) was tested. Tkip, but not DRTkip inhibited STAT1 phosphorylation (Data not show n). The lysyl-palmitate group is an L-lysine and may be affected by proteases in such a way as to affect the efficiency of DRTk ip uptake by cells. It is also possible that the D-isomer amino acids ma y result in a peptide whose conformation is different enough from Tkip to have slightly di fferent cellular function. However, since only a limited number of cellular function assays were carried out, it can not be conclusively be determined whether DRTkip has or does not ha ve intracellular SOCS-1 like function. Thus, factors currently unknown may affect DRTkip intracellular function. The observations that DRTkip, unlike Tkip, did not seem to significantly affect biological activity suggested that the use of the Tkip retro-inversion, DRTkip, may have changed the orientation of the amino acids enough to affect function. Hence, this research focused on Tkip and SOCS1-KIR and not the other SOCS-1 mimetic s. There is however, continued interest in why DRTkip is not the same as Tkip in cell functional comparisons.

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49 SOCS-1 Kinase Inhibitory Region (SOCS1-KIR ) Binds to JAK2 Autophosphorylation Site Yasukawa et al. (1999) suggested that the binding of SOCS-1 to JAK2 requires the SOCS1 SH2-domain and the extended SH2 domain (ESS) and that the kinase i nhibitory region (KIR), while not required for the initial binding is essential fo r the inhibitory acti on of SOCS-1. Here, I present results showing that a peptide corre sponding to SOCS-1 KIR region, SOCS1-KIR, specifically binds to a peptid e representing the JAK2 autophos phorylation site and inhibits STAT1 activation. Further, I show that SOCS 1-KIR, as well as Tkip, inhibit IFN -induced macrophage activation. In addition, I show th at a peptide corresponding to the ESS region, SOCS1-ESS. Does not bind to the JAK2 autophos phorylation site, the imp lication of which is that the SH2, ESS, and KIR regions may all play role in the binding of SOCS-1 to JAK2. Tkip and SOCS1-KIR Bind to JAK2 Autophosphorylation Site First I determined whether SOCS1-KIR, like th e SOCS-1 mimetic Tkip, binds to the JAK2 autophosphorylation site by carrying out dose-res ponse solid-phase binding assays with JAK2 autophosphorylation site peptide, JAK2(1001-1013). Tkip, SOCS1-KIR, or a control peptide, MuIFN (95-106), were immobilized on 96-well microtiter plates and incubated with biotinylated JAK2(1001-1013) at various concentrations. Tkip and SOCS1-KIR, but not the control peptide, bound to the JAK2(1001-1013) peptide in a dose-de pendent manner (Figure 4-5a). Thus, both Tkip and SOCS-1-KIR specifically bind to the JAK2 autophosphorylation site peptide. While this is consistent with the SOCS-1 mimetic char acter of Tkip, it also provides direct evidence that the KIR region of SOCS-1 can interact directly with JAK2 autophosphorylation site, suggesting that Tkip and KIR r ecognized a similar site on JAK2. Since phosphorylation of Y1007 is required for hi gh catalytic activity of JAK2, it is logical that SOCS-1 would bind with higher affinity to Y1007 phosphorylated JAK2. Consistent with

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50 this, it has previously been shown that Tkip binds with greater affin ity to Y1007 phosphorylated JAK2(1001) peptide, pJAK2(1001-1013), than to unphosphorylated JAK2 peptide (Flowers et al. 2004). I therefore determ ined whether SOCS1-KIR bound to pJAK2(1001) with greater affinity than to JAK2 (1001). Both SOCS1-KIR and Tkip bound to pJAK2(1001-1013) with two to three-fold greater affinity than to JAK2(1001-1013) as shown in Figure 4-5b. In addition, SOCS1-KIR bound to pJAK2(1001-1013) with higher affinity than Tkip. Thus, SOCS1-KIR recognizes JAK2 autophos phorylation similar to Tkip, the implication of which is that Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1. To determine whether Tkip and SOCS1-KIR bind to the same site on the JAK2 autophosphorylation site, binding competition assays were carried out. Tkip or SOCS1-KIR was immobilized on a 96-well plate and biotinylat ed JAK2(1001-1013) or bi otinylated pJAK2(10011013), which had been pre-incubated with Tkip, SOCS1-KIR or a control peptide, was allowed to bind to the immobilized pep tides. As shown in Figure 4-6a, soluble Tkip and soluble SOCS1KIR, but not soluble control pe ptide, inhibited the binding of biotinylated JAK2(1001-1013) to immobilized Tkip. A similar pattern of i nhibition was observed with biotinylated JAK2(10011013) binding to immobilized SOCS1-KIR (Figur e 4-6b). Homologous inhibition was slightly better for both Tkip and SOCS1-KIR, which sugge sts slight differences in recognition of JAK2 autophosphorylation site. Next, the binding competition of Tkip and SOCS1-KIR to pJAK2(1001-1013) was determined. The competition for binding to pJAK2(1001-1013) was similar to that observed in competition for binding to unphosphorylated JAK2 peptide (Figure 4-6c and 4-6d). Again, homologous competition was slightly better, wh ich again suggests slight differences in recognition of the JAK2 autophosph orylation site. These data provi de direct evidence that the

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51 mimetic effect of Tkip is applicable to th e KIR region of SOCS-1 and that Tkip and KIR recognize JAK2 autophosphorylation site similar but not exactly the same. JAK2 Kinase Activity and STAT1 Activation SOCS-1 regulates JAK2 activity at least at two levels. One involves interaction with the autophosphorylation site, which affects JAK2 phosphor ylation of substrates such as STAT1 and STAT3 (Yasukawa et al. 1999). The other level i nvolves induction of proteosomal degradation of both JAK2 and SOCS-1, requiring the SOCS box domain of SOCS-1 (Zhang et al. 2001). Obviously, neither Tkip nor SO CS1-KIR has a SOCS box, so the two peptides were compared for their relative ability to i nhibit JAK2 autophosphorylation as well as phosphorylation of the transcription factor, STAT 1. As shown in Figure 4-7a Tkip but not SOCS1-KIR inhibited JAK2 autophosphorylation. This would suggest that the si milar but slight differences in recognition of JAK2 resulted in significant differences in regulation of JAK2 autophosphorylation. Next the two peptides were compared fo r their relative ability to inhibit IFN activation of STAT1 in murine U937 cells. In contrast to i nhibition of JAK2 autophosphorylation, both Tkip and SOCS1-KIR inhibited JAK2 me diated phosphorylation of STAT1 (Figure 4-7b). Thus, Tkip inhibits JAK2 autophosphorylation as well as JAK2 mediated pho sphorylation of STAT1 in murine U937 cells, while SOCS1-KIR does not inhibit JAK2 autophosphorylation, but does inhibit JAK2 mediated phosphorylation of STAT1 transcription factor. SOCS1-KIR thus shows the same regulatory pattern as SOCS-1, in that JAK2 autophosphorylation is not inhibited, while STAT1 substrate phosphorylation by activ ated JAK2 is inhibited. It has previously showed that Tkip, like SOCS-1, also inhibited EGFR aut ophosphorylation (Flowers et al. 2004). Thus SOCS1-KIR was tested fo r ability to inhibit EGFR phosphorylation. As shown in Figure 4-7c

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52 both peptides inhibited EGFR phosphorylation with Tkip being the more effective inhibitor. SOCS1-KIR is thus similar to SOCS-1 in its kinase inhibitory function. Tkip and SOCS1-KIR Inhibit IFN -induced Activation of Macrophages IFN plays an important role in activation of macrophages for innate host defense against intracellular pathogens as well as serving to brid ge the link between innate and adaptive immune responses (Reviewed in Boehn et al. 1997). Thus, Tkip and SOCS1-KIR were examined for their ability to block IFN activation of the murine macrophage cell line Raw 264.7 as determined by inhibition of nitric oxide (NO) production using Griess r eagent (Alexis Biochemicals). Lipophilic (lipo) versions of the peptides were sy nthesized with palmitic acid for penetration of the cell membrane (Thiam et al. 1999). Both Tkip and SOCS-KIR, compared to control peptide, MuIFN (95-106), inhibited inducti on of NO by various concentrations of IFN as shown in Figure 4-8a. Dose-response with varying con centrations of the pe ptides against IFN (6 U/mL) resulted in increased inhibition of NO productio n by Tkip and SOCS1-KIR with Tkip being the more effective of the inhib itors as shown in Figure 4-8b. The control peptide, MuIFNGR1(253287) was relatively ineffective at inhibition, providing evidence fo r the specificity of Tkip and SOCS1-KIR inhibition. Tkip and SOCS1-KIR in combination (33 M each) were the most effective in inhibition of IFN induction of NO in macrophages. This synergy may reflect differences in recognition of the autophosphoryla tion site of JAK2 by the two peptides. Thus, Tkip and SOCS1-KIR both inhibited IFN induction of NO in macrophages with Tkip being the more effective inhibitor. Tkip and SOCS1-KIR Inhibit Antigenspecific Lymphocyte Proliferation Our laboratory has previously shown that Tkip inhibits antigen-speci fic proliferation of mouse splenocytes in vitro (Mujtaba et al. 2005). Specifically Tkip inhibited proliferation of

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53 splenocytes from mice immunized with bovine myelin basic prot ein (MBP). Here, I compared Tkip and SOCS1-KIR for their relative ability to inhibit prolifer ation of MBP-specific splenocytes in cell culture. Splenocytes (3 x 105 cells/well) were incubated with MBP (50 g/mL) in the presence of lipo-Tkip, lipo-SOCS1KIR, or lipo-control peptide for 48 h and proliferation assessed by testing for [3H]-thymidine incorporation. As shown in Figure 4-9, both Tkip and SOCS1-KIR inhibited MBP-induced pro liferation of splenocyt es, while the control peptide had a negligible effect on prolifer ation. Similar to inhibition of NO production by macrophages, Tkip was more effective than SOCS1-KIR in inhibition of MBP induced splenocyte proliferation with 84, 88, and 97% inhibition at 1.2, 3.7, and 11 M, respectively, compared to 61, 67, and 72% for SOCS1-KIR. Thus, both Tkip and SOCS1-KIR inhibited antigen-induced splenocyte proliferation, which is consistent with SOCS-1 protein inhibition of antigen-specific lymphocyte ac tivity (Cornish et al. 2003). An Extended SH2 Sequence (SOCS1-ESS) Pe ptide does not Bind to pJAK2 (1001-1013) Based in part on binding experiments with tr uncations of SOCS-1 protein, it has been proposed that the SH2 domain plus ESS bind to JAK2 at the activati on site represented by peptide pJAK2(1001-1013), while SOCS1-KIR binds pr imarily to the catalytic site of JAK2 (Yasukawa et al. 1999). Theref ore the SOCS1-ESS peptide, 68ITRASALLDACG, was synthesized and compared with SOCS1-KIR for binding to biotin ylated pJAK2(1001-1013). As shown in Figure 4-10 SOCS1-KIR as well as Tkip bound biotinylated pJAK2(10011013) in a dose-response manner, while SOCS1-ESS failed to bind. Hence, the SOCS1-KIR peptide (residues 56), but not the SOCS1-ESS peptide (6 8) binds to the JAK2 autophosphorylation site. The SOCS 1-KIR peptide, except for residues 53-55 is contained in the SOCS-1 ESS-SH2 construct, dN56, (Yas ukawa et al. 1999) that bound to JAK2

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54 autophosphorylation site. dN56 is a construct in which sequences N-terminal to amino acid residue 56 have been truncated, therefore it cont ains KIR, ESS, SH2 domain, SOCS box, and the C-terminal sequences. Thus, SOCS1-KIR, whic h is N-terminal and contiguous with SOCS1ESS, probably shares overlapping functional si tes with SOCS1-ESS. Clearly, SOCS1-KIR is preferentially recognized by JAK2(1001-1013) compared to the 12-mer SOCS1-ESS. The specific role of the various residues in the SH2 domain of SOCS-1 in JAK2 and pJAK2(10011013) binding remains to be determined. Tkip Cellular Targets Studies carried out in our labor atory have shown that Tkip affects the growth of cells growing in culture, as well as the progression of EAE. Specifically, Tkip inhibits LNCaP and DU145 prostate cancer cells prolifer ation and inhibits an tigen-specific cell proliferation Tkip at 63 g/mouse, given every other day prevented deve lopment of acute form of EAE, and induced stable remission in the chronic relapsing/remitting form of EAE (Flowers et al. 2004, Flowers et al. 2005, Mujtaba et al. 2005). Moreover, no toxicity was observed when Tkip at 200 g/mouse, given every other day for one week (Mujtaba et al. 2005). These data suggested that Tkip may have direct effect on cells of the immune system Hence, I attempted to define Tkip cellular targets. Data are presented showing that Tkip specifically targets antigen presenting cells (APCs), CD4+ T, CD8+ T, and B cells. In addition prel iminary data on SOCS1-KIR peptide cellular targets are also presented. Effect of Tkip and SOCS1-KIR on CD4+ T Cells In order to identify Tkip cellular targets, I first asked whether Tkip had any effect on primary splenocytes derived from myelin basic protein (MBP) se nsitized mice. Sensitization of mice with MBP, in the presence of adjuvant, results in the devel opment of experimental allergic encephalomyelitis, EAE, a disease characterized by paralysis. As shown in Figure 4-9, Tkip and

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55 SOCS1-KIR inhibit antigen-specific primary cell (splenocytes) proliferat ion in a dose-dependent manner. Next I asked, which cell populations were ta rgeted by Tkip. First the effect of Tkip and SOCS1-KIR on CD4+ T cell subset was tested. CD4+ T cells are important in generating effective cell mediated immunity and in mediating humoral immune responses. CD4+ T cells obtained from MBP sensitized mice after disease onset we re incubated with lipoph ilic peptides, in the presence or absence of APCs and MBP. Both Tkip and SOCS1-KIR (33 M), but not the control peptide, MuIFNGR1(253-287) inhibited CD4+ T cell proliferation (Fi gure 4-11a). In addition, Tkip and SOCS1-KIR (33 M), inhibited antigen-induced IFN production by CD4+ T as determined by IFN ELISPOT assays (Figure 4-11b). The presence of MBP enhanced cell proliferation and the number of IFN -producing cells, but the presence of APCs did not seem to have such effects. The data presented for pep tides were obtained in the presence of both MBP and APCs. Hence, Tkip a nd SOCS1-KIR target CD4+ T cells and inhibit antigen-specific CD4+ T cell proliferation and IFN production. Effects of Tkip and SOCS1-KIR on CD8+ T Cells CD8+ T cells play important roles in the im mune response mechanis m including cytotoxic T cell activity and production of cytokines that serve as effectors for various other immune responses. Since Tkip and SOCS1-KIR inhibited CD4+ T cell activity, I asked whether the peptides could also target CD8+ T cells. CD8+ T cells derived from MBP-sensitized mice that were showing signs of active dis ease were treated with lipophilic peptides in the presence or absence of MBP and APCs. IFN ELISPOT assays carried out. Both Tkip and SOCS1-KIR, but not the control peptide, MuIFNGR1(253-287) showed a general trend in inhibiting MBP-induced IFN -production by CD8+ T cells (Figure 4-12). The presence of MBP lead to a slight increase in the number of IFN -producing cells, but the presence of APCs did not seem to have such an effect. The data presented for peptides was obtained in the presence of both MBP and APCs.

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56 Effect of Tkip and SOCS1-KIR on B Cells B cells also play an important role in imm une response mechanism. Activation of B cells results in B cell proliferation and differentiati on into antibody producing cells and memory cells. B cells are also APCs. Tkip was tested for eff ect on B cells derived from MBP-sensitized mice, two months after disease remission. The B cells were treated with either lipophilic Tkip, lipophilic SOCS1-KIR, or lipophilic control peptide, MuIFNGR1( 253-287), or no peptide, in the presence APCs and in the presence, or absenc e of unprocessed antigen (MBP). Tkip and SOCS1KIR, in the presence of MBP and APCs, inhib it antigen-specific B cell proliferation (Figure 413a). In addition, both Tkip and SOCS1-KIR inhib it the secretion of MBPspecific antibodies in MBP-treated B cells (Figure 4-13b) The addition of APCs did not significantly affect the experimental results. The data shows that Tkip and SOCS1-KIR target B cell and inhibit both B cell proliferation and secr etion of antibodies by plasma cells, the implication of which is that Tkip and SOCS1-KIR may have an effect on B cell activity. This is of specific interest in the study of EAE and multiple sclerosis, because th e presence of MBP-specific antibodies and of inflammatory cytokines such as IFN has been shown to exacerbate disease. Hence, this may be one of the mechanisms by which Tkip inhibits disease progression in MBP-sensitized mice. Effect of Tkip on Macrophages I have described experiments showing that Tkip and SOCS1-KIR inhibit IFN -induced macrophage activation (Figure 4-8) I also tested the ability of Tkip to inhibit LPS-induced macrophage activity. Both Tkip and SOCS1-KI R inhibited LPS-induced macrophage activity (Figure 4-14). LPS signaling utilizes the Toll Li ke Receptor 4 (TLR4), a major pathway used in immune response to gram-negative bacteria. Hen ce, these results have possible implication on the effect that Tkip on TLR4 signaling and ther efore in inhibition of excessive signaling through the TLR4. Such excessive signaling has been imp licated in the development of inflammation.

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57 Additional research is being carri ed out to determine additional effects that Tkip may have on TLR4 signaling. Since macrophages are APCs, these da ta are preliminary evidence that Tkip and SOCS1-KIR target APCs. I have therefore shown that Tkip and SOCS1-KI R target and affect the activity of immune cells including CD4+ T cells, CD8+ T cells, B cells, and APCs, the implication of which Tkip and SOCS1-KIR may have effects on an tigen-induce immune responses. SOCS-1 Antagonist Activity of pJAK2 (1001) Peptide The demonstration that the KIR region of SO CS-1 could bind to the autophosphorylated JAK2 peptide raised the possibility that the phosphorylated peptide, pJAK2 (1001), may inhibit the function of endogenous SOCS-1 and thus enhance IFN and IL-6 activities that are mediated by JAK2. As shown in Figure 4-15a, the antiviral activity of a suboptimal dose of IFN (0.4 U/mL) was enhanced against encephalom yocarditis virus (EMCV) in WISH cells by pJAK2(1001). Specifically, unphos phorylated JAK2(1001) at 11 M final concentration reduced EMCV plaques relative to IFN alone by 42%, while the same concentration of pJAK2(1001) reduced plaque s by 59%. This is consistent with better binding of pJAK2(1001) by SOCS1-KIR as show n above and by previous studies showing that SOCS-1 is active against JAK2 phosphorylat ed at tyrosine 1007 (Yasukawa et al. 1999). The peptide alone had no effect on EMCV, simila r to media. Thus, pJAK2(1001) boosts the activity of a suboptimal concentration of IFN possibly interfering with endogenous SOCS-1 activity. At the level of signal transduction, I examined the effects of pJAK2(1001) on activation of STAT3 transcription factor in the LNCaP prostrate cancer cell line. The cells were treated with IL-6 to activate STAT3 signaling, whic h occurs through JAK2 kinase (Flowers et al.

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58 2005). The SOCS-1 gene was overexpressed in these cells, which resulted in reduction of the level of IL-6 induced activation of pSTAT3 as shown in Figure 4-15b. Treatment of the cells with pJAK2(1001) (20 M) resulted in approximately a two-fold increase in activated STAT3 compared to IL-6 treated cells that were transfected with SOCS -1, as per densitometry readings. Expression of SOCS-1 protein in LNCaP cells is shown in Figure 4-15b. Thus, pJAK2(1001) has an inhibitory effect on SO CS-1 at the level of signal transduction. I next determined if pJAK2(1001) could enhance GAS promoter activity of IFN Accordingly, a plasmid with the GAS promoter el ement linked to the firefly luciferase reporter gene was cotransfected along with Renilla luciferase reporter plasmi d as a control, into human WISH cells. As shown in Figure 4-16c, treatment of the WISH cells with IFN (1 U/mL) resulted in a four-fold relative increase in luciferase activity, wh ich was increased to ten-fold and five-fold by 5 and 1 M pJAK2(1001), respectively. pJ AK2(1001-1013) alone did not activate reporter gene (dat a not shown) and control pe ptide did not enhance IFN activation of reporter gene. Thus, consistent with the anti -SOCS-1 effects of pJ AK2(1001), the peptide also enhanced IFN function at the level of gene activa tion. It has recently been shown that suppression of SOCS-1 in dendri tic cells by siRNA enhanced anti -tumor immunity (Shen et al. 2004). In order to determine the effect of pJ AK2(1001-1013) on cell-mediated immune response, C56BL/6 mice were treated with pJAK2(10011013), control peptide, or PBS, following immunization with BSA. It was shown th at pJAK2(1001-1013) enhanced BSA-induced proliferation of splenocytes by four to five fo ld when compared to control peptide or PBS (Waiboci et al. 2007). Further, I showed that supernatants cont aining SOCS-1 protein competed with SOCS1-KIR for binding to pJAK2(1001-1013) (Figure 4-15d). The demonstration of SOCS-1 competition for pJAK2(1001-1013) is c onsistent with pJAK2(1001-1013) antagonism

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59 via sequestration of critical functional site(s) on SOCS-1. Hence, the data have shown that pJAK2(1001-1013) antagonizes SOCS-1 activit y at five different levels; IFN antiviral function, IL-6 signal transduction, IFN activation of reporter gene via the GAS promoter, enhancement of antigen-specific proliferati on, and possible sequestration of binding sites on SOCS-1. Expression of SOCS-1 Protein SOCS-1 is a negative regulator of immune f actors including IFNs, IL-2, IL-4, and IL-6. SOCS-1 also modulates signaling by a variety of hormones. However, in spite of the presence of SOCS-1 and other immune modulators, the host defense system can pathologically perpetuate inflammation by overproducing immune mediators, su ch as inflammatory cytokines, that cause damage to multiple organs, resulting in what are referred to as inflammatory diseases/disorders. Further, it is estimated that approximatel y 20% of human cancers result from chronic inflammation. In addition, silenc ing of the SOCS-1 gene, by me thylation, has been found in several human cancers (Hanada et al. 2006). Res earch carried our in our laboratory has shown that Tkip protects MBP sensitized mice from deve loping EAE, an inflammatory disease. Further, it has shown that Tkip inhibits proliferation of prostate cancer cells. Since Tkip is a SOCS-1 mimetic, I reasoned that recombinant SOCS-1 protein may have the same effect as Tkip in preventing inflammation and inhibiting proliferat ion of cancer cells. Therefore, recombinant SOCS-1 protein was expressed with the aim of obtaining protein to fi rst characterize SOCS-1 functional sites and second to de termine functional relationship to Tkip. The recombinant SOCS1 protein was tested for binding to JAK2 autophosphorylation site. First, primers were designed to amplify mu rine SOCS-1 (muSOCS-1) from an expression library, pEF-FLAG-I/mSOCS-1, a gift from Dr. D. Hilton (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). SOCS-1 DNA was amplified from the expression library, gel purified, and cloned into pBlueBac4.5/V5-His TOPO TA v ector (Invitrogen). Plasmid DNA

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60 was isolated and the presence of muSOCS-1 conf irmed by restriction enzyme digestion (Figure 4-16a) and DNA sequencing (Figur e 4-16b). I refer to the clone d product as pBlueBac/muSOCS1. The constructs were co-transfected, with the baculovirus vector Bac-NBlue (Invitrogen), into Sf9 insect cells and propagated. Co-transfect ion of the pBlueBac4.5/ His-V5 TOPO vector carrying muSOCS-1, with Bac-N-Blue vector en sured that generally, only recombinant virus would be viable and therefore, would grow Following transfection and propagation, viral plaques were picked and tested for presence of muSOCS-1 by PCR. As shown in Figure 4-17, several plaques had viral DNA of the correct size (650 bp). Of these, two were purified and used for protein expression. West ern blot analysis of the expression of SOCS-1 protein in the Sf9 cells is shown in Figure 4-15d. The SOCS-1 lysate s and the SOCS1-KIR peptide were used for competition for binding assays to test for ability to inhibit binding of pJAK2(1001-1013) peptide to immobilized SOCS1-KIR. Figure 4-15d, shows that SOCS1-KIR and SOCS-1 lysate, similar to Tkip and SOCS1-KIR competed for binding sites on pJAK2(1001-1013), the implications of which is that SOCS1-lysate may have characteri stics similar to Tkip. The goal of the laboratory is to obtain sufficient purified SOCS-1 protein fo r detailed characterization of functional sites.

PAGE 61

61 0.0 0.5 1.0 1.5 2.0 2.5 0.00 0.25 0.50 0.75 1.00Tkip DRTkip Contr l peptide No peptide DTkip Peptide concentration (mM)Absorbance (490 nM)A 0.0 0.5 1.0 1.5 2.0 2.5 0.00 0.25 0.50 0.75 1.00 1.25DRTkip DTkip Tkip Control peptide No peptide Peptide concentration (mM)Absorbance 490 nm Figure 4-1. JAK2 autophosphorylation site peptid es JAK2(1001-1013) and pJAK2(1001-1013) bind to SOCS-1 mimetic peptides. A) JAK2(1001-1013) peptide binds to Tkip, DTkip, and DRTkip. Biotinylated JAK2(10011013), at the indicated concentrations, was added in triplicate to 96-well plates coated with Tkip, DTkip, DRTkip, control peptide (MuIFN (95-106)), or buffer alone. The assays were developed using standard ELISA methods with neutra vidin-HRP conjugate to detect bound biotinylated JAK2(1001-1013). B) Bioti nylated pJAK2(1001-1013) binds to Tkip, DTkip and DRTkip. Biotinylated pJAK2(10011013) was added to wells coated with the peptides or buffer and binding assays were carried out as described above. The binding of JAK2(1001-1013) or pJAK2(1001-1013) to Tkip, DTkip or DRTkip, when compared to control peptide was statis tically significant as determined by MannWhitney signed rank test (P < 0.02 and P < 0.004, respectively).

PAGE 62

62 A 50300500 0 10 20 30 40 50 60 70 80Tkip DRTkip MuIFN (95 106) Competitior peptide concentration, M% inhibition of binding B 50300500 0 10 20 30 40 50 60 70 80Tkip DRTkip MuIFN (95 -106) Peptide competitor concentration, M% inhibition of binding Figure 4-2. Both soluble DRTkip and solubl e Tkip inhibit the bi nding of biotinylated JAK2(1001-1013) and biotinylated pJAK2 (1001-1013) to immobilized Tkip. A) DRTkip and Tkip inhibit the binding of JAK2(1001-1013) to immobilized Tkip. The inhibition of binding by Tkip or DRTkip is statistically significant when compared to control peptide (MuIFN 95-106) (P < 0.05 as determined by Mann-Whitney signed rank test. B) DRTkip and Tkip inhi bit the binding of pJAK2(1001-1013) to immobilized Tkip. The differences in inhi bition of binding by Tkip to pJAK2, when compared to control peptide, is statistically significant (P < 0.05) but that of DRTkip is not statistically significan t (P > 0.05) as determined by Mann-Whitney signed rank test. All experiments were carried out in tr iplicate and the data ar e representative of three independent experiments.

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63 Figure 4-3. DRTkip and Tkip but not the control peptide, MuIFN (95-106), inhibit JAK2 autophosphorylation. Kinase assays were carr ied out in the presence of JAK2 kinase, soluble IFNGR-1, and radiolab elled ATP as described in Chapter 3. To show equal protein loading, an immunoblot with JAK2 antibody of the reactions was carried out as described above, but in the presence of unlabelled ATP.

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64 Figure 4-4. Tkip, but not DRTkip inhibits superantigen-induced splenocyte proliferation. A) Tkip, but not DRTkip inhibits SEB-induced splenocyte proliferati on. Splenocytes (4 x 105 cells/mL) from nave NZW mice were inc ubated with varying concentrations of lipophilic peptides Tkip, DRTkip or control peptide MuIFN (95-106) and SEB (0.5 g/mL) for 72 h, followed by pulse labeling with 3-[H]-thymidine, and harvesting on filter discs. Cell associated radioactivity was quantified using a -scintillation counter and is reported as counts per minute (cpm ). Differences between Tkip and control peptide were statistically significant (P < 0.05) as determined by Mann-Whitney signed rank test. Differences between DR Tkip and control peptide were not statistically significant (P > 0.05). B) Tkip, but not DRTkip inhibits SEA-induced splenocyte proliferation. The experiment was carried out as described for SEB, but in the presence of SEA (0.5 g/mL). The differences between Tkip and control peptide were statistically significan t (P < 0.05), but the differe nces between DRTkip and control peptide are not sta tistically significant (P > 0.05) as determined by the MannWhitney signed rank test. The experiments we re carried out in tr iplicate and data are representative of three i ndependent experiments. 0 1000 2000 3000 4000 5000Media Media+SEB Tkip + SEB DRTkip + SEB Control peptide + SEB 1.2 3.7 11 Peptide concentration, McpmA 0 10000 20000 30000 40000Media Media + SEA Tkip + SEA DRTkip + SEA Control peptide + SEA 0.4 11Peptide concentration, McpmB

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65 A 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9buffer Tkip SOCS1-KIR 95-106 Biotinylated JAK2 (1001 1013)Absorbance 490 nm 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0 0.5 1.0 1.5 2.0 2.5Buffer Tkip SOCS1-KIR 95-106 Biotinylated pJAK2(1001 1013), mMAbsorbance 490 nmB Figure 4-5. JAK2 autophosphorylation site peptides bind to SOCS1-KIR. A) JAK2(1001-1013) peptide binds to both SOCS1-KIR and Tk ip. Biotinylated JA K2(1001-1013), at the indicated concentrations, was added in tripli cate to a 96-well plate coated with either Tkip, SOCS1-KIR, control peptide (MuIFN (95-106)), or binding buffer and binding assays carried out as described in Ch apter 3. B) Biotinylated pJAK2(1001-1013) binds to both SOCS1-KIR and Tkip. Bi otinylated pJAK2(1001-1013) was added to wells coated with either Tkip, SOCS1-KIR, MuIFN (95-106), or buffer and binding assays were carried out as described in Chapter 3. Th e binding of JAK2(1001-1013) and pJAK2(1001-1013) to Tkip or to SOCS1-KIR, when compared to control peptide were statistically significan t (P < 0.05) as determined by the Mann-Whitney signed rank test.

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66 Figure 4-6. Both soluble SOCS1KIR and soluble Tkip inhibit the binding of biotinylated JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip or SOCS1-KIR. A) SOCS1-KIR and Tkip i nhibit the binding of JAK2(1001-1013) to immobilized Tkip. B) SOCS1-KIR and Tkip inhibit the binding of JAK2(1001-1013) to immobilized SOCS1-KIR. C) SOCS1KIR and Tkip inhibit the binding of pJAK2(1001-1013) to immobilized Tkip. D ) SOCS1-KIR and Tkip inhibit the binding of pJAK2(1001-1013) to immobilized SOCS1-KIR. For all competition for binding assays the differences in inhibition of binding by Tkip or SOCS1-KIR, when compared to the control peptide, were statistically significant as Mann-Whitney signed rank test (P < 0.05). A ll experiments were carried out in triplicate and the data are representative of thre e independent experiments.

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67 A 100 300 500 0 25 50Tkip SOCS1-KIR 95 106 Soluble Peptide, M% inhibition of binding B 0 25 50 75Tkip SOCS1-KIR 95-106 100 300 500Soluble peptide, M% inhibition of binding C 100 300 500 0 25 50Tkip SOCS1-KIR 95 106 Soluble Peptide, M% inhibition of binding D 100 300 500 0 25 50 75Tkip SOCS1-KIR 95 106 Soluble peptide, M% inhibition of binding

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68 A B 0 50 100 150 200Densitometry reading C 0 50 100 150Densitometry reading Figure 4-7. Differences in the kinase inhibition patterns of SOCS1-KIR and Tkip in JAK2 autophosphorylation, STAT1 phosphorylation, and EG FR phosphorylation. A) Tkip, but not SOCS1-KIR or the control peptide, MuIFN (95-106), inhibits JAK2 autophosphorylation. B) SOCS1-KIR and Tkip, but not the control peptide MuIFGR1(253-287) inhibit IFN -induced STAT1 activation in murine U937 cells. Immunoblots with phosphorylated (pY701) STAT1 and the corresponding densitometry readings of band intensities are shown. The membrane was stripped and reprobed with STAT1 antibody. C) Both Tkip and SOCS1-KIR inhibit EGFR phosphorylation. In vitro kinase assays were carried out in which SOCS1-KIR, Tkip or control peptide was in cubated with EGF and EGFR and ATP for 30 min at 250C. The kinase reaction mixtures were reso lved on 12% SDS-PAGE, transferred onto a nitrocellulose membrane and immunobl otted with anti-phosphorylated EGFR antibody, with the densitometry readings of band intensities shown. The membrane was stripped and reprob ed with EGFR antibody.

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69 A 0 5 10 15 20 25 30Media IFN IFN + Tkip IFN + SOCS1-KIR IFN + 95-106 Media 3.12 6.25 12.5 25IFN u/mlNitrite concentration,M 0 11 22 33 44 55 66 77 88 99 110 0 5 10 15 20 25 30Media Media + IFN Tkip + IFN SOCS1-KIR + IFN 253-287 + IFN SOCS1-KIR + Tkip + IFN Peptide, MNitrite concentration,M Figure 4-8. SOCS1-KIR and Tkip inhibit IFN -induced macrophage activation. A) SOCS1-KIR and Tkip, but not the control pe ptide significantly inhibited IFN -induced macrophage activation as determined by test ing for nitrite concentration using Greiss reagent. The inhibition by Tkip, or SOCS 1-KIR compared to control peptide was statistically significant as determined by Mann-Whitney rank test (P < 0.05). B ) Tkip, and SOCS1-KIR, but not the control pe ptide show dose-dependent synergy in inhibiting IFN -induced induction of NO. Raw 264.7 cells were treated with IFN in the presence of varying concentrations of Tkip and SOCS1-KIR and screened for NO production as described above The differences between Tkip or SOCS1-KIR, compared to the control peptide were st atistically significant as determined by Wilcoxon matched pairs test (P < 0.05). B

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70 0 1000 2000 3000 4000 5000 6000 7000 8000 9000MBP MBP + Tkip MBP + SOCS1-KIR MBP + 253 287 0 1. 2 3.7 11 Peptide concentration, Mcpm Figure 4-9. Both SOCS1-KIR and Tk ip inhibit proliferation of mu rine splenocytes. Splenocytes (1 x 105 cells/well) were obtained from MBP sensitized SJL/J mice that had developed EAE and were in remission. The splenocytes were incubated with RPMI 1640 complete media containing MBP (50 g/mL) and varying concentrations of lipophilic SOCS1-KIR, lipophilic Tkip or lipophilic control peptide, MuIFNGR1(253-287), for 48 h. Cultures were then incubated with [3H]-thymidine for 18 h before harvesting. Ra dioactivity was counted on a -scintillation counter and data reported as cpm above background (media only). Both lipo-SOCS1-KIR and lipo-Tkip, but not the control peptide inhi bited splenocyte pro liferation in a dosedependent manner. The inhibition of pro liferation by lipo-SOCS1-KIR or lipo-Tkip, compared to the control peptide, was stat istically significant as determined by MannWhitney signed rank test (P < 0.05). The data are representative of two independent experiments, each carried out in triplicate.

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71 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.5 1.0 1.5 2.0SOCS1-KIR Tkip SOCS1-ESS 106 Biotinylated pJAK2(1001-1013), mMAbsorbance (490 nm) Figure 4-10. Biotinylated pJAK2(1001-1013) bi nds to SOCS1-KIR but not to SOCS1-ESS. Biotinylated pJAK2(1001-1013) was added to wells coated with either Tkip, SOCS1KIR, SOCS1-ESS, control peptide MuIFN (95-106), or buffer and binding assays were carried out as described in Ch apter 3. The binding of pJAK2(1001-1013) to SOCS1-KIR when compared to control pe ptide was statistically significant as determined by Mann-Whitney signed rank test (P < 0.01), while no significant binding was observed between SOCS1-ESS and the control peptide (P > 0.05).

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72 A 0 50000 100000 150000Media Media + MBP MBP + Tkip MBP + SOCS1-KIR MBP + 253-287 11 33Peptide concentration, Mcpm B 0 10 20 30 40Media MBP Tkip SOCS1-KIR 253-287 3.7 11 33 100Peptide concentration, MSpots/well Figure 4-11. Tkip and SOCS1-KIR inhibit antigen-specific CD4+ T cell proliferation and CD4+ T cell-induced IFN production. A) Tkip and SOCS1-KIR inhibit CD4+ T cell proliferation. Splenocytes obtained from MBP sensitized SJL/J mice, in remission, were enriched for CD4+ T cells and incubated (5 x 105 cells/well) with varying concentrations of lipophilic peptide in the presence or absence of MBP (50 g/mL) and APCs for 72 h. The cultures were pulsed with 3[H]-thymidine for 18 h before harvesting. Radioactivity was counted and is reported as cpm. Differences in inhibition of proliferation by Tkip or SO CS1-KIR, compared to control peptide, MuIFNGR1(253-287) were statistically sign ificant (P < 0.05) as determined by Mann-Whitney signed rank test. B) Tkip and SOCS1-KIR inhibit CD4+ T cell induced IFN production. CD4+ T cells were incubated with lipophilic peptides in the presence or absence of MBP and APCs The cells were transferred onto IFN ELISPOT plate, and incubated fo r 48 h. Spots, representing IFN -producing cells, were detected using HRP-conjugated sec ondary antibody. Differences in reduction of the number of IFN -producing cells by Tkip or SOCS 1-KIR, compared to control peptide, were not statistically signifi cant as determined by Mann-Whitney signed rank test (P < 0.05). The experiments were car ried out in triplicate and the data are representative of two independent experiments.

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73 0 2 4 6 8 10 12 14 16 18Media MBP MBP + Tkip MBP + SOCS1-KIR MBP + 253 287 1.2 3.7 11 33Peptide concentration, MSpots/well Figure 4-12. Tkip and SOCS1-KIR inhibit CD8+ T cell-induced IFN production. Splenocytes were obtained from MBP sensit ized SJL/J mice that a few days after signs of active disease. The splenocytes were enriched for CD8+ T cells (5 x 105 cells/well) and incubated with varying concentrations of lipophilic peptides in the presence or absence of MBP (50 g/mL) and APCs. The cells were transferred into IFN -Elispot plates and incubated for 48 h. The spots, representing IFN -producing CD8+ T cells, were detected using HRP-conjugated secondary antibody. The differences in inhibition of IFN production by Tkip-treated or SO CS1-KIR treated cells, compared to control peptide-tr eated cells ( 11 and 33 M) were statistically significant as determined by Mann-Whitney signed rank test (P < 0.03). The experiment was carried out in triplicate and the data are representative of two independent experiments.

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74 A 0 1000 2000 3000 4000 5000 6000 7000 8000 9000Media Media + MBP Tkip SOCS1-KIR 253-287 3.7 11 33 Peptide concentration, Mcpm B 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Media MBP Tkip SOCS1-KIR 253 287 3.7 11 33Peptide concentration, MAbsorbance, 490 nm Figure 4-13. Tkip and SOCS1-KIR inhibit an tigen-induced B cell prol iferation and antibody production. A) Tkip and SOCS1-KIR inhib it B cell proliferati on. B cells (5 x 105 cells/well) obtained from MBP sensitized SJL/J mice 2 months after last remission were incubated with lipophilic peptides in the presence or absence of MBP and APCs. The data presented for peptides was in the presence of MBP and APCs. The cultures were pulse labeled with 3[H]-thymidine for 18 h and harvested. Radioactivity was counted and is reported as cpm. The differe nces in inhibition of proliferation by Tkip or SOCS1-KIR (3.7, 11, and 33 M), compared to the control peptide were statistically significant as determined by Mann-Whitney test (P < 0.01). B) Tkip and SOCS1-KIR inhibit the production of MB P-specific antibodies. B cells (5 x 105 cells/well) from MBP sensitized SJL/J mice were incubated with lipophilic peptides in the presence of MBP (50 g/mL) and APCs for 48 h. Culture supernatants were then harvested and tested for MBP-speci fic antibodies by ELISA. The difference recorded for Tkip or SOCS1-KIR (33 M), compared to control peptide were statistically significant (P < 0.01) as determined by Wilcoxon matched pairs test. The experiments were carried out in triplicat e and data is representative of two independent experiments.

PAGE 75

75 0.0 2.5 5.0 7.5 10.0Media LPS LPS + Tkip LPS + SOCS1-KIR LPS + 95-106 0.1 0. 5 1.0LPS concentration, g/mlNitrite,M Figure 4-14. Tkip inhibits LPS-induced m acrophage activity. Murine macrophage cells, Raw 264.7, were incubated with varying concentr ations of LPS alone or with either lipophilic Tkip, lipophilic SOCS1-KIR or lipophilic control peptide, MuIFN (95106), each at 24 M final concentration, for 48 h. Cultu re supernatants were collected and nitrite concentration determined us ing Greiss assay. The experiments were carried out in triplicate and data are representative of two independent experiments. There was statistically significant differen ce between Tkip and control peptide (P < 0.05), but none between SOCS1-KIR and control peptide (P > 0.05), at the concentrations tested, as determ ined by Wilcoxon matched pairs test.

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76 Figure 4-15. pJAK2(1001-1013) peptide has SOCS-1 an tagonist properties. A) pJAK2(10011013) enhances suboptimal IFN -induced antiviral activity against EMC virus in human fibroblast WISH cells as determ ined using a cytopathic assay. B) pJAK2(1001-1013) reverses SOCS-1 inhibiti on of STAT3 phosphorylation in human prostate cancer cells (LNCaP) transfected with a SOCS-1 plasmid DNA, pEF-FLAGI/mSOCS-1, provided by Dr. David Hilton. Th e results of an immunoblot assay with phosphorylated STAT3 (pY705) and the co rresponding densitometry readings of band intensities are shown. Also shown ar e results of an immunoblot assay with unphosphorylated STAT3 antibody as well as SOCS-1 expression in LNCaP cells. The data are representative of two i ndependent experiments. C) pJAK2(1001-1013) enhances GAS promoter activity. WISH ce lls were transfected with a vector expressing firefly luciferase driven by a GAS promoter, along with a vector expressing Renilla luciferase as a control vector. IFN (1 U/mL) and lipopJAK2(1001-1013) at 5 or 1 M final concentration, or a control peptide, MuIFN (95-125) (5 M) were added to the cells. After 48 h incubation, the cell extracts were assayed for relative lucife rase activities using a luminometer. D) Soluble SOCS-1 protein, similar to SOCS1KIR inhibits the bindi ng of biotinylated pJAK2(1001-1013) to SOCS1-KIR. Biotinyl ated pJAK2(1001-1013) that had been preincubated with varying concentrations of soluble SOCS1-KIR, SOCS-1 lysate or control lysates, was added in triplicate to a 96 well plate coated with SOCS1-KIR. Bound biotinylated pJAK2(1001-1013) was detected using neutravidin-HRP conjugate as described in Chapter 2. Also shown is an immunoblot lysate showing expression of SOCS-1 in Sf9 insect cells. Al iquots of the cell lysate were used for the competition for binding assay.

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77 A 0 50 100 150 200Media IFN IFN + JAK2 IFN + pJAK2Number of viral plaques B 0 50 100 150Densitometry readin g C 0.0 2.5 5.0 7.5 10.0Relative Luciferase UnitTreatment None IFN IFN+ IFN+ IFN+ pJAK2 pJAK2 95-125 M5 1 5 0.0 2.5 5.0 7.5 10.0Relative Luciferase UnitTreatment None IFN IFN+ IFN+ IFN+ pJAK2 pJAK2 95-125 M5 1 5 D 0 10 20 30 40 50 60 70 80SOCS-1 lysate Control lysate SOCS1-KIR 95-106 Peptide ( M) 100 300 500 Protein ( g/ml) 1.2 110 330% inhibition of binding SOCS-1 SOCS-1

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78 A L a n e 1 2 3 4 5 6 7 8 9 1 0 11 12 1 3 14 B 10 20 30 40 50 0 MVARNQVAAD NAISPAAEPR RRSEPSSSSS SSSPAAPVRP RPCPAVPAPA 50 PGDTHFRTFR SHSDYRRITR TSALLDACGF YWGPLSVHGA HERLRAEPVG 100 TFLVRDSRQR NCFFALSVKM ASGPTSIRVH FQAGRFHLDG SRETFDCLFE 150 LLEHYVAAPR RMLGAPLRQR RVRPLQELCR QRIVAAVGRE NLARIPLNPV 200 LRDYLSSFPF QIKGNSKLRP Figure 4-16. SOCS-1 protein was expressed in baculovi rus infected Sf9 insect cells. A) The restriction enzyme digesti on pattern of pBlueBac/muSOCS 1 plasmid DNA with Lane 2 showing the correct restriction enzyme digestion product. B) Amino acid sequence of the cloned plasmid showing full-length SO CS-1 sequence, sequences derived from the cloning vector are underlined.

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79 Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Figure 4-17. Recombinant baculovirus contai ning muSOCS-1 DNA. PCR amplification of recombinant plaques (blue) with Bacul ovirus forward and baculovirus reverse primers. Lanes 1, 4, 5, 6, 9, and 10 ar e carrying inserts of the expected size. Constructs represented by lane 4 and 6 were chosen for further analysis.

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80 CHAPTER 5 DISCUSSION SOCS-1 is absolutely essential for surviv al of the individual Although SOCS-1 knockout mice appear to be normal at birth, they exhib it stunted growth and die neonately by three weeks of age (Alexander et al. 1999). These mice exhibit a syndrome characterized by severe lymphopenia, activation of T lymphocytes, fatt y degradation and necrosis of the liver, hematopoetic infiltration of multiple orga ns, and high levels of constitutive IFN as well as abnormal sensitivity to IFN (Alexander et al. 1999, Review ed in Alexander and Hilton 2004, Yoshimura 2005). IFN plays a central role in the pathol ogy as SOCS-1 knockout mice that are deficient in IFN or IFN receptor do not die as neonates. Similar pathology and lethality is observed in normal neonates th at are injected with IFN. It is worth noting that SOCS-1-/-, IFN -/double knockout mice die by 6 months of age of se vere inflammatory disease (Metcalf et al. 2002) indicating that SOCS-1 regul ation is not specific for IFN The dynamics of induction of SOCS-1 by IFN in cells and the activation of STAT1 is illustrative of how SOCS-1 attenuates IFN functions under physiological conditions. For example, treatment of monocyt es or astrocytes with IFN was followed by activation of the SOCS-1 gene at approximately 90 min (Dickensh eets et al. 1999, Brysha et al. 2001). Low doses of IFN resulted in transient increases in SOCS-1 mR NA that returned to baseline after 4 h, while high concentrations of IFN resulted in increases of SOCS-1 mRNA up to 24 h. Thus, the SOCS-1 response appears to be induced by the IFN signal. Treatment of hepatocytes from SOCS-1+/+ mice with IFN resulted in STAT1 activation within 15 min, which peaked by 2 h before declining (Brysha et al. 2001). Although STAT1 is similarly activated in SOCS-1

PAGE 81

81 deficient livers, it persists for 8 h. SOCS-1 thus appears to attenuate IFN persistent activation of STAT1, which nonetheless allows the beneficial effects of IFN-induced signaling. Given the critical importance of SOCS-1 in modulating the activities of IFN and other inflammatory cytokines that use tyrosine kinases such as JAK2 in their signaling pathways, our laboratory developed the small tyro sine kinase inhibitor peptide, Tkip, which is a mimetic of SOCS-1 (Flowers et al. 2004). Tk ip was designed to recognize the autophos phorylation site on JAK2 involving residues 1001 to 1013 containing th e critical tyrosine at 1007 (Yasukawa et al. 1999). Flowers et al (2004) showed that Tkip blocked JAK2 autophosphor ylation as well as tyrosine phosphorylation of substrates such as STAT1 and IFN receptor chain, IFNGR-1. The authors also showed that like SOCS-1, Tkip blocked EGFR autophos phorylation, while not affecting the tyrosine kinase function of c-Src a nd vascular endothelial gr owth factor receptor. Additional experiments showed that Tkip bl ocked IL-6 induced activation of the STAT3 oncogene in LNCaP prostate cancer cells, which involved inhibition of JAK2 activation (Flowers et al. 2005). These studies pres ented a proof-of-concept demonstr ation of a peptide mimetic of SOCS-1 that regulates JAK2 tyrosine kinase function. Because of its potential for regulation of in flammatory conditions where tyrosine kinases such as JAK2 play a role in the resultant pa thology, Tkip was tested in a mouse model of multiple sclerosis called experimental allergic encephalomyelitis (EAE) (Mujtaba et al. 2005). SJL/J mice were immunized with myelin basic protein (MBP) for induction of the relapsing/remitting form of EAE. Tkip, 63 g every other day, given intraperitoneally, completely protected the mice against relapses when compared to control groups in which greater that 70% of the mice re lapsed after primary incidence of disease. Protection of mice correlated with lower MBP antibody titers in Tkip-t reated groups as well as suppression of MBP-

PAGE 82

82 induced proliferation of splenocyt es taken from EAE-afflicted mi ce. Consistent with its JAK2 inhibition function, Tkip also inhibited th e activity of inflammatory cytokine TNF-, which uses the STAT1 transcription factor. Thus, Tkip, like SOCS-1, possesses anti-inflammatory activity that protects mice against ongoi ng relapsing/remitting EAE. The design of Tkip was independent of any knowledge of the structural and functional domains of SOCS-1. Given that the design fo cused on Tkip binding to the autophosphorylation site of JAK2, I compared Tkip with regions of SOCS-1 that have been proposed to be either directly involved in such bindi ng or to be involved in enha ncement of SOCS-1 binding to the autophosphorylation site. Yasukawa et al. (1999) identified thr ee regions that were directly involved in SOCS-1 binding to JAK2, the la rge SH2 domain, a N-terminal 12-amino acid sequence called extended SH2 (ESS) and an additional N-termin al 12-amino acid region called the kinase inhibitor region (KIR). The ESS and SH2 domains were felt to bind to the autophosphorylation or activation si te of JAK2, while KIR bound to the catalytic site in this model. The 12-amino acid ESS sequence consists of residues 68, wh ile KIR consists of residues 56-67. The SOCS1-KIR peptide sequence compared to the KIR above consists of residues 53-68, sharing just the I68 with the ESS and containing three addi tional residues in its N-terminus. In comparative binding, th e SOCS1-KIR peptide bound to pJAK2(1001-1013), while SOCS1-ESS peptide failed to bind (Figur e 4-10). Close examination of N-terminal truncated SOCS-1 expressed proteins, designated dN51 (missing residues N-terminal to 51) and dN68, in Yasukawa et al (1999) showed that removal of the KIR region resulted in loss of binding to JAK2 in transfected cells. Further, in direct binding to pJ AK2 autophosphorylation site peptide by truncat ed dN56 SOCS-1 protei n, the SOCS1-KIR peptide sequence except for residues 53-55, was present along with ESS and SH2 (Yasukawa et al. 1999). Although the

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83 region of SOCS-1 that binds to the autophosphoryl ation site of JAK2 as per Yasukawa et al. (1999) is referred to as SOCS-1 SH2 plus ESS, th e bindings that they refe r to also contained our SOCS1-KIR sequence. Additionally, I have show n that SOCS-1 protein competed with SOCS1KIR for pJAK2(1001-1013), suggesting that the tw o recognized the JAK2 autophosphorylation site similarly. Thus, I feel th at the binding data with our SOCS 1-KIR are consistent with the binding studies of these au thors. It should be note d that the peptide bindi ng approach used here does not involve assessment of collaboration and/or synergism among the KIR, ESS, and the SH2 domains of SOCS-1. Thus, based on the studies reported here, along with those by others (Yasukawa et al. 1999), KIR, ESS and SH2 may all be involved in binding to JAK2 autophosphorylation site. It remains to be determined as to the extent of their relative roles. I have shown in this study that SOCS1-KIR, independent of other domains of SOCS-1, can bind directly to a peptide, JAK2(1001-101 3), that corresponds to the autophosphorylation site of JAK2. Further, I show ed that SOCS1-KIR competed wi th Tkip for binding to JAK2(10011013). The competitions suggest that the peptides recognized JAK2 similarly but not exactly the same way. Phosphorylation of tyrosine 1007 on th e JAK2 peptide enhanced binding of Tkip and SOCS1-KIR. Tkip blocked JAK2 autophosphory lation as well as JA K2 phosphorylation of STAT1, while SOCS1-KIR did not block autophos phorylation but did block phosphorylation of STAT1, similar to the pattern or profile of SOCS-1 inhibiti on of phosphorylation (Alexander and Hilton 2004). The peptides were also functionally similar in inhibiting IFN activation of macrophages to produce NO and inhibiting antige n-specific induction of proliferation of splenocytes, with Tkip being the more effective i nhibitor. Thus, I have shown here that the KIR region of SOCS-1 can directly bind to the au tophosphorylation site of JAK2. These data are consistent with the observation made by Babon et al. (2006) on SOCS-3, which like SOCS-1 has

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84 KIR. These authors showed that th e SH2, ESS, and the C-terminal half of KIR directly contacted the phosphotyrosine binding loop of IL-6 receptor, gp130, and that the N-terminal half of KIR likely bound the JAK kinase. Hence, SOCS-1 KIR likely binds directly to JAK2 autophosphorylation site. Tkip is relatively hydrophobic, while SOCS1-KI R is hydrophilic. However, both peptides have hydropathic profiles that are complement ary to that of pJAK2(1001-1013). ESS however, had a hydropathic profile different from Tkip. Tk ip was designed to have a hydrophathic profile complementary to that of pJAK2(1001-1013) (Flo wers et al. 2004, Weat hington and Blalock 2003). Thus, Tkip would recognize primarily hydrophobic residues or groups in the pJAK2 peptide, while SOCS1-KIR would recognize primarily hydrophilic residues or groups. The binding competition could thus be due to a steric interference, which is consistent with differential effects of Tkip and SO CS1-KIR on JAK2 kinase activity. It has previously been shown that Tkip has potential anti-t umor (Flowers et al. 2005) and anti-inflammatory (Mujtaba et al. 2005) properties. Hence, Tk ip may have potential as a therapeutic agent. Here, I have shown that Tkip and SOCS1-KIR directly affect the activity of CD4+ T cells, CD8+T cells, B cells, and macrophages, whic h provides additiona l insights of the direct effect that these peptides have on the cells of the immune system. These data further show the probable therapeutic potential of Tkip. The fact that the KIR region of SOCS-1 can bind directly to pJAK2(1001-1013) raises the possibility that pJAK2(1001-1013) can function as an antagonist of SOCS -1. It has thus been shown here under four different types of experiments that pJAK2(1001-1013) possesses SOCS-1 antagonist properties. First, pJAK2 (1001-1013) enhanced suboptimal IFN activity. Second, prostate cancer cells transfected for constitutive production of SOCS-1 protein had reduced

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85 activation of STAT3 by IL-6 treatment. pJAK2( 1001-1013) reversed the SOCS-1 effect. Third, pJAK2(1001-1013) enhanced IFN activation of the luciferase reporter gene via the GAS promoter. Fourth, pJAK2(1001-1013) enhanced antig en-specific splenocyte proliferation. As indicated above, treatment of cells with IFN resulted in activation of the SOCS-1 gene in approximately 90 min and it has been proposed th at it is associated with the physiological attenuation of the IFN response by SOCS-1 (Dickensheets et al. 1999, Brysha et al. 2001) Consistent with this, it has recently been repo rted that small-interf ering RNA inhibition of SOCS-1 expression in bone marro w dendritic cells resulted in enhanced CTL activity and IFN production by ELISPOT, culminating in enhancemen t of anti-tumor immunity (Shen et al. 2004). I have thus shown here that SOCS1-KIR binds directly to th e autophosphorylation site of JAK2, similar to the binding of Tkip SOCS-1 mi metic, which results in inhibition of JAK2 phosphorylation of substrate. This di rectly identifies a region of SOCS-1 that possesses intrinsic anti-kinase function. Related to this, the autophosphorylation site peptide, pJAK2(1001-1013), functioned as an antagonist of SOCS-1. These findings with SOCS-1 mimetics and antagonists have implications for novel therapeutic approaches to mimicking SOCS-1 for treatment of inflammatory diseases and for suppressing SOCS -1 in order to enhance the immune response against cancer and infectious diseases.

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86 CHAPTER 6 FUTURE WORK Future work will involve expr essing and purifying large quanti ties of murine SOCS-1 and using the purified protein to characterize the func tional regions of SOCS-1 protein. I have also designed additional experiments to determine speci fically the role of the SOCS-1 KIR and ESS regions. This would provide additional insight on the minimum domain essential for SOCS-1 activity. Mouse studies are currently unde rway attempting to determine whether Tkip can be used for treatment of ongoing relapsing/remitting form of EAE, with implications for treatment of multiple sclerosis. Further, additional experiment s are being designed to determine whether Tkip binds to the other JAK kinases, JAK1, JAK3 a nd TYK2, implication of which Tkip would be used to regulate signa ling by these kinases. For SOCS-1 antagonist studies, mouse studies ar e being carried out in which attempts are being made to determine whether pJAK2( 1001-1013) can enhance protection against ongoing infectious disease.

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87 APPENDIX: VECTOR MAP OF THE TRANSFER (CLONING) VECTOR Figure A-1. A map of the pBlueBac4.5/V5-H is vector. Adapted from pBlueBac4.5/V5-His TOPO Expression Kit manual (Invitrogen).

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88 LIST OF REFERENCES Ahmed, C. M., M. A. Burkhart, M. G. Mujtaba, P. S. Subramaian, and H. M. Johnson. 2003. The role of IFN nuclear localization sequence in intracellular function. J. Cell Sci. 116: 30893098. Ahmed, C. M. I., and H. M Johnson 2006. IFN and its receptor subunit IFNGR-1 are recruited to the IFN -activated genes: Evidence for tran sactivational activity of IFNGR-1. J. Immunol. 177: 315-321. Alexander, W.S., R. Starr, J. E. Fenner, C. L Sco tt, E. Handman, N. S. Sprigg, J. E Corbin, A. L Cornish. R. Darwiche, C. M. Owczarek. T. W. Kay, N. A. Nicola, P. J. Hertzog, D. Metcalf, and D. J. Hilton. 1999. SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fa tal neonatal actions of cytokines. Cell 98: 597 608. Alexander W.S. 2002. Suppressors of cytokine signaling (SOCS) in the immune system. Nat. Rev. Immunol. 2:410. Alexander, W.S. and D. J. Hilton 2004. The role of suppressors of cy tokine signaling (SOCS) proteins in regulation of the immune response. Ann. Rev. Immunol. 22: 503. Babon, J. J., E. J. MaManus, S. Yao, D. P. DeSou za, L. A. Mielke, N. S. Sprigg, T. A. Willson, D. J. Hilton, N. A. Nicola, M. Baca, S. E Ni cholson, and R. S. Norton. 2006. The structure of SOCS3 reveals the basis of the extende d SH2 domain function and identifies an unstructured insertion that regulates stability. Molecular Cell 22: 205-216. Boehn, U., T. Klamp, M. Groot, and J. C. Ho war. 1997. Cellular respons es to interferon-gamma. Ann. Rev. Immunol. 15:74 -795. Brysha, M., J. G. Zhang, P. Bertolino, J. E. Corb in, W. S. Alexander, N. A. Nicola, D. J. Hilton, and R. Starr. 2001. Suppressor of cytokine sign aling-1 attenuates the duration of interferon signal transduction in vitro and in vivo. JBC. 276(25): 22086. Bromberg, J. and Darnell J.E. Jr. 2000. The role of STATs in transcript ional control and their impact on cellular function. Oncogene 19: 2468. Cal V., M. Migliavacca M, V. Bazan, M. Macaluso M. Buscemi, N. Gebbia, and A. Russo A. 2003. STAT Proteins: From normal control of cellular events to tumorigenesis. J. Cell. Physiol. 197: 157. Cornish, A. L., M. M. Chong, G. M. Davey, R. Da rwiche, N. A. Nicola, D. J. Hilton, T. W. Kay, R. Starr, and W. S. Alexander. 2003. Suppr essor of Cytokine Si gnaling-1 regulates signaling in response to interleukin-2 and other c-dependent cytokine s in peripheral T cells. JBC 278: 22755-22761.

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91 Metcalf, D., S. Mitsud, L. Rago, N. A. Nicola D. J. Hilton, and W. S. Alexander. 2002. Polycystic kidneys and chronic inflammatory lesions are the delayed consequences of loss of the suppressor of cytokine signaling-1 (SOCS-1). 2002. Proc. Natl. Acad. Sci. U.S.A.. 99(2): 943-948. Motulsky, H. Intuitive Biostatics: Oxford University Press, Inc, New York 1995 pp 217-229. Mujtaba M.G., F. O. Flowers, C. B. Patel, R. Patel, M. I. Haider, and H. M. Johnson 2005. Treatment of mice with suppressor of cytoki ne signaling-1 mimetic peptide, tyrosine kinase inhibitor peptide, prev ents development of acute form of experimental allergic encephalomyelitis and induces stable remi ssion in chronic relapsing/remitting form. J. Immunol. 175: 5077. Naka, T., M. Narazaki, M. Hirata, T. Matsumot o, S. Minamoto, A. Anono, N. Nashimoto, T. Kajita, T. Taga, K. Yoshizaki, S. Akira, and T. Kashimoto 1997. Structure and function of a new STAT-induced STAT inhibitor. Nature 387: 924. Naka, T., T. Matsumoto, M. Narazaki, M. Fuji moto, Y. Morita, Y. Ohsawa, H. Saito, T. Nagasawa, Y. Uchiyama, and T. Kishimoto 1998. Accelerated apoptos is of lymphocytes by augumented induction of Bax in SSI-1 (S TAT-Induced STAT Inhibitor-1) deficient mice. Proc. Natl. Acad. Sci. U.S.A.. 95:15577. Parganas, E., D. Wang, D. Stravapodis, D. Topha m, J. C. Marine, S. Teglund, E. Vanin, S. Bodner, O. Calamonici, J. M. Deursen, G. Groveld, and J. Ihle. 1998. JAK2 is essential for signaling through a variety of cytokine receptors. Cell 93: 385. Rico-Bautista, E., A. Flores-Morales, and L. Fe rnandez-Perez. 2006. Suppressors of cytokine signaling (SOCS) 2 proten with multiple functions. Cytokines & Ggrowth Factors Rev. 17: 431-439. Shawver, L. K., D. Slamon, and A. Ullrich 2002. Smart drugs: tyrosine kinase inhibitors in cancer therapy. Cancer Cell 1(2): 117-123. Shen, L., K. Evel-Kabler, R. Strude R, and S. Chen 2004. Silencing of SOCS-1 enhances antigen presentation by dendritic cells and an tigen specific anti-tumor immunity. Nature Biotech. 22(12): 1546. Shouda, T., T. Yoshida, T. Hanada, T. Wakioka, M. Oishi, K. Miyoshi, S. Komiya, K. I. Kosai, Y. Hanakawa, K. Hoshimoto, K. Nagata, and A. Yoshimura 2001. Induction of the cytokine signal regulator SOCS 3/CIS3 as atheurapeutic stra tegy for treating inflammatory arthritis. J. Clin. Investig. 108: 178 Shuai, K. 1999. The STAT family of proteins in cytokine signaling. Prog. Biophy. Molec. Biol. 71:405. Shuai, K. 2006. Regulation of cytokine signaling pathways by PIAS proteins. Cell research 16: 196-202.

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92 Song, E., S. K. Lee, J. Wang, N. Ince, N. Ouyang, J. Min, J. Chen, P. Shankar, and J. Lierberman 2003. RNA interference targe ting Fas protects mice from fulminant hepatitis. Nat. Med. 9: 347 351. Starr, R., T. A. Willson, E. M. Viney, L. J. L. Murray, J. R. Rayner, B. J. Jenkins, T. J. Gonda, W. S. Alexander, D. Metcalf, N. A. Nicola, and D. J. Hilton. 1997. A family of cytokineinducible inhibito rs of signaling. Nature 387: 917. Starr, R., D. Metcalf, A. G. Elefanty, M. Brysha, T. A. Willson, N. A. Nicola, D. J. Hilton, and Alexander W.S. 1998. Liver degeneration a nd lymphoid deficienci es in mice lacking suppressor of cytokine sinaling-1. Proc. Natl. Acad. Sci. U.S.A. 95: 14395 14399. Subramaniam, P. S., B. A. Torres, and H. M. Johnson 2001. So many ligands, so few transcription factors: a new pa radigm for signaling through the STAT transcription factors. Cytokine 15(4): 175. Suzuki, A. T. Hanada, K. Mitsuyama, T. Yoshida, S. Kamizono, T. Hoshina, M. Kubo, A. Yamashita, M. Okabe, K. Takeda, S. Akira, S. Matsumoto, A. Toyanag, M. Sata, and A. Yoshimura 2001. CIS3/SOC3/SSI3 pays a negative regulatory role in STAT3 activation and intestinal inflammation. J. Exp. Med. 193: 471. Szente, B. E., I. J. Weiner, M. J. Jablonsky, N. P. Krishmna, B. A.Torres, and H. J. Johnson 1996. Structural requirement for agon ist activity of a murine interferon-peptide. J. Interferon Cytokine Res. 16: 813. Tan, J. C. and R. Rabkin. 2005. Suppressors of cytokine signaling in health and disease. Pediatric Nephrol. 20(5): 567-575. Thiam, K., E. Long, C. Verwaerde, C. Auriault, and H. Gras-Masse. 1999. IFN -derived lipopeptide: Influence of lipid modification on the conformation and the ability to induce MHC class II expression on murine and human cells. J. Med. Chem. 42: 3732 Thompson, J. E. 2005. JAK pr otein kinase inhibitors. Drug News Persp. 18(5): 305-310. Torres, B.A., S. L. Kominsky, G. Q. Perrin, A. C. Hobeika, and H. M. Johnson. 2001. Superantigens: The good, the bad and the ugly. Exp. Biol. Med. 226: 164. Ungureanu, D., P. Saharinen, I. Junttila, D. J. Hilton, and O. Silvennoinen. 2002. Regulation of JAK2 through the ubiquitin-proteosome path way involves phosphoryl ation of JAK2 on Y1007 and interaction with SOCS1. Mol. and Cell. Biol. 22(10): 3316-3326. Vincentini, C., C. Festuccia, G. L. Gravina, A. Angelucci, A. Marronaro, and M. Bologna 2003. Prostate cancer proliferation is strongly re duced by the epidermal gr owth factor receptor kinase inhibitor ZD1839 in vitro in human cell lines and primary cultures. J. Res. Clin. Oncol. 129: 165.

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93 Waiboci, L.W., C. M. Ahmed, M. G. Mujtaba, L. O. Flowers, J. P. Martin, M. I. Haider, and H. M. Johnson. 2007. Both suppressors of cytoki ne signaling 1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: Implications for development of a SOCS-1 antagonist. J. Immunol. 178: 5058-5068. Weathington, N. M., and J. E. Blalock, 2003. Rational design of peptide vaccines for autoimmune disease: harnessing molecula r recognition to fix a broken network. Exp. Rev. Vac. 2: 61-73. Yamoaka, K., P. Saharinen, M. Pesu, V. E. Ho lt III, O. Silvennoinen, and J. J. OShea. 2004. The Janus kinaes. Genome Biology 5: 253. Yasukawa, H., H. Misawa, H. Sakamoto, M. Ma suhara, A. Sasaki, T. Wakioka,S. Ohtsuka, T. Imaizuni, J. N. Ihle, and A.Yoshimura 1999. The JAK-binding protei n JAB inhibits Janus tyrosine kinase activity through binding to the activation loop. EMBO Journal. 18: 1309 1320. Yoshimura, A. 2005. Negative regul ation of cytokine signaling. Clin. Rev. All. Immunol. 28: 205. Yu, .L., D. J. Meyer, G. S. Campbell, A. C. Larn er, C. Carter-Su, J. Schwartz, and R. Jove 1995. Science 269 (5220): 81-83. Zhang, J., G. D. Metcalf, S. Rakar, M. Asimakis, C. J. Greenhalgh, T. A. Willson, R. Starr, S. E. Nicholson, W. Carter, W. S. Alexander, D. J. Hilton, and N. A. Nicola. 2001. The SOCS box of Suppressor of Cytokine Signaling-1 is important for inhibition of cytokine action in vivo. Proc. Natl. Acad. Sci. U.S.A. 98(23): 13261-13265.

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94 BIOGRAPHICAL SKETCH Lilian Wangechi Waiboci was born on October 5, 1971, in Nyeri, Kenya to Mr. Francis Waiboci Matu and Mrs. Esther K. Waiboci. She grew up in Nyeri, graduating from Bishop Gatimu Ngandu Girls High School in 1989. She ear ned her B.S. in Biochemistry and Zoology and her M.S. in Biochemistry from the Un iversity of Nairobi, Kenya in 1995 and 2001, respectively. Upon graduating in 1995, Lilian worked as a high school teacher, teaching Chemistry and Biology. During her M.S. program, she was a resear ch assistant at the In ternational Livestock Research Institute (ILRI) and upon completion of her degree requirements worked as a part-time lecturer at Jomo Kenyatta Univ ersity of Agriculture and Tec hnology (JKUAT), and later for the Walter Reed Army Medical Research Project, HIV Laboratory in Kericho, Kenya as a Laboratory Manager and Research Technician. Lilian earned her Ph.D. from the Department of Microbiology and Cell Science, University of Florida in May 2007. She will pursue a carrier that in cludes both aspects of research and teaching in a University or a Resear ch Institute. Lilian is married to Dr. George Muhia Kariuki and they have a son, Victor Kariuki Muhia.


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SUPPRESSORS OF CYTOKINE SIGNALING-1 (SOCS-1) MIMETIC AND ANTAGONIST
PEPTIDES: POTENTIAL AS THERAPEUTIC AGENTS FOR TREATMENT OF
IMMUNOLOGICAL DISEASES



















By

LILIAN WANGECHI WAIBOCI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007






























O 2007 Lilian Wangechi Waiboci

































To the memory of my grandmother Mrs. Priscilla Wangechi Matu and my grandfather Mr.
Richard Mwaniki wa Nyangi









ACKNOWLEDGMENTS

I wish to thank my maj or supervisor Dr. Howard M. Johnson for funding this research, for

helpful suggestions and discussions, and for providing a conducive environment for learning. I

also wish to thank the members of my graduate committee Drs. Ayalew Mergia, Edward

Hoffmann, Peter Kima, and Janet Yamamoto for their helpful suggestions, and time.

I wish to thank member' s of the Johnson laboratory who helped with various aspects of

this research. I wish to specially thank Drs. Chulbul Ahmed and Mustafa Mujtaba for teaching

me many of the techniques used in this study. I wish to thank Mr. Mohammed Haider for

synthesizing all the peptides used in this study, Dr. Levy Omara-Opyene, Dr. Lawrence Flowers,

James Martin, Ezra Noon-Song, Rea Dabelic, Lindsey Jager, and Lauren Thornton for helpful

suggestions and assistance.

I also wish to thank Janet Lyles and Mary Ann Soncrant for helping make sure that my

studies and stay at the Microbiology Department ran smoothly and the staff of the International

Student Center for ensuring that my stay in the USA went smoothly.

I wish to thank my parents Mr. Francis Waiboci and Mrs. Esther Waiboci for all they have

done for my education, for instilling the love for learning, and discipline in my siblings and I. I

also wish to thank my siblings for their constant encouragement. Last, but not least, I wish to

thank my family, my husband Dr. George Kariuki for his love, understanding, and constant

encouragement through out the rough tides and my son Victor Kariuki for his love,

understanding, and patience.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ............ ..... .__ ...............7...


LI ST OF FIGURE S .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 INTRODUCTION ................. ...............12.......... ......


Rationale ................. ...............14.................

Specific Obj ectives ................. ...............16................

2 LITERATURE REVIEW ................. ...............18................


Janus Kinases ............... ... ... ... ....... .. .. ....... ........................1

Signal Transducers and Activators of Transcription (STAT) Proteins .............. ................. 19
Interferon Signaling Through the JAK/STAT Signaling Pathway............_._. .........._._. ...21
Regulation of the JAK/STAT Signaling Pathway ........._._........__. ......__. .........2
Suppre ssors of Cytokine Signaling (SOC S) ................. ...............23........... ...
Physiological Role of SOCS-1 Protein ................. ...............25...............
SOC S- 1 Mimetic Peptides ................. .. ........ ... ...... ........ .......2
Inhibition of SOCS-1 Activity and Immunological Relevance ................. ......._._. ........28

3 MATERIALS AND METHODS .............. ...............33....


Cell Culture............... ...............33

Peptides ........._.___..... ._ __ ...............33.....
Binding Assays ........._.___..... .__. ...............34....
In vitro Kinase As says ........._.___..... .___ ...............35....
Immunoblot Analysis............... ...............36
Macrophage Activity .............. ...............37....
Tkip Cellular Targets ................... ............ .. ...............37.....
Antiviral Assays for SOC S- 1 Antagoni st Function ................. ...............39........... .
Transfections of LNCaP Cells with SOC S- 1 DNA ................. ...............40.............
GAS Promoter Activity .............. .... ............ ...... ...............4
Primer Design and PCR Amplification of Murine SOCS-1 DNA ................ ................ ...42
Cloning SOC S-1 into pBlueB ac4.5/V5 -Hi s TOPO TA Expression Vector .........................42
Expression of SOCS-1 in Sf9 Cells............... ...............43.
Statistical Analysis............... ...............44

4 RE SULT S .............. ...............46....











Tkip Family Members Bind to JAK2 Autophosphorylation Site.............__ ..........___.....46
Tkip Family Members Inhibit JAK2 Kinase Activity ........._._ ......... ... ........._.....47
Tkip Inhibits Superantigen-induced Proliferation of Mouse Splenocytes ......................47
SOCS-1 Kinase Inhibitory Region (SOCS1-KIR) Binds to JAK2 Autophosphorylation
S ite ......................... .. .. .. ........ ..... .. ... ..................4
Tkip and SOCS1-KIR Bind to JAK2 Autophosphorylation Site ............. ..............49
JAK2 Kinase Activity and STATlot Activation .............. ..... ... ..............5
Tkip and SOCS1-KIR Inhibit IFNy-induced Activation of Macrophages ................... ...52
Tkip and SOCS1-KIR Inhibit Antigen-specific Lymphocyte Proliferation ................... .52
An Extended SH2 Sequence (SOCS 1-ESS) Peptide does not Bind to pJAK2 (1001-
1013) .............. ...............53....
Tkip Cellular Targets ........._._........... ..._._............._._ ............5
Effect of Tkip and SOCS1-KIR on CD4' T Cells ................ ...............54...........
Effects of Tkip and SOCS1-KIR on CD8' T Cells .....__.___ ..... .._. __ ........_.......55
Effect of Tkip and SOCS1-KIR on B Cells .....__.___ ..... ........ ...............56
Effect of Tkip on Macrophages .................. ... ......__............. ........ ....... 5
SOCS-1 Antagonist Activity of pJAK2 (1001-1013) Peptide .........__......... .._.... .........57
Expression of SOCS-1 Protein .............. ...............59....

5 DI SCUS SSION ............ ..... ..__ ............... 0...

6 FUTURE WORK............... ...............86..

APPENDIX: Vector Map of the Transfer (Cloning) Vector ................. ............................87

LIST OF REFERENCES ................. ...............88................

BIOGRAPHICAL SKETCH .............. ...............94....










LIST OF TABLES


Table page

2-1 A table showing the JAK kinases and STAT proteins utilized by some cytokines,
growth factors, hormones, and oncogenes. .............. ...............32....

3-1 List of peptides used in this study. ................ .......... ......... ........ ............45










LIST OF FIGURES


Figure page

2-1 A schematic representation of the domain structure of the JAK kinase family. ........._.....30

2-2 A schematic diagram showing the domain structure of SOCS-1 protein. .........................31

4-1 JAK2 autophosphorylation site peptides JAK2(1001-1013) and pJAK2(1001-1013)
bind to SOCS-1 mimetic peptides............... ...............61

4-2 Both soluble DRTkip and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip. ........._......62

4-3 DRTkip and Tkip but not the control peptide, MulFNy(95-106), inhibit JAK2
autophosphorylation............... ..........6

4-4 Tkip, but not DRTkip inhibits superantigen-induced splenocyte proliferation. ................64

4-5 JAK2 autophosphorylation site peptides bind to SOCS1-KIR. A) JAK2(1001-1013)
peptide binds to both SOCS1-KIR and Tkip. ................ ...............65.............

4-6 Both soluble SOC S1-KIR and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip or
SOCS1-KIR. ............. ...............66.....

4-7 Differences in the kinase inhibition patterns of SOCS1-KIR and Tkip in JAK2
autophosphorylation, STATlot phosphorylation, and EGFR phosphorylation. ...............68

4-8 SOCS1-KIR and Tkip inhibit IFNy-induced macrophage activation. ............. ..... ..........69

4-9 Both SOCS1-KIR and Tkip inhibit proliferation of murine splenocytes.. ................... ......70

4-10 Biotinylated pJAK2(1001-1013) binds to SOCS1-KIR but not to SOCS1-ESS............_...71

4-11 Tkip and SOCS1-KIR inhibit antigen-specific CD4+ T cell proliferation and CD4+ T
cell-induced IFNy production. ............. ...............72.....

4-12 Tkip and SO C S1-KIR inhibit CD 8 T cell-induced IFNy product on............... ..... ........._.73

4-13 Tkip and SOCS1-KIR inhibit antigen-induced B cell proliferation and antibody
production. ............. ...............74.....

4-14 Tkip inhibits LP S-induced macrophage activity ........._.._........_. ......._.. .......7

4-15 pJAK2(1001-1013) peptide has SOCS-1 antagonist properties. ............. ....................76

4-16 SOCS-1 protein was expressed in baculovirus infected Sf9 insect cells. ..........................78











4-17 Recombinant baculovirus containing muSOCS-1 DNA ................. ................. ......79

A-1 A map of the pBlueBac4.5/V5-His vector. ................. ....___.....__ ..........8









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SUPPRESSORS OF CYTOKINE SIGNALING -1 (SOCS-1) MIMETIC AND ANTAGONIST
PEPTIDES: POTENTIAL AS THERAPEUTIC AGENTS FOR TREATMENT OF
IMMUNOLOGICAL DISEASES

By

Lilian Wangechi Waiboci

May 2007

Chair: .Howard M. Johnson
Major: Microbiology and Cell Science

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by interferon

gamma (IFNy) by binding to the autophosphorylation site of Janus kinase 2 (JAK2) and by

targeting bound JAK2 to the proteosome for degradation. Studies on a tyrosine kinase inhibitor

peptide, Tkip, which is a SOCS-1 mimetic, are described. Tkip was synthesized from the

physiological form of amino acids, L-amino acids. We synthesized two additional SOCS-1

mimetic peptides, DTkip and DRTkip, from D-amino acids, which are potentially more resistant

to degradation and therefore would likely be better therapeutic agents. DTkip and DRTkip bound

to the unphosphorylated JAK2 autophosphorylation site peptide, JAK2(1001-1013) and the

tyrosine 1007 phosphorylated peptide, pJAK2(1001-1013). Further, DTkip and DRTkip inhibited

JAK2 autophosphorylation and JAK2 phosphorylation of IFNy receptor-1 (IFNGR-1).

Tkip was also compared with the kinase inhibitory region (KIR) of SOCS-1 for JAK2

recognition, inhibition of kinase activity, and regulation of IFNy-induced biological activity.

Tkip and a peptide corresponding to the KIR region of SOCS-1, 53DTHFRTFRSHSDYRRI

(SOCS1-KIR), bound similarly to JAK2(1001-1013) and to pJAK2(1001-1013). Dose-response

competitions suggested that Tkip and SOCS1-KIR similarly recognized the autophosphorylation









site of JAK2. While Tkip inhibited JAK2 autophosphorylation as well as IFNy-induced STATla

phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but

inhibited STATla activation. Both Tkip and SOCS1-KIR inhibited IFNy activation of murine

macrophages and antigen-specific splenocyte proliferation.

The fact that SOCS1-KIR bound to pJAK2(1001-1013) suggested that the JAK2 peptide

could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFNy

activity, blocked SOCS-1 induced inhibition of STAT3 phosphorylation in IL-6-treated cells,

enhanced IFNy activation site (GAS) promoter activity, and enhanced antigen-specific

proliferation. Further, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the

KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide

corresponding to this site can function as an antagonist of SOCS-1.

In summary, Tkip and SOCS1-KIR recognized the autophosphorylation site of JAK2

similarly and pJAK2(1001-1013) peptide functioned as a SOCS-1 antagonist. Thus, we have

developed peptides that function as SOCS-1 agonists and antagonists, which have potential for

suppressing or enhancing the immune response.









CHAPTER 1
INTTRODUCTION

Janus tyrosine kinases (JAKs) are an enzyme family that mediates the biological effects of

cytokines, hormones, and growth factors by tyrosine phosphorylation of signal transducers and

activators of transcription, STATs (Reviewed in Yamoaka et al. 2004; Parganas et al. 1998). The

interferons (IFNs), including types I and type II, hormones such as growth hormone and

angiotensin, and growth factors such as thrombopoietin, are all among 50 related factors

dependent on JAK tyrosine phosphorylation of appropriate STAT transcription factors for their

physiological functions (Subramaniam et al. 2001; Johnson et al. 2004).

The immediate early signal transduction events associated with IFNy and its receptor

subunits involve the obligatory action of two tyrosine kinases, JAK1 and JAK2 (Reviewed in

Kotenko and Peska 2000). The IFNy receptor (IFNGR) system is a heterodimeric complex

consisting of an a-subunit (IFNGR-1) and a P subunit (IFNGR-2), both of which are essential for

the biological activity of IFNy (Kotenko and Peska 2000). JAK1 is associated with the IFNGR-1

chain, whereas JAK2 is associated with the IFNGR-2 chain. The interaction of IFNy, primarily

with the IFNGR-1 subunit, initiates a sequence of events that results in increased binding of

JAK2 to IFNGR-1. This has important consequences for subsequent critical phosphorylation

events. JAK2, in the process of binding to IFNGR-1, undergoes autophosphorylation, and at the

same time IFNGR-1 is phosphorylated. These events occur in concert with JAK1 function,

resulting in recruitment and tyrosine phosphorylation of the IFNy transcription factor, STAT 1

(Reviewed in Kotenko and Peska 2000; Bromberg and Darnell 2000).

A family of proteins called suppressors of cytokine signaling (SOCS) negatively regulates

JAK/STAT signaling (Starr et al. 1997; Endo et al. 1997; Naka et al. 1997). SOCS proteins are

also negative regulators of signaling by other cytokines, growth factors, and hormones









(Reviewed in Alexander et al. 2002; Larsen and Ropke 2002; Alexander and Hilton 2004). There

are currently eight identified members of the SOCS family, SOCS-1 to SOCS-7 and cytokine-

induced SH2 domain containing protein (CIS). SOCS-1 is of particular interest, because it is a

negative regulator of the JAK kinases, as well as several cytokines, and hormone receptor

systems including epidermal growth factor receptor (EGFR) (Reviewed in Calo et al. 2003).

Our laboratory recently designed and synthesized a tyrosine kinase inhibitor peptide, Tkip

(WLVFFVIFYFFR) (Flowers et al. 2004). The characteristics of the Tkip 12-mer are

summarized in Chapter 2. In this study we show that Tkip inhibits IFNy-induced macrophage

activation and define Tkip cellular targets in antigen-induced immune responses. We also

describe a family of Tkip-related peptides, DTkip (WLVFFVIFYFFR) and DRTkip

(RFFYFIVFFVLW). DTkip and DRTkip, which like Tkip, bind to the JAK2

autophosphorylation site peptide, inhibit JAK2 autophosphorylation, and JAK2 phosphorylation

of IFNy receptor subunit, IFNGR-1.

It has been suggested that the binding of SOCS-1 to JAK2 requires the SOCS-1 SH2-

domain and that the kinase inhibitory region (KIR), while not required for the binding, is

essential for the inhibitory action of SOCS-1 (Yasukawa et al. 1999). We show here that a

peptide corresponding to SOCS-1 KIR region, SOCS 1-KIR, specifically binds to a peptide

representing the JAK2 autophosphorylation site and inhibits STATla activation. Further, we

show that SOCS1-KIR, as well as Tkip, inhibit IFNy-induced macrophage activation. We also

present data on a novel SOCS-1 antagonist peptide, pJAK2(1001-1013), which corresponds to

the JAK2 autophosphorylation site. pJAK2(1001-1013) enhances suboptimal IFNy-induced

antiviral activity, enhances IFNy-activated sequences (GAS) promoter activity, and inhibits









SOCS-1 suppression of STAT3 activation of LNCaP prostate cancer cells, thus functioning as a

SOCS-1 antagonist peptide.

Rationale

SOCS proteins play an important role in the regulation of JAK/STAT signaling. Disruption

of the normal SOCS function may contribute to disease onset, progression or death. It has been

shown that deletion of SOCS-1-'- or SOCS-3- genes in mice results in death of the mice either as

neonates (SOCS-1-'-) or embryos (SOCS-3- -) (Naka et al. 1998; Starr et al. 1998). SOCS proteins

regulate immune response by inhibiting JAK kinases. Uregulated JAK kinases signaling may

result in inflammation and cancers.

Several cancers are characterized by constitutive activation of the JAK/STAT signaling

pathway. SOCS proteins may play a role in inhibiting malignant transformation of cells by

regulating JAK/STAT signaling, thereby preventing cancer onset and progression. This has been

shown in a number of cancers including leukemia (T cell acute lymphoblastic leukemia (ALL),

Pre-B ALL, and atypical chronic myelogenous leukemia (CML)), and hepatocellular carcinoma

(Alexander and Hilton 2004). These examples raise the interesting possibility of the role that

SOCS proteins and/or SOCS mimetics may play in the management of these cancers.

Dysregulation of JAK/STAT signaling also plays important roles in the pathogenesis of

some inflammatory diseases including rheumatoid arthritis (Suzuki et al. 2001), inflammatory

diseases of the gastrointestinal tract (SOCS2), (Suzuki et al. 2001; Lovato et al. 2003; Shouda et

al. 2001) as well as inflammations of the central nervous system for example in experimental

allergic encephalomyelitis (EAE), an animal model for multiple sclerosis in humans (Maier et al.

2002). Hence, dysregulation of JAK/STAT signaling plays an important role in the pathology of

some inflammatory diseases and therefore raises the possibility that SOCS proteins and/or SOCS









mimetics that negatively regulate JAK/STAT signaling may be possible therapeutics for the

control and treatment of the inflammatory disease.

There are several tyrosine inhibitors that are currently undergoing clinical trials for

treatment of various cancers, especially cancers resistant to chemotherapy- and radiation.

Herceptin (Trastuzumab) is a humanized monoclonal antibody specific for a member of the

epidermal growth factor receptor (EGFR) family called HER2, which binds to the extracellular

binding domain of HER2/neu on tumor cells inducing receptor internalization and inhibiting cell

cycle progression (Reviewed in Shawyer et al. 2002). The discovery of Herceptin resulted in the

treatment of aggressive forms of breast cancer, in which cancer cells overexpressed HER2.

These cancers are usually less responsive to chemotherapy (Shawyer et al. 2002). A second drug,

GleevacTM, iS a small molecule inhibitor of the oncogenes BCR-Abl, Abl, PDGFR, and c-kit.

GleevacTM blocks ATP binding to the kinase, thereby preventing phosphorylation events that are

required for signal transduction. GleevacTM has been shown to increase the effectiveness of

interferon therapy on chronic myelogenous leukemia patients (Shawyer et al. 2002). Other small

molecule tyrosine kinases targeted therapies include Erbitux (EGFR) for treatment of colorectal

cancer, Tarceva (EGFR) for treatment of pancreatic cancer, and Iressa (EGFR) for treatment of

non-small-cell lung cancer (Shawyer et al. 2002; Vincentini et al. 2003). This provides additional

evidence that tyrosine kinases targeted approaches may have potential as anti-cancer therapy.

Hence, it is possible that the SOCS-1 mimetic peptides developed in our laboratory, which

are tyrosine kinase inhibitors of STAT transcription factors such as the STAT3 oncogene, may

have potential as anti-cancer and anti-inflammatory disease agents. The mimetics would likely

augment the effect of endogenous SOCS-1. We will discuss these mimetic peptides and present

data on their effect on the JAK/STAT signaling pathway, IFNy-signaling, and cell proliferation.









SOCS-1 regulates signaling by a variety of cytokines including IFNy, IFNa, and several

interleukins. Put differently, SOCS-1 reduces the immune response mechanisms initiated by

these cytokines. In an immune competent individual, this is desirable because it prevents

excessive signaling, which would likely result in inflammation. However, in an

immunocompromised individual, there may be a need to enhance cytokine-induced immune

response to infection. One way of enhancing immune response to pathogen infection may be by

inhibition of SOCS-1 activity. It has recently been shown that silencing of SOCS-1 in dendritic

cells promoted cell activation, which led to enhancement of effective antigen-specific anti-tumor

immunity (Shen et al. 2004). This implies that reagents that negatively regulate SOCS-1 may

have potential in control and/or treatment of diseases arising from inadequate immune response.

Hence, we designed a SOCS-1 inhibitor peptide, pJAK2(1001-1013), that is derived from the

JAK2 autophosphorylation site. I will present data showing that pJAK2(1001-1013) is a SOCS-1

antagomist.

Specific Objectives

In this dissertation Tkip and other SOCS-1 mimetic peptides, which I hypothesized, would

function similar to Tkip and therefore similar to SOCS-1 are described. The first obj ective of this

study therefore was to determine whether these peptides; DTkip and DRTkip have Tkip-related

characteristics. Specifically, whether the peptides bind to JAK2 autophosphorylation site, inhibit

JAK2 autophosphorylation, and inhibit activated JAK2 activity.

It has previously been stated that the SOCS-1 domains that bind directly to JAK2

autophosphorylation site are the SH2 domain and the extended SH2 region (ESS), and that the

kinase inhibitory region (KIR) is not essential in the initial binding (Yasukawa et al. 1999). The

second objective of this study was therefore to determine whether a peptide corresponding to









SOCS-1 KIR, SOCS1-KIR, binds directly to JAK2 autophosphorylation site, inhibits JAK2

autophosphorylation, and inhibits activated JAK2 activity.

Since Tkip has been shown to be a SOCS-1 mimetic with possible therapeutic potential,

the third objective of this study was to define Tkip cellular targets. Specifically, to determine

whether Tkip targeted B cells, CD4+ T cells, CD8+ T cells, and antigen presenting cells. This

would provide insights on the direct effect of Tkip on specific cells of the immune system, which

is important if Tkip proves be a potential therapeutic agent.

The fourth objective of this study was to define a novel way of addressing inadequate

cytokine-induced immune response mechanism. Here, a novel SOCS-1 antagonist peptide,

pJAK2(1001-1013), is described and preliminary data showing that this peptide reverses SOCS-1

inhibition is presented.









CHAPTER 2
LITERATURE REVIEW

Janus Kinases

Janus tyrosine kinases (JAKs) are a small but indispensable enzyme family of nonreceptor

tyrosine kinases that mediates the biological effects of cytokines, hormones, and growth factors

by tyrosine phosphorylation of signal transducers and activators of transcription, STAT,

(Reviewed in Ihle 1995; Parganas et al. 1998). The JAK family consists of four members (JAKl,

JAK2, JAK3, and TYK2) that are differentially activated in response to various cytokines (Ihle

1995). JAK proteins are approximately 120 to 130 kDa cytosolic proteins expressed in many

types of tissue with the exception of JAK3 whose expression is restricted to cells of the

hematopoietic system (Ihle 1995, Reviewed in Thompson 2005).

Members of the JAK protein family contain highly conserved structural domains

designated JAK homology domains (JH). There are currently seven known JH domains (JH1-

JH7) of which JH1 is the functional catalytic kinase. For JAK2, this domain possesses a critical

activation loop that becomes phosphorylated in order to activate the kinase. The phosphorylation

of tyrosine residue (Y1007) in the activation loop of JAK2 is essential for activation and

downstream signaling events (Feng et al. 1997). It has recently been suggested that the

phosphorylation of other tyrosines may also be necessary for JAK2 activation (Kurzer et al.

2004). Phosphorylation of Y1007 results in a conformational change in the activation loop,

which allows substrate access to specific binding sites in the catalytic groove (Yasukawa et al.

1999).

JH2 is a non-functional catalytic kinase domain but it bears sequence homology to typical

tyrosine kinase domains, hence it is referred to as a pseudokinase domain. The functional role of

the JH2 domain is unclear, however studies have suggested that it may have a kinase inhibitory









function. Both the JH1 and JH2 domains are located near the carboxyl terminus and comprise the

maj or portion of the JAK molecule (Reviewed in Leonard 2001; Kisseleva et al. 2002). The JH3,

JH4, and JH5 domains are poorly understood and require additional work to elucidate their

functions. JH6 and JH7 amino terminal domains have been implicated in the association between

the JAK molecule and the specific cytokine receptor (Leonard 2001; Kisseleva et al. 2002).

Figure 2-1 shows a diagrammatic representation of the domain structure of the JAK kinases.

Comparisons of the JAK2 autophosphorylation site amino acid sequences

(LPQDKEYYKVKEP) revealedl00% sequence homology among different mammalian species

including of the human JAK2 (Homo sapiens, genebank accession number NM_004972) mouse

(M~us musculus, AAH54807), rat (Rattus norvegicus, NP_113702), and pig (Sus scrofa,

BAA21662) as determined using the basic local alignment search tool (BLAST search,

http://www.ncbi .nlm.nih.gov/blast/). In addition, the amino acid sequence of the human JAK 1

(IETDKEYYTVKDD, accession number NP_002218) was 100% homologous to the mouse

JAK1 (NP_666257), as was human TYK2 (VPEGHEYYRVRED, accession number

NP_003322) and mouse TYK2 (NP_061263). Human JAK3 (LPLDKDYYVVREP,

NP 000206) and mouse JAK3 (LPLGKDYYVVREP, NP 034719 are nearly identical with a

one amino acid substitution, underlined. It is worth noting that in all four JAK kinases, the

sequences are similar, but not identical. The conservation in this region indicates the importance

of the autophosphorylation site in JAK function.

Signal Transducers and Activators of Transcription (STAT) Proteins

STAT proteins are a family of cytoplasmic transcription factors that participate in a variety

of cellular events, including differentiation, proliferation, cell survival, apoptosis, and

angiogenesis involving cytokines, growth factors, oncogenes, and hormones. Some of the









cytokines, growth factors, oncogenes, and hormones that utilize STAT proteins are shown in

Table 2-1.

There are currently seven known STAT family members: STAT1, STAT2, STAT3,

STAT4, STAT5A, STAT5B, and STAT6 (Darnell 1997). We are particularly interested in

STAT 1 and STAT3, both of which are negatively regulated by SOCS-1. It is worth noting that

other SOCS proteins may also negatively regulate STAT1 and STAT3.

The STAT proteins are comprised of six domains. These are oligomerization, coiled-coil,

DNA binding, linker, SH2, and transcription activation domains. The binding of STAT to the

receptors occurs through interaction of the SH2 to the receptor-docking site. The critical tyrosine

residues required for SH-phosphotyrosine interaction are STAT1-Y701; STAT2-Y690; STAT3-

Y705; STAT4-Y693; STAT5-Y694, and STAT6-Y641. These phosphotyrosines are located near

the SH2 domain (Calo et al. 2003).

The linker domain, which is alpha helical is the bridge between the DNA binding and the

SH2 domains. The transcription activation domain, located on the carboxyl terminus is involved

in communication with transcription complexes. The domain has a conserved serine residue

(except in STAT2 and STAT6) that when phosphorylated regulates STAT transcription activity

(Reviewed in Imada and Leonard 2000). The amino terminus region of STAT proteins is highly

conserved and provides protein to protein interaction, such as dimer interaction of STAT

molecules, which contributes to the stability of STAT-DNA binding, thereby increasing

transcription activity (Imada and Leonard 2000). The coiled-coil domain may be involved in

regulatory function and may be responsible for nuclear export of STAT.

Although some constitutively activated STATs have been observed in some human cancer

cell lines and primary tumors, STAT proteins are generally activated by tyrosine phosphorylation









(Cal6 et al. 2003). The STAT proteins become activated by a variety of receptors, such as

cytokine, growth factor, and hormone receptors, which may activate STATs directly or indirectly

through JAK kinases. In JAK/STAT signaling, the binding of a cytokine to its receptor results in

the associated JAK kinase becoming phosphorylated, and hence activated. The activated JAK

kinase phophorylates the receptor's cytoplasmic domain, at specific tyrosine residues, and opens

docking sites for STAT proteins. The STAT proteins docked on the receptor, are phosphorylated,

and thus become activated. Some models of JAK/STAT signaling indicate that the activated

STATs dissociate from the receptor, dimerize, and translocate to the nucleus where they activate

the transcription of specific genes (Bromberg and Darnell 2000). However, it has been shown

that in the case of IFNy signaling, IFNy, IFNGR-1, and the bound phospho-STAT1 dimer are

translocated to the nucleus as a complex (Subramaniam et al. 2001, Ahmed et al. 2003, Ahmed

and Johnson 2006), which implies that IFNGR-1 likely plays additional roles in signal

transduction.

Interferon Signaling Through the JAK/STAT Signaling Pathway

There are two classes of interferon, type I and type II, both of which utilize the JAK/STAT

signaling pathway. Although type I and type II IFNs signaling pathways are similar, distinct

receptors, JAK kinases, and STAT proteins are utilized. IFNy, the type II IFN, signaling utilizes

IFNGR, which is a heterodimeric complex comprised of two subunits, oc (IFNGR-1) and P

(IFNGR-2), both of which are essential for biological activity of IFNy (Kotenko and Pestka

2000).

IFNGR-1 is associated with JAKl, while the IFNGR-2 is associated with JAK2.

Interaction of IFNy, primarily with IFNGR-1, causes the receptor subunits to dimerize and brings

the associated JAK1 and JAK2 into close proximity. The JAK kinases undergo









autophosphorylation at specific tyrosine residues (1007 for JAK2) and become activated

(Kotenko and Pestka 2000). The activated JAKs phosphorylate and activate the IFNGR subunits,

which results in a cascade of events including the phosphorylation of STATla. Two

phosphorylated STAT1 monomers dimerize and are phosphorylated at specific serine residues to

form an activated STAT1 transcription factor. According to some models of IFNy signaling, the

STAT 1 dimers dissociate from the IFNGR-1, translocate to the nucleus, bind to IFNy activation

sites (GAS), and induce expression of target genes (Bromberg and Darnell 2000). It has however

been shown that IFNGR-1 accumulates in the nucleus and colocalizes with STATla in a time

and dose dependent manner, implying that IFNGR-1 is likely also translocated into the nucleus

(Subramanian et al. 2001, Ahmed et al. 2003, Ahmed and Johnson 2006). Thus, IFNGR-1 may

play an active role in signal transduction events subsequent to binding of the receptor complex.

Studies have indicated that IFNGR-2 likely is not translocated to the nucleus (Ahmed and

Johnson 2006).

Type I interferons, IFNa for example, utilize the interferon alpha-receptor (IFNAR) for

signal transduction. The IFNAR is comprised of two subunits, IFNAR-1 (associated with TYK2)

and IFNAR-2 (associated with JAKl) (Kotenko and Pestka 2000). The signal transduction

pathway is initiated when IFNa binds to the receptor, which results in IFNAR-1 and IFNAR-2

forming a heterodimer with subsequent autophosphorylation and activation of both JAK1 and

TYK2. The activated JAK kinases phosphorylate IFNAR-2, providing docking sites for STAT2,

which binds to the receptor and becomes phosphorylated at tyrosine 690. The phosphorylation

favors the binding of STAT1 to the phosphorylated STAT2 (Durbin et al. 1996). STAT1 is

phosphorylated (tyrosine 701) by the JAK kinases and the STAT2/STAT1 heterodimer is

released from the receptor and translocates to the nucleus. The STAT2/STAT1 heterodimer









associates with p48 nuclear factor to form the IFN-stimulated gene factor (ISGF3) complex

(Horvath et al. 1996), which stimulates activation of target genes within the IFN-stimulated

response elements (IRSE) (Li et al. 1998).

Regulation of the JAK/STAT Signaling Pathway

The JAK/STAT signaling pathway is carefully regulated. Unregulated JAK/STAT

signaling may result in excessive cytokine-induced immune response, which would likely result

in inflammation and be harmful to cells, and ultimately to the organism. Three classes of

regulators of the JAK/STAT pathway are currently known. These are the protein inhibitors of

activated STATs (PIAS), tyrosine phosphatases that include the Src-homology 2 (SH2)-

containing protein tyrosine phosphatases (SHPs), and the suppressors of cytokine signaling

protein family (SOCS) (Reviewed in Kile et al. 2001; Kisseleva 2002; Larken and Roipke, 2002,

Alexander and Hilton 2004). PIAS proteins regulate transcription through several mechanisms,

including blocking the DNA-binding activity of transcription factors, recruiting transcriptional

co-repressors and promoting protein sumoylation (Shuai 2006). SHPs regulate JAK/STAT

pathway by dephosphorylating activated phosphotyrosine (Reviewed in Rico-Bautista et al.

2006). SOCS can block cytokine signaling by acting as (i) kinase inhibitors of JAK proteins

(SOCS1 and SOCS3), (ii) binding competitors against STATs (SOCS3 and CIS) and (iii) by

acting as ubiquitin ligases, thereby promoting the degradation of their partners (SOCS1, SOCS3,

and CIS) (Rico-Bautista et al. 2006). I will discuss the SOCS proteins, in detail, as an example of

negative regulators of JAK/STAT signaling, because SOCS proteins are essential for the

regulation of JAK/STAT signaling and other signaling pathways.

Suppressors of Cytokine Signaling (SOCS)

SOCS proteins are a family of cytoplasmic proteins that negatively regulate signal

transduction of cytokines, hormones, and growth factors that utilize the JAK/STAT signaling










pathway (Naka et al. 1997, Starr et al. 1997, Endo et al. 1997). Loss or insufficient expression of

SOCS proteins may result in diseases including several immune disorders, inflammatory

diseases, and cancers (Reviewed in Alexander and Hilton 2004; Tan and Rabkin 2005). Mouse

studies have shown that deletion of the SOCS-1 gene results in neonatal death (Naka et al. 1998;

Starr et al. 1998), while deletion of the SOCS-3 gene results in embryonic death (Croker et al.

2003). In the case of human disease, it has been shown that a number of hematological

malignancies are characterized by constitutive activation of JAK/STAT signaling pathway.

These malignancies include T cell acute leukemia and atypical chronic myelogenous leukemia

(Alexander and Hilton 2004). Further, SOCS-1 is likely a tumor suppressor since aberrant DNA

methylation of SOCS-1 gene, resulting in transcriptional silencing, has been observed in human

hapatocellular carcinomas and hepatoblastomas. The restoration of SOCS-1 expression in cells in

which SOCS-1 gene had been silenced led to reduction in the transformed phenotype (Alexander

and Hilton 2004), providing direct evidence that lack of SOCS-1 expression may have played a

role in the development of the malignancies. In addition, constitutive activation of STAT3 and

deregulation of SOCS3 expression have been observed in a variety of inflammatory diseases

(Alexander and Hilton 2004). Thus, SOCS proteins play a fundamental role in maintaining

health.

There are eight members of the SOCS family. These are the cytokine-induced SH2 domain

containing protein (CIS), SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7

(Reviewed in Alexander 2002; Larsen and Roipke, 2002; Alexander and Hilton, 2004). The

SOCS proteins are also known as JAK-binding protein (JAB), STAT-induced STAT inhibitor

(SSI), and cytokine-inducible SH2 containing (CIS) proteins (Larsen and Roipke, 2002). All

SOCS proteins have three shared domains, which are an N-terminal domain of varying length









and sequences, a central SH2 domain, which is essential for binding to the JH1 region of JAK

kinases (Yasukawa et al. 1999), and a C-terminal SOCS box. The SOCS box couples substrate-

specific interactions of the SH2 domain to the ubiqutination machinery, resulting in proteosomal

degradation of associated JAK (Alexander 2002). SOCS-1 and SOCS-3 also have the kinase

inhibitory region, KIR, which is hypothesized to be involved in catalytic activity of SOCS-1, but

not in the actual binding to JAK2 autophosphorylation site (Yasukawa et al. 1999, Giordanetto

and Kroemer 2003). In this manuscript, data will be provided indicating that the KIR region is

likely involved in direct binding to JAK2 autophosphorylation site. Figure 2-2 contains a

schematic representation of the domain structure of SOCS-1.

The physiological roles of four SOCS family members (CIS, SOCS-1, SOCS-2, and

SOCS-3) are well defined. I will briefly discuss the physiological role of SOCS-1 as an example

of the important role that SOCS proteins play in maintaining homeostasis.

Physiological Role of SOCS-1 Protein

SOCS-1 plays a vital role in negative regulation of IFNy signaling as shown by both in

vivo studies and in vitro assays. SOCS-1 double knockout mice (SOCS-1- -) die as neonates,

displaying low body weight and liver damage including necrosis. Diseased livers are

characterized by the presence of aggregates of granulocytes, eosinophils, and macrophages. The

SOCS-1--~ mice also exhibit monocytic invasion of the pancreas, the lung, and the heart (Starr et

al. 1998; Naka et al. 1998) and have a marked reduction in blood and spleen lymphocytes, as

well as severe deficiencies in both mature B- and T lymphocytes. In addition, mice thymuses are

reduced in size and show increased numbers of apoptotic cells both in the spleen and the thymus

when compared to normal mice. These symptoms indicate severe deficiencies in the immune

system (Naka et al. 1998; Starr et al. 1998) and show that SOCS-1 is indispensable for normal

neonatal development.









The pathologies observed in SOCS-1-'- gene knock out mice were similar to those observed

in wild-type mice administered excess IFNy, which led to the hypothesis that the disease

observed in the SOCS-1-'- gene knock out mice was likely due to excessive response to IFNy.

Direct evidence that IFNy was required for the development of the lethal disease observed in

SOCS-1- gene knock out mice was obtained when the mice were treated from birth with IFNy

neutralizing antibodies. After three weeks of IFNy treatment the anti-IFNy treated mice remained

healthy, while all the untreated mice had succumbed to disease (Alexander et al. 1999). In

addition, the SOCS-1- -/IFNy- double knockout mice did not exhibit the lethal phenotype

observed in SOCS-1-'- knockout mice (Alexander et al. 1999).Thus, SOCS-1 is a key

physiological regulator of IFNy signaling. It is worth noting that SOCS-1- -/IFNy- double

knockout mice eventually died 6 months after birth with inflammation and polycystic kidneys

(Metcalf et al. 2002), which suggested that SOCS-1 regulation was not specific for IFNy.

The prediction that SOCS-1 regulates IFNy signaling was confirmed using biochemical

studies. Upon inj section of IFNy into mice, STAT1 phosphorylation was evident in the livers of

the SOCS-1+/ mice within 15 min but declined after 2 h. However, in the SOCS-1-'- gene knock

out mice, phosphorylated STAT1 remained and was detectable 8 h after IFNy administration

(Brysha et al. 2001), indicating continued IFNy signaling. These observations, taken together,

indicated that SOCS-1 is a physiological negative regulator of IFNy signaling (Alexander and

Hilton 2004) and that unregulated IFNy activity contributed to the pathology observed in the

SOCS-1- gene knock out mice. Thus, SOCS-1 plays a fundamental role in regulating IFNy

signal transduction. It has also been shown that SOCS-1 plays a fundamental role in the

regulation of IFNa and IFNP (Fenner et al. 2006) and other cytokines signaling through the

JAK/STAT pathway.









SOCS-1 Mimetic Peptides

Our laboratory has designed a family of SOCS-1 mimetic peptides, which are being tested

for SOCS-1-like activity. The first of these peptides was tyrosine kinase inhibitor peptide (Tkip,

WLVFFVIFYFFR), which was designed using a complementary peptide approach for

complementarity to the JAK2 autophosphorylation site (Flowers et al 2004). Tkip binds to the

JAK2 autophosphorylation site and inhibits JAK2 autophosphorylation and JAK2 mediated

phosphorylation of the IFNGR-1 (Flowers et al. 2004). Tkip also inhibits the

autophosphorylation of the epidermal growth factor receptor (EGFR), consistent with the fact

that EGFR is regulated by SOCS-1 and SOCS-3. In contrast, Tkip does not bind or inhibit

tyrosine phosphorylation of the vascular endothelial growth factor receptor (VEGFR) or the

substrate peptide of the protooncogene, c-Src (Flowers et al. 2004), both of which are not

regulated by SOCS-1 suggesting specifieity of Tkip-mediated inhibition. Although Tkip binds to

unphosphorylated JAK2 autophosphorylation site peptide, JAK2(1001-1013), it binds

significantly better to phosphorylated JAK2 autophosphorylation site peptide, pJAK2(1001-

1013). It has been suggested that SOCS-1 recognizes and binds only to phosphorylated JAK2,

therefore Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not in

precisely the same way. Consistent with inhibition of JAK2, Tkip also inhibits the ability of

IFNy to induce an antiviral state as well as upregulation of IVHC class I molecules, and blocks

the phosphorylation of both STAT1 and STAT3 (Flowers et al. 2004). Tkip also inhibits the

proliferation of the prostate cancer cell lines DUl45 and LNCaP, in a dose dependent manner

(Flowers et al. 2005). In addition, Tkip has been shown to protect mice from EAE, an animal

model for the human inflammatory disease, multiple sclerosis (Mujtaba et al. 2005) via blockage

activation of inflammatory cytokines. Hence, Tkip appears to have both anti-inflammatory and

anti-tumor properties.









Tkip is synthesized from L-amino acids, which are the physiological form of amino acids

in nature and therefore are potentially readily degraded by proteases. Hence, if Tkip were to be

used as a therapeutic agent, there is potential that it may be readily degraded. This would likely

limit Tkip usefulness as a therapeutic agent. To address this limitation, we synthesized other

Tkip-related mimetic peptides. These were DTkip (WLVFFVIFYFFR) and DRTkip

(RFFYFIVFFVLW), which were synthesized from D-amino acids, which from a medicinal

chemistry view, were likely to be more resistant to proteolytic digestion and therefore likely to

be better therapeutics. DTkip has the same amino acid sequence as Tkip, while in DRTkip the

sequence is reversed, in other words DRTkip is a retro-inversion of Tkip. DTkip and DRTkip

were tested for SOCS-1-like activity such as the ability to bind to JAK2, inhibit JAK2

autophosphorylation, and inhibit JAK2-mediated phosphorylation of the substrates of the

JAK/STAT pathway. In addition, the peptides were also tested for effects on cell proliferation,

IFNy-induced antiviral activity and upregulation of IVHC class I molecules. In addition some

Tkip cellular targets were identified. I will present data describing a family of SOCS-1 mimetic

peptides and present a proof-of-concept for the therapeutic potential of Tkip.

Inhibition of SOCS-1 Activity and Immunological Relevance

As stated earlier, SOCS-1 is a negative regulator of immune factors including IFNs,

interleukins -2, -3, -4, -6, -7, and -12, tumor necrosis factor (TNFa) as well as a variety of

hormones such as growth hormone (Tan and Rabkin 2005). This raises the possibility that one

way of enhancing cytokine-mediated immune response to pathogen infection may be by

inhibition of SOCS-1 activity. It has recently been shown that silencing of SOCS-1 in dendritic

cells promoted cell activation, which led to enhancement of effective antigen-specific anti-tumor

immunity (Shen et al. 2004). This implies that reagents that regulate SOCS-1 may enhance

immune responses and therefore have potential in control and/or treatment of diseases arising









from inadequate immune responses. A SOCS-1 inhibitor peptide, pJAK2(1001-1013), that is

derived from the JAK2 autophosphorylation site was designed and synthesized. I will present

data showing that pJAK2(1001-1013) is a SOCS-1 antagonist.










JH7 JH6 JH5


JH4 JH3 JH2


N-l 65CI 125a Ra 65C 150 aa CI 6 I 245 aa _I 215 aa


Receptor binding Pseudo-kinase Kinase domain,
domain domain includes Y1007

Figure 2-1. A schematic representation of the domain structure of the JAK kinase family. The
JAK tyrosine kinase protein family contains four members: JAKl, JAK2, JAK3, and
TYK2. Each JAK molecule contains seven distinct regions: JH1-JH7. The JH1
domain is the catalytic domain, which includes the activation loop, in which
autophosphorylation site (Y1007) is located. The JH2 domain is the pseudo-kinase
domain and is believed to play a role in the autoregulatory activities of JAK2. The
JH6-JH7 domains mediate binding of JAK molecules to cytokine receptor proteins.
Adapted with modifications from Imada and Leonard 2000.









N-terminus


KIR ESS SH~2 domain


SOCS Box


SOCS-1 I



Figure 2-2. A schematic diagram showing the domain structure of SOCS-1 protein. All SOCS
proteins have an N-terminal region of varying length and sequence, a central SH2
domain, and a C-terminal SOCS box. SOCS-1 and SOCS-3 have a kinase inhibitory
domain (KIR), which lies between the N-terminal and the SH2-domain. In SOCS-1,
the 12-amino acids N-terminal and contiguous to the SH2 domain form the extended
SH2 (ESS) region, and the 12-amino acid residues N-terminal and contiguous with
the ESS form the kinase inhibitory region (KIR). The SOCS1-KIR peptide is derived
from the KIR, while the SOCS1-ESS peptide is derived from the ESS region.

































Adapted with modification from Subramaniam et al. 2001


Table 2-1. A table showing the JAK kinases and STAT proteins utilized by some cytokines,
growth factors, hormones, and oncogenes.
Cytokines/hormones/oncogenes/ JAK STAT
Growth factors


Cytokines
IFN-y
IFN-a/P
IL-2
IL-3
IL-4
IL-6
IL-10
IL-12
Growth factors/hormones
EGF
Growth hormone
Insulin
Oncogenes
V-abl
V-src


JAK1 and JAK 2
JAK1 and TYK-2
JAK 1 and JAK 3
JAK 2
JAK 1 and JAK 3
JAK 1
JAK 1 and TYK-2
JAK 2 and TYK-2


STAT 1
STAT 1, 2, 3, 4, 5A, 6
STAT 1, 3, 5A/5B
STAT 1, 3, 5A/B, 6
STAT 6
STAT 3, 5A/B
STAT 1, 3
STAT 1, 3, 4, 5

STAT 1, 2, 3, 5
STAT 1, 3, 5A/B
STAT3, STAT 5B


JAK2
JAK2
JAK1


JAK1
JAK1 and JAK2


STAT 1, 3 and 5
STAT 3









CHAPTER 3
MATERIALS AND METHODS

Cell Culture

All the cell lines, except Sf9 insect cells, were obtained from the American Type Culture

Collection (Manassas VA). The human prostate cancer cells, LNCaP, and the murine

macrophage cells, Raw 264.7, were maintained in RPMI (JRH Biosciences, Lenexa, KS)

supplemented with 10% FBS (Hyclone, Logan, CT), 100 U/mL penicillin, 100 U/mL

streptomycin (complete media). Murine fibroblast cells, L929, were maintained in DMEM (JRH

Biosciences), supplemented with FBS, penicillin, streptomycin, non-essential amino acids,

sodium bicarbonate, and sodium pyruvate. The murine monocyte cell line, U937, was maintained

in RPMI complete media supplemented with 10 mM HEPES (Sigma-Aldrich, St. Louis, MO),

and 1 mM sodium pyruvate (Sigma-Aldrich). All the cell types were cultured at 37oC and 5 %

carbon dioxide humidified incubator. The Sf9 cells obtained from Invitrogen (Invitrogen

Corporation, Carlsbald, CA) were maintained at 27oC as adhesion cultures in complete TNM-FH

media (Grace Insect Medium, Supplemented, containing 10% FBS, 100 U/mL penicillin, and

100 U/mL streptomycin) or as suspension cultures in Sf-900 SFM media containing 5% FBS,

100 U/mL penicillin, and 100 U/mL streptomycin.

Peptides

The peptides used in this study are listed in Table 3-1, and were synthesized by Mr.

Mohammed Haider, in our laboratory on an Applied Biosystems 9050 automated peptide

synthesizer (Foster City, CA) using conventional fluorenylmethyoxycarbonyl (Fmoc) chemistry

as previously described (Szente et al. 1996). A lipophilic group (palmitoyl-lysine) was added to

the N-terminus of peptides, to facilitate entry into cells, as a last step using a semi-automated

protocol (Thiam et al. 1999). Peptides were characterized by mass spectrometry and where









possible, HPLC purified. Peptides were dissolved in water or in DMSO (Sigma-Aldrich, St.

Louis, MO). The control peptides used in this proj ect did not show significant biological activity

in the systems tested.

Binding Assays

Binding assays were performed as previously described (Flowers et al. 2004) with minor

modifications. Tkip, SOCS1-KIR, and control peptide, MulFNy(95-106) at 3 Gig/well, were

bound to 96-well plates, in binding buffer (0.1 M sodium carbonate and sodium bicarbonate, pH

9.6). The wells were washed three times in wash buffer (0.9% NaCl and 0.05% Tween-20 in

PBS) and blocked in blocking buffer (2% gelatin and 0.05% Tween-20 in PB S) for 1 h at room

temperature. Wells were then washed three times and incubated with various concentrations of

biotinylated JAK2(1001-1013) or biotinylated pJAK2(1001-1013), in blocking buffer, for 1 h at

room temperature. Following incubation, wells were washed five times and bound biotinylated

peptides were detected using HRP-conjugated neutravidin (Molecular Probes) and color detected

using o-phenylenediamine in stable peroxidase buffer (Pierce, Rockford, 1L). The chromogenic

reaction was stopped by the addition of 2 M H2SO4 (50 CIL) to each well. Absorbance was

measured at 490 nm using a 450-microplate reader (Bio-Rad Laboratories, Hercules, CA).

Peptide competition assays were carried out as described above except following peptide

immobilization, washing, and blocking, biotinylated JAK2(1001-1013) or biotinylated

pJAK2(1001-1013), which had been pre-incubated with various concentrations of soluble

peptide competitors (Tkip, SOCS1-KIR or control peptide) was added. Detection of bound

biotinylated peptide was conducted as described above. Data obtained from binding assays was

plotted using Graph Pad Prism 4.0 software (Graph Pad Software, San Diego, CA).









In2 vitro Kinase Assays

Autophosphorylation activity of JAK2 was measured in a reaction mixture containing

GST-JAK2 kinase fusion protein (Cell Signaling Technology, Danvers, MA), ATP (20 CIM, Cell

Signaling) and the appropriate peptide in kinase buffer (10 mM HEPES, pH 7.4, 50 mM sodium

chloride, 0.1 mM sodium orthovanadate, 5 mM magnesium chloride, and 5 mM manganese

chloride). It had previously been determined that soluble IFNGR-1 enhanced JAK2 activity

(Flowers et al. 2004), therefore IFNGR-1 (2 Cpg/ reaction) was added and the reaction mix

incubated at 250C for 30 min with intermittent agitation. The assays were carried out according

to a JAK2 kinase protocol obtained from Cell Signaling (Cell Signaling Technology, Danvers,

MA), but with modifications, derived in part from Flowers et al. (2004). The reactions were

terminated by addition of appropriate volume of 6 X SDS-PAGE loading buffer (0.5 M Tris-HCI

(pH 6.8), 36% glycerol, 10% SDS, 9.3% DTT, 0.012% bromophenol blue), and heating at 950C

for 5 min. The proteins were separated on a 12% SDS-polyacrylamide gel (Bio-Rad

Laboratories), transferred onto nitrocellulose membrane (Amersham Biosciences, Piscataway,

NJ), and probed with anti-pJAK2 antibodies (Santa Cruz Biotechnology, San Diego, CA).

Membranes were then stripped and re-probed with anti-JAK2 antibody (Santa Cruz

Biotechnology). Detection of proteins was accomplished using ECL protein detection reagents

(Amersham Biosciences).

Autophosphorylation activity of epidermal growth factor receptor (EGFR, Upstate) was

measured in a 50 Cl1 reaction containing kinase buffer, 0.2 Cpg EGFR (Upstate Biotechnology),

0.1 Cpg EGF (Upstate Biotechnology), 20 CLM ATP (Cell Signaling), and the appropriate peptide

(50 CLM) as described in Flowers et al. (2004). The reaction mix was incubated for 30 min at

250C, resolved by 12% SDS-PAGE, transferred onto nitrocellulose membrane, and









immunoblotted with anti-pEGFR antibody. The membranes were then stripped and reprobed

with anti-EGFR antibody. Detection of proteins was accomplished using ECL protein detection

reagents (Amersham Biosciences).

Immunoblot Analysis

U937 murine fibroblast cells were plated on 6-well plates at a cell density of 1 x 106 CellS/

well and after an overnight incubation at 370C and 5% CO2, the cells were cultured in complete

media containing varying concentrations of lipophilic Tkip, lipophilic SOCS1-KIR, or lipophilic

control peptide for 18 h at 370C in a 5% CO2 incubator. To activate the JAK/STAT signaling

pathway, U937 cells were then incubated in the presence or absence of 1000 U/mL IFNy (PBL

Biochemical Laboratories, Piscataway, NJ) for an additional 30 min. The media was aspirated

out and the cells washed twice with cold PBS to remove media and cell debris. Cell lysates were

prepared by adding 250 CIL of cold lysis buffer (50 mM Tris-HCI (pH 7.4), 250 mM NaC1, 50

mM NaF, 5 mM EDTA, and 0. 1% NP40) containing protease inhibitor cocktail (Amersham

Bioscience) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Lysis was allowed to

proceed for 1 h at 40C (rocking) to ensure complete cell lysis. Lysates were then centrifuged and

supernatant transferred into fresh microcentrifuge tubes, protein concentrations determined, and

protein lysates resolved by SDS-PAGE on a 12% polyacrylamide gel (Bio-Rad Laboratories).

Proteins were transferred onto nitrocellulose membranes (Amersham Biosciences), placed in

blocking buffer (5% nonfat dry milk and 0.1% Tween-20 in TBS), and washed in 0.1% Tween-

20 in TB S. To detect phosphorylated STAT lo, membranes were incubated with pY701-STAT1 a

antibody (1:400 dilution; Santa Cruz) in blocking buffer for 2 h at room temperature. After four

washes, the membranes were incubated in HRP-conjugated goat anti-rabbit IgG secondary

antibody (1:2000 dilution; Santa Cruz) in blocking buffer for 1 h at room temperature, washed









four times, incubated for 1 min with ECL detection reagents (Amersham Biosciences) and

exposed to photographic film (Amersham Biosciences) to visualize protein bands.

Macrophage Activity

Murine macrophage cells, Raw 264.7, were seeded on 24-well plates at a concentration of

3 x 105 cells/ well and allowed to adhere. Varying concentrations of the lipophilic peptides, Tkip,

SOCS1-KIR or MulFNy(95-106), were then added to the wells and the cells incubated for 2 h at

370C in a 5% CO2 incubator. Recombinant IFNy, in varying concentrations, was then added and

the cells incubated for an additional 72 h at 370C in a 5% CO2 incubator, after which

supernatants were transferred into fresh tubes and assayed for nitrite levels as a measure of nitric

oxide production using Gniess reagent, according to manufacturer' s instructions (Alexis

Biochemicals, San Diego, CA). To test for synergy between Tkip and SOCS1-KIR, the cells

were incubated in the presence of IFNy and varying concentrations of peptides as described

above and also in the presence of both lipophilic Tkip and lipophilic SOCS1-KIR or lipophilic

Tkip and lipophilic MulFNy(95-106). Supernatants were collected after 48 h and tested for nitric

oxide production as described above.

Tkip Cellular Targets

SJL/J mice were immunized with bovine myelin basic protein (MBP) as previously

described (Mujtaba et al. 2005). Briefly, 6 to 8 week old female SJL/J mice were immunized

subcutaneously at two sites on the base of the tail, with MBP (300 Cpg/mouse) in complete

Freund' s adjuvant. At the time of MBP immunization and 48 h later, pertusis toxin (400

ng/mouse) was administered (i.p). This protocol was approved by IACUC at the University of

Florida. The mice were observed daily for signs of EAE, and severity of disease was graded

using the following scale: 1) loss of tail tone; 2) hind limb weakness; 3) paraparesis; 4)









paraplegia; and 5) moribund/death. The first physical signs of disease were generally observed

beginning on day 18 to 21 after MBP immunization. Spleens were extracted after disease onset

and homogenized into a single cell suspension. Splenocytes (1 x 105 cells/well) were incubated

with medium, MBP (50 Clg/mL), lipo-Tkip, lipo-SOCS1-KIR or control peptide lipo-

MulFNGR(253-287) for 48 h at 370C in 5% CO2. To test the effect of the peptides on cell

proliferation, the cultures were pulsed with [3H]-thymidine (1.0 CICi/well; Amersham

Biosciences) for 18 h before harvesting onto filter paper discs using a cell harvester. Cell

associated radioactivity was quantified using a beta scintillation counter and data are reported as

counts per minute (cpm).

To test for the effect of Tkip on specific cellular targets, splenocytes obtained as described

above were enriched for the desired cell type using negative isolation kits purchased from Dynal

Biotech (Dynal Biotech, Oslo, Norway). The enrichments were carried out according to the

manufacturer' s instructions. Mouse B cells negative isolation kit, mouse CD4+ T cells negative

isolation kit, or mouse CD8' T cells negative isolation kit was used for B cells, CD4' T cells, or

CD8+ T cells, respectively. The use of the isolation kits results in cells enriched for the specific

cell type, but to confirm the purity level, the cells may be stained with cell-type specific

antibodies and FACS (fluorescence activated cell sorter) analysis carried out. This would have

given a better indication of the actual purity level, and would provide an indication what

contaminants (if any) were present.

Enriched cells were incubated with varying concentrations of appropriate lipophilic

peptides in the presence or absence of antigen presenting cells (APCs), and in the presence or

absence of antigen (MBP). The APCs were derived from splenocytes obtained from naive SJL/J

mice, which were incubated with MBP for 48 h, fixed with 2% paraformaldehyde for 30 min,









and washed extensively to remove residual paraformaldehyde. The cells were then transferred

into mouse IFNy-ELISPOT plates (Mabtech Inc, USA) and incubated for 48 h, after which the

wells were washed, incubated with secondary antibody, washed, and spots developed according

to manufacturer' s instructions. The plates were then blotted dry, spots counted, and the data

plotted using Graph Pad Prism 4.0 software (Graph Pad Software, San Diego, CA).

To test for the effect of Tkip on antibody production, B cells (5 x 105 cells/well) from

MBP sensitized mice obtained two months after disease remission were incubated with varying

concentrations of lipophilic peptides in the presence of MBP (50 Cpg/mL) and APCs and

incubated for 48 h. Culture supernatants were then harvested and tested for MBP-specific

antibodies by enzyme-linked immunoabsorbent assay (ELISA).

Antiviral Assays for SOCS-1 Antagonist Function

Antiviral activity was determined using the standard viral cytopathogenic effect assay

described previously with minor modifications (Langford et al. 1981). Briefly, human fibroblast

WISH cells at 70-80% confluency, were incubated in media alone or 0.4 U/mL human IFNy

(PBL Biomedical Laboratories) or both 0.4 U/mL human IFNy and lipo-pJAK2(1001-1013) or

lipo-JAK2(1001-1013) for 22 h in DMEM containing 2% FBS (maintenance media). Following

incubation, WISH cells were washed once with maintenance media and infected with

encephalomyocarditi s virus (EMCV) (200 pfu/well) for 1 h at 3 70C. The WISH cells were then

washed once to remove unbound viral particles and incubated in fresh maintenance media for an

additional 24 h at 370C in a 5% CO2 incubator. Plates were subsequently blotted dry and stained

with 0.1% crystal violet solution for 5 min. Unbound crystal violet was aspirated and the plates

thoroughly rinsed with deionized water, blotted, and air-dried. Viral plaques were counted using









a dissecting microscope and antiviral activity was determined by comparing experimental

treatment groups with the virus only control group.

Transfections of LNCaP Cells with SOCS-1 DNA

Transfections were carried out to introduce SOCS-1 DNA into mammalian cells and test

the ability of pJAK2(1 00 1-1 013) peptide to reverse SOC S- 1 inhibition of STAT3

phosphorylation. Human prostate cancer cells, LNCaP, were plated in a 6-well plate and allowed

to grow to 60% confluency. SOCS-1 plasmid DNA (1.6 Gig/well) (pEF-FLAG-I/mSOCS1), a gift

from Dr. David Hilton (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia)

or an empty vector, was transfected into the LNCaP cells using lipofectamine (Invitrogen

Corporation, Carlsbad, CA) according to manufacturers instructions, but with modifications. The

cells were incubated for 4 h, after which the transfection media was aspirated, fresh complete

media (DMEM supplemented with 10% FBS and 100 U/mL streptomycin and 100 U/mL

penicillin) added, and the cells incubated for an additional 72 h at 370C. The complete media was

then aspirated out, fresh media containing lipo-pJAK2(1001-1013) (20 CIM) or control peptide,

lipo-MulFNy(95-125), added to the transfected cells, and the JAK/STAT signaling pathway

activated by adding IL-6 (50 ng/mL). Cells were incubated for 30 min prior to harvesting. Cell

extracts were resolved by SDS-PAGE on a 12% polyacrylamide gel, transferred onto

nitrocellulose membrane (Amersham Biosciences), and probed with phosphorylated (pY705)

STAT3 antibody (Santa Cruz Biotechnology). The membranes were stripped and reprobed with

unphosphorylated STAT3 antibody (Santa Cruz Biotechnology). Detection of proteins was

accomplished using ECL detection reagents (Amersham Biosciences).

For immunoprecipitations, LNCaP cells growing on 60 mm plates and at 50% confluency

were transfected with either SOCS-1 plasmid DNA (8 Cpg/plate) or empty vector (8 Cpg/plate) in









lipofectamine and incubated for 4 h after which 5 mL complete media was added and cells

allowed to grow for 72 h. The cells were harvested in lysis buffer as described for western blot

analysis, and the cell lysates centrifuged at 10000 x g to remove cellular debris and nuclei.

Supernatants were transferred into fresh tubes and incubated with 2 Cpg/mL anti-Flag antibody

(Sigma-Aldrich) for 2 h at 4oC while rotating. Protein G PLUS-agarose beads (Santa Cruz

Biotechnology) were added to the supernatants and allowed to incubate for 2 h at 4oC while

rotating, followed by centrifugation to pellet protein G immune complexes. Supernatants were

discarded and the immune complexes washed three times with lysis buffer and once with PBS.

The immune complexes were then heated (95oC/5 min) in 50 CLL of 1 X SDS sample buffer,

resolved on a 12% polyacrylamide gel, transferred onto nitrocellulose, and immunoblotted with

anti-SOCS-1 antibody (Santa Cruz Biotechnology). Detection of proteins was accomplished

using ECL detection reagents (Amersham Biosciences).

GAS Promoter Activity

A plasmid, pGAS-Luc, that contains the promoter for IFNy-activated sequence (GAS)

linked to firefly luciferase gene was obtained from Statagene (La Jolla, CA). A constitutively

expressed thymidine kinase promoter-driven Renilla luciferase gene (pRL-TK) (Promega

Corporation, Madison, WI) was used as internal control in reporter gene transfections. WISH

cells (1 x 105 cells/well) were seeded in a 12-well plate and incubated overnight at 37oC,

following which 3 Clg GAS promoter-driven firefly luciferase expressing plasmid DNA and 10

ng pRL-TK were cotransfected into the WISH cells, using Fugene 6 (Roche Diagnostics

Corporation, Indianapolis, IN). Two days later, the cell lysates were used to assay for firefly

luciferase and Renilla luciferase, using a dual luciferase assay kit (Promega Corporation).









Luciferase activity, in relative luciferase units, was calculated by dividing firefly luciferase

activity by Renilla luciferase activity in each sample.

Primer Design and PCR Amplification of Murine SOCS-1 DNA

Primers were designed to amply full-length SOCS-1 from the mouse SOCS-1 intracellular

expression vector, pEF-FLAG-I/mSOCS-1 previously described. Primers were designed for

compatibility with the multiple cloning site (MCS) of pBlueBac4.5/V5-His TOPO TA vector

(Invitrogen). The primers were purchased from Integrated DNA Technology Inc and the primer

sequences are shown below.

Forward primer 1

5'- AGG ATG GTA GCA CGC AAC CAG GT 3'

Reverse primer

5'- GAT CTG GAA GGG GAA GGA AC 3'

PCR reactions were carried out in 50 CLL reactions containing dNTPs (0.2 mM each),

MgCl2 (1.5 mM), primers (0.2 CLM each), template DNA (1 Cpg), platinum Taq DNA polymerase

(1 unit), platinum Pfxc polymerase (1 unit), 10 X PCR buffer (5 pIL) and DNAse free water. The

thermocycler was programmed for 30 cycles at 94oC for 30 sec, 50oC for 30 sec, 72oC for 1 min,

with initial denaturation at 94oC for 4 min and final extension at 72oC for 7 min. The PCR

products were resolved on a 1% TAE agarose gel, stained with ethidium bromide, and

photographed.

Cloning SOCS-1 into pBlueBac4.5/V5-His TOPO TA Expression Vector

The PCR products of correct size (650 bp) were excised out of the gel, purified using

Wizard PCR prep DNA purification system (Promega, Madison WI) and cloned into the

pBlueBac4.5/V5-His TOPO TA expression vector according to the manufacturer's instructions

(Invitrogen). Following cloning, the recombinant vector was used to transform TOP10









chemically competent cells according to manufacturer' s instructions (Invitrogen), and the

transformed cells grown on LB-ampicilin nutrient agar plates overnight at 370C. Positive and

negative control cloning reactions were carried out similar to those for SOCS-1 DNA, but in the

absence of SOCS-1 DNA. Positive clones were identified by PCR, and plasmid DNA isolated

from the PCR positive clones. Restriction enzyme digestion and DNA sequencing were used to

confirm the presence of SOCS-1 DNA.

Expression of SOCS-1 in Sf9 Cells

The recombinant pBlueBac4.5/His-V5 TOPO vector carrying the murine SOCS-1 gene

(pBlueBac4.5/muSOCS-1) was transfected into the expression vector Bac-N-Blue (Invitrogen)

according to the manufacturer' s instructions. Briefly, the Bac-N-Blue vector contains a triple-cut,

linearized AcMNPV (Autographa californica multiple nuclear polyhedrosis virus). The

linearized virus lacks sequences essential for efficient propagation, specifically sequences in the

ORFl1629. Hence, for successful propagation and isolation of viable virus, the essential

ORFl1629 sequences need to be supplied by a transfer vector. The pBlueBac4.5/His-V5 TOPO

vector contains these essential sequences. The pBlueBac/muSOCS-1 and Bac-N-Blue were co-

transfected into Sf9 (Spodoptera Jfrugiperda)) insect cells growing at 50% confluency (Invitrogen)

in the presence of cellfectin reagent (Invitrogen) in unsupplemented Grace Insect Medium

(Invitrogen). The transfection was carried out at room temperature and the transfection

complexes incubated with the cells for 6 h following which complete TNM-FH medium was

added and the cells allowed to grow for an additional 72 h. Half the volume of the culture media

was harvested and used for plaque assay. The same volume of fresh TNM-FH media was added

to the cells, and the cells allowed to grow for an additional 72 h at which point significant cell

lysis was observed. The virus (Pl stock) was then collected by centrifugation and tested for the

presence of murine SOCS-1 DNA by PCR using the Baculovirus primers.









Forward primer 5'- TTTACTGTTTTCGTAACAGTTTTG 3'

Reverse primer 5'- CAACAACGCACAGAATCTAGC 3'

In order to generate high titer recombinant virus, PCR positive clones were propagated

further and used to infect fresh Sf9 cells growing in suspension culture. The cells were allowed

to grow and harvested at different time points to determine the best time for harvesting cells

expressing SOCS-1 protein. It was determined that 96 h infection provided the highest SOCS-1

protein yield, and subsequently Sf9 cells were harvested 96 h post infection. Cell lysates were

harvested using native conditions as described in the ProBond purification manual (Invitrogen)

and the expression of murine SOCS-1 confirmed by immunoblot analysis using anti-SOCS-1

antibody (Santa Cruz).

Statistical Analysis

The data were not normally distributed therefore nonparametric statistical analyses tests,

the Mann-Whitney and Wilcoxon tests were used. The Mann-Whitney signed rank sum test

compares two groups and performs calculations on the rank of the values, rather than the actual

data (Motulsky 1995). It is considered to be similar to the t-test, except the data are not normally

distributed. The Wilcoxon signed rank sum test compares two paired groups of nonparametric

data (Motulsky 1995). It is similar to the paired t-test, but the data are not normally distributed.

All calculations were performed using the GraphPad Prism statistical package (GraphPad

Software Inc, San Diego, CA).









Table 3-1. List of peptides used in this study. DTkip and DRTkip were synthesized from D-
amino acids. All the other peptides were synthesized from L-amino acids. The Y1007
in pJAK2(1001-1013), in italics, is phosphorylated.
Peptide Sequences
Tkip WLVFFVIFYFFR
DTkip WLVFFVIFYFFR
DRTkip RFFYFIVFFVLW
JAK2(1001-1013) 1001LPQDKEYYKVKEP
pJAK2(1001-1013) 00LPQDKEYYKVKEP
MulFNy(95-106) 95AK M/ulFNy(95-125) 95AKFEVNNP QVQRQAFNELIRVVHQLLPE SSL
SOCS1-KIR 53DTHFRTFRSHSDYRRI
SOCS1-ESS 68ITRASALLDACG
MulFNGR1 (253 -287) 2 53 TKKN SFKRK SIMLPK SLL SVVK SATLETKPE SKY S
MulFNy(95-106), MulFNy(95-125), and MulFNR1(253-287) were used as control peptides.
These peptides do not show significant biological activity in the assays for which they have
been used as control peptides.









CHAPTER 4
RESULTS

Tkip Family Members Bind to JAK2 Autophosphorylation Site

It has previously been shown that Tkip binds to the autophosphorylation site of JAK2 and

inhibits JAK2 autophosphorylation and phosphorylation of IFNGR-1 (Flowers et al. 2004). Here

I show that DTkip and DRTkip but not the control peptide, MulFNy(95-106), bound to the

JAK2(1001-1013) peptide in a dose-dependent manner (Figure 4-la). Thus, these SOCS-1

mimetic peptides bind JAK2 autophosphorylation site peptide in a dose-dependent manner.

Current literature suggests that phosphorylation of JAK2 tyrosine 1007 is important in

SOCS-1 mediated JAK2 ubiquitin-proteosome-dependent degradation (Ungureanu et al. 2002).

Hence, I wanted to determine whether these peptides, like SOCS-1, bind to phosphorylated

JAK2, pJAK2(1001-1013). Dose response solid-phase ELISA were carried out with biotinylated

pJAK2(1001-1013) peptide in place of biotinylated JAK2(1001-1013) peptide. DTkip and

DRTkip bound with two to threefold greater affinity to pJAK2(1001-1013) than to JAK2(1001-

1013) as shown in Figure 4-1b. This is consistent with previous data showing that Tkip binds

with a greater affinity to pJAK2(1001-1013) than to JAK2(1001-1013) (Flowers et al. 2004).

The binding observed was dose dependent, implying specificity in binding. Hence, the SOCS-1

mimetic peptides recognize JAK2 autophosphorylation site similar to Tkip, implication of which

is that they recognize JAK2 autophosphorylation similar to SOCS-1.

In order to determine whether Tkip and DRTkip bind to the same site on JAK2

autophosphorylation site, competition for binding assays were carried out. Soluble Tkip and

soluble DRTkip, but not the control peptide (MulFNy95-106) inhibited binding of biotinylated

JAK2(1001-1013) to immobilized Tkip in a dose dependent manner (Figure 4-2a). Next, the

competition for binding of Tkip with DRTkip to pJAK2(1001-1013) was determined. As shown









in Figure 4-2b, Tkip and DRTkip inhibited the binding of pJAK2(1001-1013) to immobilized

Tkip. Hence, Tkip and DRTkip likely bind to the same site on JAK2 autophosphorylation site,

the implication of which is that DRTkip may have similar binding characteristics as Tkip to

JAK2. The D amino acid isomer thus, binds similar to the L isomer, suggesting a stable form of

Tkip for functional studies.

Tkip Family Members Inhibit JAK2 Kinase Activity

SOCS-1 regulates JAK2 activity by interacting with the autophosphorylation site and

inhibiting JAK2 kinase activity. Therefore, DRTkip was tested for its ability to inhibit JAK2

autophosphorylation and JAK2 phosphorylation of substrate (IFNGR-1). DRTkip but not the

control peptide, MulFNy(95-106) inhibited JAK2 autophosphorylation and JAK2 induced

phosphorylation of IFNGR-1 (Figure 4-3). These results are similar to what has been previously

shown with Tkip (Flowers et al. 2004). The data suggest that DRTkip may inhibit both JAK2

activation and JAK2-mediated phosphorylation of IFNGR-1, the implication of which is that the

SOCS-1 mimetic peptides, like SOCS-1, may potentially regulate IFNy signaling. It is worth

noting that like Tkip, DRTkip did not inhibit tyrosine phosphorylation of c-Src kinase (data not

shown), which is consistent with the fact that c-Src kinase is not inhibited by SOCS-1.

Tkip Inhibits Superantigen-induced Proliferation of Mouse Splenocytes

The staphylococcus superantigens are potent T cell mitogens that exert their effects by

forming complexes with 1VHC class II molecules on antigen presenting cells, and binding to the

T cell receptor (TCR) via the Vp-element of the (TCR), resulting in activation of the T cells

(Reviewed in Torres et al. 2001). Our laboratory has shown that superantigens such as

staphylococcus entrotoxin A and B (SEA and SEB) can exacerbate immunological disease and

induce relapses in the mouse model for multiple sclerosis, experimental allergic

encephalomyelitis, EAE, (Torres et al. 2001). It has previously shown that Tkip inhibits antigen-









induced proliferation of splenocytes (Mujtaba et al. 2005). Here the ability of Tkip and DRTkip

to inhibit superantigen-induced proliferation of primary cells was tested. Mouse splenocytes

were stimulated with SEB (500 ng/mL) in the presence of lipophilic Tkip, lipophilic DRTkip, or

lipophilic control peptide, MulFNy(95-106) (a lipophilic group, lysyl-palmitate, is added to the

N-terminal ends of the peptides to facilitate entry into the cells) and incubated for 72 h prior to

pulsing with 3-[H]-thymidine. As shown in Figure 4-4a, Tkip but not DRTkip inhibited SEB

induced proliferation of the splenocytes. Next I tested the ability of Tkip and DRTkip to inhibit

SEA-induced splenocytes proliferation. Tkip, but not DRTkip inhibited SEA-induced

splenocytes proliferation (Figure 4-4b). Next the ability of Tkip and DRTkip to inhibit STATla

phosphorylation in murine fibroblast cells (U937) was tested. Tkip, but not DRTkip inhibited

STAT la phosphorylation (Data not shown). The lysyl-palmitate group is an L-lysine and may be

affected by proteases in such a way as to affect the efficiency of DRTkip uptake by cells. It is

also possible that the D-isomer amino acids may result in a peptide whose conformation is

different enough from Tkip to have slightly different cellular function. However, since only a

limited number of cellular function assays were carried out, it can not be conclusively be

determined whether DRTkip has or does not have intracellular SOCS-1 like function. Thus,

factors currently unknown may affect DRTkip intracellular function.

The observations that DRTkip, unlike Tkip, did not seem to significantly affect biological

activity suggested that the use of the Tkip retro-inversion, DRTkip, may have changed the

orientation of the amino acids enough to affect function. Hence, this research focused on Tkip

and SOCS1-KIR and not the other SOCS-1 mimetics. There is however, continued interest in

why DRTkip is not the same as Tkip in cell functional comparisons.









SOCS-1 Kinase Inhibitory Region (SOCS1-KIR) Binds to JAK2 Autophosphorylation Site

Yasukawa et al. (1999) suggested that the binding of SOCS-1 to JAK2 requires the SOCS-

1 SH2-domain and the extended SH2 domain (ESS) and that the kinase inhibitory region (KIR),

while not required for the initial binding is essential for the inhibitory action of SOCS-1. Here, I

present results showing that a peptide corresponding to SOCS-1 KIR region, SOCS1-KIR,

specifically binds to a peptide representing the JAK2 autophosphorylation site and inhibits

STAT la activation. Further, I show that SOCS1-KIR, as well as Tkip, inhibit IFNy-induced

macrophage activation. In addition, I show that a peptide corresponding to the ESS region,

SOCS 1-ES S. Does not bind to the JAK2 autophosphorylation site, the implication of which is

that the SH2, ESS, and KIR regions may all play role in the binding of SOCS-1 to JAK2.

Tkip and SOCS1-KIR Bind to JAK2 Autophosphorylation Site

First I determined whether SOCS1-KIR, like the SOCS-1 mimetic Tkip, binds to the JAK2

autophosphorylation site by carrying out dose-response solid-phase binding assays with JAK2

autophosphorylation site peptide, JAK2(1001-1013). Tkip, SOCS1-KIR, or a control peptide,

MulFNy(95-106), were immobilized on 96-well microtiter plates and incubated with biotinylated

JAK2(1001-1013) at various concentrations. Tkip and SOCS1-KIR, but not the control peptide,

bound to the JAK2(1001-1013) peptide in a dose-dependent manner (Figure 4-5a). Thus, both

Tkip and SOCS-1-KIR specifically bind to the JAK2 autophosphorylation site peptide. While

this is consistent with the SOCS-1 mimetic character of Tkip, it also provides direct evidence

that the KIR region of SOCS-1 can interact directly with JAK2 autophosphorylation site,

suggesting that Tkip and KIR recognized a similar site on JAK2.

Since phosphorylation of Y1007 is required for high catalytic activity of JAK2, it is logical

that SOCS-1 would bind with higher affinity to Y1007 phosphorylated JAK2. Consistent with









this, it has previously been shown that Tkip binds with greater affinity to Y1007 phosphorylated

JAK2(1001-1013) peptide, pJAK2(1001-1013), than to unphosphorylated JAK2 peptide

(Flowers et al. 2004). I therefore determined whether SOCS1-KIR bound to pJAK2(1001-1013)

with greater affinity than to JAK2(1001-1013). Both SOCS1-KIR and Tkip bound to

pJAK2(1001-1013) with two to three-fold greater affinity than to JAK2(1001-1013) as shown in

Figure 4-5b. In addition, SOCS1-KIR bound to pJAK2(1001-1013) with higher affinity than

Tkip. Thus, SOCS1-KIR recognizes JAK2 autophosphorylation similar to Tkip, the implication

of which is that Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1.

To determine whether Tkip and SOCS1-KIR bind to the same site on the JAK2

autophosphorylation site, binding competition assays were carried out. Tkip or SOCS1-KIR was

immobilized on a 96-well plate and biotinylated JAK2(1001-1013) or biotinylated pJAK2(1001-

1013), which had been pre-incubated with Tkip, SOCS1-KIR or a control peptide, was allowed

to bind to the immobilized peptides. As shown in Figure 4-6a, soluble Tkip and soluble SOCS1-

KIR, but not soluble control peptide, inhibited the binding of biotinylated JAK2(1001-1013) to

immobilized Tkip. A similar pattern of inhibition was observed with biotinylated JAK2(1001-

1013) binding to immobilized SOCS1-KIR (Figure 4-6b). Homologous inhibition was slightly

better for both Tkip and SOCS1-KIR, which suggests slight differences in recognition of JAK2

autophosphorylation site.

Next, the binding competition of Tkip and SOCS1-KIR to pJAK2(1001-1013) was

determined. The competition for binding to pJAK2(1001-1013) was similar to that observed in

competition for binding to unphosphorylated JAK2 peptide (Figure 4-6c and 4-6d). Again,

homologous competition was slightly better, which again suggests slight differences in

recognition of the JAK2 autophosphorylation site. These data provide direct evidence that the









mimetic effect of Tkip is applicable to the KIR region of SOCS-1 and that Tkip and KIR

recognize JAK2 autophosphorylation site similar but not exactly the same.

JAK2 Kinase Activity and STATla Activation

SOCS-1 regulates JAK2 activity at least at two levels. One involves interaction with the

autophosphorylation site, which affects JAK2 phosphorylation of substrates such as STAT 1 and

STAT3 (Yasukawa et al. 1999). The other level involves induction of proteosomal degradation

of both JAK2 and SOCS-1, requiring the SOCS box domain of SOCS-1 (Zhang et al. 2001).

Obviously, neither Tkip nor SOCS1-KIR has a SOCS box, so the two peptides were compared

for their relative ability to inhibit JAK2 autophosphorylation as well as phosphorylation of the

transcription factor, STAT1. As shown in Figure 4-7a, Tkip but not SOCS1-KIR inhibited JAK2

autophosphorylation. This would suggest that the similar but slight differences in recognition of

JAK2 resulted in significant differences in regulation of JAK2 autophosphorylation.

Next the two peptides were compared for their relative ability to inhibit IFNy activation of

STATla in murine U937 cells. In contrast to inhibition of JAK2 autophosphorylation, both Tkip

and SOCS1-KIR inhibited JAK2 mediated phosphorylation of STAT la (Figure 4-7b). Thus,

Tkip inhibits JAK2 autophosphorylation as well as JAK2 mediated phosphorylation of STATla

in murine U937 cells, while SOCS1-KIR does not inhibit JAK2 autophosphorylation, but does

inhibit JAK2 mediated phosphorylation of STAT la transcription factor. SOCS1-KIR thus shows

the same regulatory pattern as SOCS-1, in that JAK2 autophosphorylation is not inhibited, while

STATla substrate phosphorylation by activated JAK2 is inhibited. It has previously showed that

Tkip, like SOCS-1, also inhibited EGFR autophosphorylation (Flowers et al. 2004). Thus

SOCS1-KIR was tested for ability to inhibit EGFR phosphorylation. As shown in Figure 4-7c,









both peptides inhibited EGFR phosphorylation with Tkip being the more effective inhibitor.

SOCS1-KIR is thus similar to SOCS-1 in its kinase inhibitory function.

Tkip and SOCS1-KIR Inhibit IFNy-induced Activation of Macrophages

IFNy plays an important role in activation of macrophages for innate host defense against

intracellular pathogens as well as serving to bridge the link between innate and adaptive immune

responses (Reviewed in Boehn et al. 1997). Thus, Tkip and SOCS1-KIR were examined for their

ability to block IFNy activation of the murine macrophage cell line Raw 264.7 as determined by

inhibition of nitric oxide (NO) production using Griess reagent (Alexis Biochemicals).

Lipophilic (lipo) versions of the peptides were synthesized with palmitic acid for penetration of

the cell membrane (Thiam et al. 1999). Both Tkip and SOCS-KIR, compared to control peptide,

MulFNy(95-106), inhibited induction of NO by various concentrations of IFNy as shown in

Figure 4-8a. Dose-response with varying concentrations of the peptides against IFNy (6 U/mL)

resulted in increased inhibition of NO production by Tkip and SOCS1-KIR with Tkip being the

more effective of the inhibitors as shown in Figure 4-8b. The control peptide, MulFNGR1(253-

287) was relatively ineffective at inhibition, providing evidence for the specificity of Tkip and

SOCS1-KIR inhibition. Tkip and SOCS1-KIR in combination (33 CIM each) were the most

effective in inhibition of IFNy induction of NO in macrophages. This synergy may reflect

differences in recognition of the autophosphorylation site of JAK2 by the two peptides. Thus,

Tkip and SOCS 1-KIR both inhibited IFNy induction of NO in macrophages with Tkip being the

more effective inhibitor.

Tkip and SOCS1-KIR Inhibit Antigen-specific Lymphocyte Proliferation

Our laboratory has previously shown that Tkip inhibits antigen-specific proliferation of

mouse splenocytes in vitro (Mujtaba et al. 2005). Specifically, Tkip inhibited proliferation of









splenocytes from mice immunized with bovine myelin basic protein (MBP). Here, I compared

Tkip and SOCS1-KIR for their relative ability to inhibit proliferation of MBP-specific

splenocytes in cell culture. Splenocytes (3 x 105 cells/well) were incubated with MBP (50

Clg/mL) in the presence of lipo-Tkip, lipo-SOCS 1-KIR, or lipo-control peptide for 48 h and

proliferation assessed by testing for [3H]-thymidine incorporation. As shown in Figure 4-9, both

Tkip and SOCS1-KIR inhibited MBP-induced proliferation of splenocytes, while the control

peptide had a negligible effect on proliferation. Similar to inhibition of NO production by

macrophages, Tkip was more effective than SOC S1-KIR in inhibition of MBP induced

splenocyte proliferation with 84, 88, and 97% inhibition at 1.2, 3.7, and 11 CIM, respectively,

compared to 61, 67, and 72% for SOCS1-KIR. Thus, both Tkip and SOCS1-KIR inhibited

antigen-induced splenocyte proliferation, which is consistent with SOCS-1 protein inhibition of

antigen-specific lymphocyte activity (Cornish et al. 2003).

An Extended SH2 Sequence (SOCS1-ESS) Peptide does not Bind to pJAK2 (1001-1013)

Based in part on binding experiments with truncations of SOCS-1 protein, it has been

proposed that the SH2 domain plus ESS bind to JAK2 at the activation site represented by

peptide pJAK2(1001-1013), while SOCS1-KIR binds primarily to the catalytic site of JAK2

(Yasukawa et al. 1999). Therefore the SOCS1-ESS peptide, 68ITRASALLDACG, was

synthesized and compared with SOCS1-KIR for binding to biotinylated pJAK2(1001-1013).

As shown in Figure 4-10, SOCS1-KIR as well as Tkip bound biotinylated pJAK2(1001-

1013) in a dose-response manner, while SOCS1-ESS failed to bind. Hence, the SOCS1-KIR

peptide (residues 56-68), but not the SOCS1-ESS peptide (68-79) binds to the JAK2

autophosphorylation site. The SOCS1-KIR peptide, except for residues 53-55 is contained in the

SOCS-1 ESS-SH2 construct, dN56, (Yasukawa et al. 1999) that bound to JAK2









autophosphorylation site. dN56 is a construct in which sequences N-terminal to amino acid

residue 56 have been truncated, therefore it contains KIR, ESS, SH2 domain, SOCS box, and the

C-terminal sequences. Thus, SOCS1-KIR, which is N-terminal and contiguous with SOCS1-

ESS, probably shares overlapping functional sites with SOCS1-ESS. Clearly, SOCS1-KIR is

preferentially recognized by JAK2(1001-1013) compared to the 12-mer SOCS1-ESS. The

specific role of the various residues in the SH2 domain of SOCS-1 in JAK2 and pJAK2(1001-

1013) binding remains to be determined.

Tkip Cellular Targets

Studies carried out in our laboratory have shown that Tkip affects the growth of cells

growing in culture, as well as the progression of EAE. Specifically, Tkip inhibits LNCaP and

DUl45 prostate cancer cells proliferation and inhibits antigen-specific cell proliferation Tkip at

63 Cpg/mouse, given every other day prevented development of acute form of EAE, and induced

stable remission in the chronic relapsing/remitting form of EAE (Flowers et al. 2004, Flowers et

al. 2005, Mujtaba et al. 2005). Moreover, no toxicity was observed when Tkip at 200 Cpg/mouse,

given every other day for one week (Mujtaba et al. 2005). These data suggested that Tkip may

have direct effect on cells of the immune system. Hence, I attempted to define Tkip cellular

targets. Data are presented showing that Tkip specifically targets antigen presenting cells

(APCs), CD4' T, CD8' T, and B cells. In addition preliminary data on SOCS1-KIR peptide

cellular targets are also presented.

Effect of Tkip and SOCS1-KIR on CD4+ T Cells

In order to identify Tkip cellular targets, I first asked whether Tkip had any effect on

primary splenocytes derived from myelin basic protein (MBP) sensitized mice. Sensitization of

mice with MBP, in the presence of adjuvant, results in the development of experimental allergic

encephalomyelitis, EAE, a disease characterized by paralysis. As shown in Figure 4-9, Tkip and









SOCS1-KIR inhibit antigen-specific primary cell (splenocytes) proliferation in a dose-dependent

manner. Next I asked, which cell populations were targeted by Tkip. First the effect of Tkip and

SOCS1-KIR on CD4+ T cell subset was tested. CD4' T cells are important in generating effective

cell mediated immunity and in mediating humoral immune responses. CD4' T cells obtained

from MBP sensitized mice after disease onset were incubated with lipophilic peptides, in the

presence or absence of APCs and MBP. Both Tkip and SOCS1-KIR (33 CLM), but not the control

peptide, MulFNGR1(253-287) inhibited CD4+ T cell proliferation (Figure 4-11a). In addition,

Tkip and SOCS1-KIR (33 CLM), inhibited antigen-induced IFNy production by CD4' T as

determined by IFNy ELISPOT assays (Figure 4-11b). The presence of MBP enhanced cell

proliferation and the number of IFNy-producing cells, but the presence of APCs did not seem to

have such effects. The data presented for peptides were obtained in the presence of both MBP

and APCs. Hence, Tkip and SOCS1-KIR target CD4' T cells and inhibit antigen-specific CD4

T cell proliferation and IFNy production.

Effects of Tkip and SOCS1-KIR on CD8+ T Cells

CD8+ T cells play important roles in the immune response mechanism including cytotoxic

T cell activity and production of cytokines that serve as effectors for various other immune

responses. Since Tkip and SOCS1-KIR inhibited CD4+ T cell activity, I asked whether the

peptides could also target CD8' T cells. CD8' T cells derived from MBP-sensitized mice that

were showing signs of active disease were treated with lipophilic peptides in the presence or

absence of MBP and APCs. IFNy ELISPOT assays carried out. Both Tkip and SOCS1-KIR, but

not the control peptide, MulFNGR1(253-287) showed a general trend in inhibiting MBP-induced

IFNy-production by CD8' T cells (Figure 4-12). The presence of MBP lead to a slight increase in

the number of IFNy-producing cells, but the presence of APCs did not seem to have such an

effect. The data presented for peptides was obtained in the presence of both MBP and APCs.









Effect of Tkip and SOCS1-KIR on B Cells

B cells also play an important role in immune response mechanism. Activation of B cells

results in B cell proliferation and differentiation into antibody producing cells and memory cells.

B cells are also APCs. Tkip was tested for effect on B cells derived from IVBP-sensitized mice,

two months after disease remission. The B cells were treated with either lipophilic Tkip,

lipophilic SOCS1-KIR, or lipophilic control peptide, MulFNGR1(253-287), or no peptide, in the

presence APCs and in the presence, or absence of unprocessed antigen (1VBP). Tkip and SOCS1-

KIR, in the presence oflVIBP and APCs, inhibit antigen-specific B cell proliferation (Figure 4-

13a). In addition, both Tkip and SOCS1-KIR inhibit the secretion oflVIBP-specific antibodies in

IVBP-treated B cells (Figure 4-13b). The addition of APCs did not significantly affect the

experimental results. The data shows that Tkip and SOCS1-KIR target B cell and inhibit both B

cell proliferation and secretion of antibodies by plasma cells, the implication of which is that

Tkip and SOCS1-KIR may have an effect on B cell activity. This is of specific interest in the

study of EAE and multiple sclerosis, because the presence of 1VBP-specific antibodies and of

inflammatory cytokines such as IFNy has been shown to exacerbate disease. Hence, this may be

one of the mechanisms by which Tkip inhibits disease progression in IVBP-sensitized mice.

Effect of Tkip on Macrophages

I have described experiments showing that Tkip and SOCS1-KIR inhibit IFNy-induced

macrophage activation (Figure 4-8). I also tested the ability of Tkip to inhibit LPS-induced

macrophage activity. Both Tkip and SOCS1-KIR inhibited LPS-induced macrophage activity

(Figure 4-14). LPS signaling utilizes the Toll Like Receptor 4 (TLR4), a maj or pathway used in

immune response to gram-negative bacteria. Hence, these results have possible implication on

the effect that Tkip on TLR4 signaling and therefore in inhibition of excessive signaling through

the TLR4. Such excessive signaling has been implicated in the development of inflammation.









Additional research is being carried out to determine additional effects that Tkip may have on

TLR4 signaling. Since macrophages are APCs, these data are preliminary evidence that Tkip and

SOCS1-KIR target APCs.

I have therefore shown that Tkip and SOCS1-KIR target and affect the activity of immune

cells including CD4+ T cells, CD8+ T cells, B cells, and APCs, the implication of which Tkip and

SOCS1-KIR may have effects on antigen-induce immune responses.

SOCS-1 Antagonist Activity of pJAK2 (1001-1013) Peptide

The demonstration that the KIR region of SOCS-1 could bind to the autophosphorylated

JAK2 peptide raised the possibility that the phosphorylated peptide, pJAK2 (1001-1013), may

inhibit the function of endogenous SOCS-1 and thus enhance IFNy and 1L-6 activities that are

mediated by JAK2. As shown in Figure 4-15a, the antiviral activity of a suboptimal dose of IFNy

(0.4 U/mL) was enhanced against encephalomyocarditi s virus (EMCV) in WISH cells by

pJAK2(1001-1013). Specifically, unphosphorylated JAK2(1001-1013) at 11 CIM final

concentration reduced EMCV plaques relative to IFNy alone by 42%, while the same

concentration of pJAK2(1001-1013) reduced plaques by 59%. This is consistent with better

binding of pJAK2(1001-1013) by SOCS1-KIR as shown above and by previous studies showing

that SOCS-1 is active against JAK2 phosphorylated at tyrosine 1007 (Yasukawa et al. 1999). The

peptide alone had no effect on EMCV, similar to media. Thus, pJAK2(1001-1013) boosts the

activity of a suboptimal concentration of IFNy, possibly interfering with endogenous SOCS-1

activity.

At the level of signal transduction, I examined the effects of pJAK2(1001-1013) on

activation of STAT3 transcription factor in the LNCaP prostrate cancer cell line. The cells were

treated with IL-6 to activate STAT3 signaling, which occurs through JAK2 kinase (Flowers et al.









2005). The SOCS-1 gene was overexpressed in these cells, which resulted in reduction of the

level of IL-6 induced activation of pSTAT3 as shown in Figure 4-15b. Treatment of the cells

with pJAK2(1001-1013) (20 CIM) resulted in approximately a two-fold increase in activated

STAT3 compared to IL-6 treated cells that were transfected with SOCS-1, as per densitometry

readings. Expression of SOCS-1 protein in LNCaP cells is shown in Figure 4-15b. Thus,

pJAK2(1001-1013) has an inhibitory effect on SOCS-1 at the level of signal transduction.

I next determined if pJAK2(1001-1013) could enhance GAS promoter activity of IFNy.

Accordingly, a plasmid with the GAS promoter element linked to the firefly luciferase reporter

gene was cotransfected along with Renilla luciferase reporter plasmid as a control, into human

WISH cells. As shown in Figure 4-16c, treatment of the WISH cells with IFNy (1 U/mL)

resulted in a four-fold relative increase in luciferase activity, which was increased to ten-fold and

five-fold by 5 and 1 CIM pJAK2(1001-1013), respectively. pJAK2(1001-1013) alone did not

activate reporter gene (data not shown) and control peptide did not enhance IFNy activation of

reporter gene. Thus, consistent with the anti-SOCS-1 effects of pJAK2(1001-1013), the peptide

also enhanced IFNy function at the level of gene activation. It has recently been shown that

suppression of SOCS-1 in dendritic cells by siRNA enhanced anti-tumor immunity (Shen et al.

2004). In order to determine the effect of pJAK2(1001-1013) on cell-mediated immune response,

C56BL/6 mice were treated with pJAK2(1001-1013), control peptide, or PBS, following

immunization with BSA. It was shown that pJAK2(1001-1013) enhanced BSA-induced

proliferation of splenocytes by four to five fold when compared to control peptide or PBS

(Waiboci et al. 2007). Further, I showed that supernatants containing SOCS-1 protein competed

with SOCS1-KIR for binding to pJAK2(1001-1013) (Figure 4-15d). The demonstration of

SOCS-1 competition for pJAK2(1001-1013) is consistent with pJAK2(1001-1013) antagonism









via sequestration of critical functional site(s) on SOCS-1. Hence, the data have shown that

pJAK2(1001-1013) antagonizes SOCS-1 activity at five different levels; IFNy antiviral function,

IL-6 signal transduction, IFNy activation of reporter gene via the GAS promoter, enhancement of

antigen-specific proliferation, and possible sequestration of binding sites on SOCS-1.

Expression of SOCS-1 Protein

SOCS-1 is a negative regulator of immune factors including IFNs, IL-2, IL-4, and IL-6.

SOCS-1 also modulates signaling by a variety of hormones. However, in spite of the presence of

SOCS-1 and other immune modulators, the host defense system can pathologically perpetuate

inflammation by overproducing immune mediators, such as inflammatory cytokines, that cause

damage to multiple organs, resulting in what are referred to as inflammatory diseases/disorders.

Further, it is estimated that approximately 20% of human cancers result from chronic

inflammation. In addition, silencing of the SOCS-1 gene, by methylation, has been found in

several human cancers (Hanada et al. 2006). Research carried our in our laboratory has shown

that Tkip protects IVBP sensitized mice from developing EAE, an inflammatory disease. Further,

it has shown that Tkip inhibits proliferation of prostate cancer cells. Since Tkip is a SOCS-1

mimetic, I reasoned that recombinant SOCS-1 protein may have the same effect as Tkip in

preventing inflammation and inhibiting proliferation of cancer cells. Therefore, recombinant

SOCS-1 protein was expressed with the aim of obtaining protein to first characterize SOCS-1

functional sites and second to determine functional relationship to Tkip. The recombinant SOCS-

1 protein was tested for binding to JAK2 autophosphorylation site.

First, primers were designed to amplify murine SOCS-1 (muSOCS-1) from an expression

library, pEF-FLAG-I/mSOCS-1, a gift from Dr. D. Hilton (Walter and Eliza Hall Institute of

Medical Research, Victoria, Australia). SOCS-1 DNA was amplified from the expression library,

gel purified, and cloned into pBlueBac4.5/V5-His TOPO TA vector (Invitrogen). Plasmid DNA









was isolated and the presence of muSOCS-1 confirmed by restriction enzyme digestion (Figure

4-16a) and DNA sequencing (Figure 4-16b). I refer to the cloned product as pBlueBac/muSOCS-

1. The constructs were co-transfected, with the baculovirus vector Bac-N-Blue (Invitrogen), into

Sf9 insect cells and propagated. Co-transfection of the pBlueBac4.5/His-V5 TOPO vector

carrying muSOCS-1, with Bac-N-Blue vector ensured that generally, only recombinant virus

would be viable and therefore, would grow. Following transfection and propagation, viral

plaques were picked and tested for presence of muSOCS-1 by PCR. As shown in Figure 4-17,

several plaques had viral DNA of the correct size (650 bp). Of these, two were purified and used

for protein expression. Western blot analysis of the expression of SOCS-1 protein in the Sf9 cells

is shown in Figure 4-15d. The SOCS-1 lysates and the SOCS1-KIR peptide were used for

competition for binding assays to test for ability to inhibit binding of pJAK2(1001-1013) peptide

to immobilized SOCS1-KIR. Figure 4-15d, shows that SOCS1-KIR and SOCS-1 lysate, similar

to Tkip and SOCS1-KIR competed for binding sites on pJAK2(1001-1013), the implications of

which is that SOCS1-lysate may have characteristics similar to Tkip. The goal of the laboratory

is to obtain sufficient purified SOCS-1 protein for detailed characterization of functional sites.











A 1~


S0.75-1 a DTkip
o 0 DRTkip
I /1 0 Tkip
8 0.501 *, Control peptide
1 / r No peptide





0.0 0.5 1.0 1.5 2.0 2.5
Peptide concentration (mM)







o o DRTkip
0 .7 a DTkip
II ;d Tkip
S0.50-9 Control peptide
I~l r No peptide




0.0 0.5 1.0 1.5 2.0 2.5
Peptide concentration (mM)


Figure 4-1. JAK2 autophosphorylation site peptides JAK2(1001-1013) and pJAK2(1001-1013)
bind to SOCS-1 mimetic peptides. A) JAK2(1001-1013) peptide binds to Tkip,
DTkip, and DRTkip. Biotinylated JAK2(1001-1013), at the indicated concentrations,
was added in triplicate to 96-well plates coated with Tkip, DTkip, DRTkip, control
peptide (MulFNy(95-106)), or buffer alone. The assays were developed using
standard ELISA methods with neutravidin-HRP conjugate to detect bound
biotinylated JAK2(1001-1013). B) Biotinylated pJAK2(1001-1013) binds to Tkip,
DTkip and DRTkip. Biotinylated pJAK2(1001-1013) was added to wells coated with
the peptides or buffer and binding assays were carried out as described above. The
binding of JAK2(1001-1013) or pJAK2(1001-1013) to Tkip, DTkip or DRTkip, when
compared to control peptide was statistically significant as determined by Mann-
Whitney signed rank test (P < 0.02 and P < 0.004, respectively).















-- 60 DTkip

I 8 I M Mul FNy(95 106)







50 300 500
Competitior peptide concentration, CLM





S70- Tkip
S60-( D IDRTkip
50 III Mul FNy(95 -106)

o



10

50 300 500
Peptide competitor concentration, tLM


Figure 4-2. Both soluble DRTkip and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip. A)
DRTkip and Tkip inhibit the binding of JAK2(1001-1013) to immobilized Tkip. The
inhibition of binding by Tkip or DRTkip is statistically significant when compared to
control peptide (MulFNy 95-106) (P < 0.05 as determined by Mann-Whitney signed
rank test. B) DRTkip and Tkip inhibit the binding of pJAK2(1001-1013) to
immobilized Tkip. The differences in inhibition of binding by Tkip to pJAK2, when
compared to control peptide, is statistically significant (P < 0.05), but that of DRTkip
is not statistically significant (P > 0.05) as determined by Mann-Whitney signed rank
test. All experiments were carried out in triplicate and the data are representative of
three independent experiments.











JAK2
IFNGR-1
Tkip
D RTkip
Control peptide








p32-lFNGRI


JAK2


+ + + + +
-+ + + +


Figure 4-3. DRTkip and Tkip but not the control peptide, MulFNy(95-106), inhibit JAK2
autophosphorylation. Kinase assays were carried out in the presence of JAK2 kinase,
soluble IFNGR-1, and radiolabelled ATP as described in Chapter 3. To show equal
protein loading, an immunoblot with JAK2 antibody of the reactions was carried out
as described above, but in the presence of unlabelled ATP.


rlM~ :"" ~111













C Media
I~~~ '"""IMedia+SEB
4000*= HHMTkip + SEB
IIIIIl DRTkip + SEB
sooo. O Control peptide + SEB








1.2 3.7 11
Peptide concentration, CLM

I Media
30000* Medla + SEA
555Tki + SEA
E ,,,,,III I IIIII DR C;T kip+ SEA
a 20000* 1m Co iintrol peptide + SEA





0.4 11

Peptide concentration, CLM

Figure 4-4. Tkip, but not DRTkip inhibits superantigen-induced splenocyte proliferation. A)
Tkip, but not DRTkip inhibits SEB-induced splenocyte proliferation. Splenocytes (4 x
105 cells/mL) from naive NZW mice were incubated with varying concentrations of
lipophilic peptides Tkip, DRTkip or control peptide MulFNy(95-106) and SEB (0.5
Cpg/mL) for 72 h, followed by pulse labeling with 3-[H]-thymidine, and harvesting on
filter discs. Cell associated radioactivity was quantified using a p-scintillation counter
and is reported as counts per minute (cpm). Differences between Tkip and control
peptide were statistically significant (P < 0.05) as determined by Mann-Whitney
signed rank test. Differences between DRTkip and control peptide were not
statistically significant (P > 0.05). B) Tkip, but not DRTkip inhibits SEA-induced
splenocyte proliferation. The experiment was carried out as described for SEB, but in
the presence of SEA (0.5 Cpg/mL). The differences between Tkip and control peptide
were statistically significant (P < 0.05), but the differences between DRTkip and
control peptide are not statistically significant (P > 0.05) as determined by the Mann-
Whitney signed rank test. The experiments were carried out in triplicate and data are
representative of three independent experiments.
















f 0.6-
O SOCS1-KIR
e~ 0.5
a Tkip
a 0.4*
2 + 95-106
0.3 a buffer

0.1-

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Biotinylated JAK2 (1001 1013)

B



E2.0-1 O SOCS1-KIR
a Tkip
+95-106
91.5 Buffer








0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Biotinylated pJAK2(1001 1013), mM

Figure 4-5. JAK2 autophosphorylation site peptides bind to SOCS1-KIR. A) JAK2(1001-1013)
peptide binds to both SOCS1-KIR and Tkip. Biotinylated JAK2(1001-1013), at the
indicated concentrations, was added in triplicate to a 96-well plate coated with either
Tkip, SOCS1-KIR, control peptide (MulFNy(95-106)), or binding buffer and binding
assays carried out as described in Chapter 3. B) Biotinylated pJAK2(1001-1013)
binds to both SOCS1-KIR and Tkip. Biotinylated pJAK2(1001-1013) was added to
wells coated with either Tkip, SOCS1-KIR, MulFNy(95-106), or buffer and binding
assays were carried out as described in Chapter 3. The binding of JAK2(1001-1013)
and pJAK2(1001-1013) to Tkip or to SOCS1-KIR, when compared to control peptide
were statistically significant (P < 0.05) as determined by the Mann-Whitney signed
rank test.











Figure 4-6. Both soluble SOCS1-KIR and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip or
SOCS1-KIR. A) SOCS1-KIR and Tkip inhibit the binding of JAK2(1001-1013) to
immobilized Tkip. B) SOCS1-KIR and Tkip inhibit the binding of JAK2(1001-1013)
to immobilized SOCS1-KIR. C) SOCS1-KIR and Tkip inhibit the binding of
pJAK2(1001-1013) to immobilized Tkip. D) SOCS1-KIR and Tkip inhibit the
binding of pJAK2(1001-1013) to immobilized SOCS1-KIR. For all competition for
binding assays the differences in inhibition of binding by Tkip or SOCS1-KIR, when
compared to the control peptide, were statistically significant as Mann-Whitney
signed rank test (P < 0.05). All experiments were carried out in triplicate and the data
are representative of three independent experiments.















50 Tklp
mm, SOCS1-KIR
mm~95-106








100 300 500
Soluble Peptide, pM

B


CD Tklp
ImSOCS1-KIR









100 300 500
Soluble peptide, pM

C

50 Tklp
mmSOCS1-KIR
mm95 -106








100 300 500

Soluble Peptide, pM

D


STkip
r' m SOCS 1-KIR
mm~ 95 106


Soluble peptide, pLM
















~II ~I


Lane 1
JAK2 +
slFNGRI -
Tkip -
SOCSI-KIR -
95-106 -


2 3 45
+ + + +
+ + ++


6 7 8 9 10





Peptide, 100 uM

p- L'


Peptide, 50 uM
pJAK2 *

JAK2 so ~r C


Lane
EGJFR
EGF
Tkip
95-106
SOCS 1-KIR


pEGFR



EGFR


+ + + +


100
50*


1155


I_ _


1 2 7

- + -


ae

Tkip (ll uM)
Tkip (33 uM)
SOCS l-KIR(ll uM)
SOCS l-KIR (33 uM)
253-287 (ll uM)
253-287 (33 uM)


pSTATl I


.. anm em


STAT1 il



Figure 4-7. Differences in the kinase inhibition patterns of SOCS1-KIR and Tkip in JAK2
autophosphorylation, STATla phosphorylation, and EGFR phosphorylation. A)
Tkip, but not SOCS1-KIR or the control peptide, MulFNy(95-106), inhibits JAK2
autophosphorylation. B) SOCS1-KIR and Tkip, but not the control peptide
MulFGR1(253-287) inhibit IFNy-induced STATla activation in murine U937 cells.
Immunoblots with phosphorylated (pY701) STATla and the corresponding
densitometry readings of band intensities are shown. The membrane was stripped and
reprobed with STATla antibody. C) Both Tkip and SOCS1-KIR inhibit EGFR
phosphorylation. In vitro kinase assays were carried out in which SOCS1-KIR, Tkip
or control peptide was incubated with EGF and EGFR and ATP for 30 min at 250C.
The kinase reaction mixtures were resolved on 12% SDS-PAGE, transferred onto a
nitrocellulose membrane and immunoblotted with anti-phosphorylated EGFR
antibody, with the densitometry readings of band intensities shown. The membrane
was stripped and reprobed with EGFR antibody.






























Media 3.12 6.25 12.5 25
IFNy, ulml

& Media+ IFRy
B
-@ 253-287 + IFRy
-# SOCS1-KIR + IFRy
Tkip+ IFRy
SOCS1-KIR + Tkip + IFRy
X Media









8 11 22 33 44 55 66 77 88 99 110
Peptide, pM

Figure 4-8. SOCS1-KIR and Tkip inhibit IFNy-induced macrophage activation. A) SOCS1-KIR
and Tkip, but not the control peptide significantly inhibited IFNy-induced
macrophage activation as determined by testing for nitrite concentration using Greiss
reagent. The inhibition by Tkip, or SOCS1-KIR compared to control peptide was
statistically significant as determined by Mann-Whitney rank test (P < 0.05). B) Tkip,
and SOCS1-KIR, but not the control peptide show dose-dependent synergy in
inhibiting IFNy-induced induction of NO. Raw 264.7 cells were treated with IFNy, in
the presence of varying concentrations of Tkip and SOCS1-KIR and screened for NO
production as described above. The differences between Tkip or SOCS1-KIR,
compared to the control peptide were statistically significant as determined by
Wilcoxon matched pairs test (P < 0.05).











I MBP
SMBP + Tkip
900. IIIIIIII MBP + SOCS1-KIR
SMBP + 253 287









0 4000*7 1




Peptide concentration, pCM

Figure 4-9. Both SOCS1-KIR and Tkip inhibit proliferation of murine splenocytes. Splenocytes
(1 x 105 cells/well) were obtained from MBP sensitized SJL/J mice that had
developed EAE and were in remission. The splenocytes were incubated with RPMI
1640 complete media containing MBP (50 Clg/mL) and varying concentrations of
lipophilic SOCS1-KIR, lipophilic Tkip or lipophilic control peptide,
MulFNGR1(253-287), for 48 h. Cultures were then incubated with [3H]-thymidine
for 18 h before harvesting. Radioactivity was counted on a p-scintillation counter and
data reported as cpm above background (media only). Both lipo-SOCS 1-KIR and
lipo-Tkip, but not the control peptide inhibited splenocyte proliferation in a dose-
dependent manner. The inhibition of proliferation by lipo-SOCS1-KIR or lipo-Tkip,
compared to the control peptide, was statistically significant as determined by Mann-
Whitney signed rank test (P < 0.05). The data are representative of two independent
experiments, each carried out in triplicate.
















c O SOCS1-KIR
V Tkip
+ 106
~~1 SOCS1-ESS






0.0 0.1 0.2 0.3 0.4 0.5 0.6
Biotinylated pJAK2(1001-1013), mM

Figure 4-10. Biotinylated pJAK2(1001-1013) binds to SOCS1-KIR but not to SOCS1-ESS.
Biotinylated pJAK2(1001-1013) was added to wells coated with either Tkip, SOCS1-
KIR, SOCS 1-ES S, control peptide MulFNy(95-106), or buffer and binding assays
were carried out as described in Chapter 3. The binding of pJAK2(1001-1013) to
SOCS1-KIR when compared to control peptide was statistically significant as
determined by Mann-Whitney signed rank test (P < 0.01), while no significant
binding was observed between SOCS1-ESS and the control peptide (P > 0.05).












Media
I C~ Media + MBP
mMBP +Tkip
looooo. 1 IC II MBP + SOCS1-KIR
E I MBP +253-287





11 33
Peptide concentration, pM




I ~Media
I I"""""IMBP
30 m Tkip
T I IIIIIIIIIII SOCS1-KIR
I I I TI 1 253-287






3.7 11 33 100
Peptide concentration, pM

Figure 4-11. Tkip and SOCS1-KIR inhibit antigen-specific CD4+ T cell proliferation and CD4+
T cell-induced IFNy production. A) Tkip and SOCS1-KIR inhibit CD4+ T cell
proliferation. Splenocytes obtained from MBP sensitized SJL/J mice, in remission,
were enriched for CD4+ T cells and incubated (5 x 105 cells/well) with varying
concentrations of lipophilic peptide in the presence or absence of MBP (50 Cpg/mL)
and APCs for 72 h. The cultures were pulsed with 3[H]-thymidine for 18 h before
harvesting. Radioactivity was counted and is reported as cpm. Differences in
inhibition of proliferation by Tkip or SOCS1-KIR, compared to control peptide,
MulFNGR1(253-287) were statistically significant (P < 0.05) as determined by
Mann-Whitney signed rank test. B) Tkip and SOCS1-KIR inhibit CD4+ T cell
induced IFNy production. CD4' T cells were incubated with lipophilic peptides in the
presence or absence of MBP and APCs. The cells were transferred onto IFNy-
ELISPOT plate, and incubated for 48 h. Spots, representing IFNy-producing cells,
were detected using HRP-conjugated secondary antibody. Differences in reduction of
the number of IFNy-producing cells by Tkip or SOCS1-KIR, compared to control
peptide, were not statistically significant as determined by Mann-Whitney signed
rank test (P < 0.05). The experiments were carried out in triplicate and the data are
representative of two independent experiments.











g, Mlledia
I MBP
mmMBP + Tkip
1411 I mmIr MBP + SOCS1-KIR
12,1 1III MBP + 253 287









1.2 3.7 11 33

Peptide concentration, CIM

Figure 4-12. Tkip and SOCS1-KIR inhibit CD8' T cell-induced IFNy production. Splenocytes
were obtained from MBP sensitized SJL/J mice that a few days after signs of active
disease. The splenocytes were enriched for CD8+ T cells (5 x 105 cells/well) and
incubated with varying concentrations of lipophilic peptides in the presence or
absence of MBP (50 Cpg/mL) and APCs. The cells were transferred into IFNy-Elispot
plates and incubated for 48 h. The spots, representing IFNy-producing CD8' T cells,
were detected using HRP-conjugated secondary antibody. The differences in
inhibition of IFNy production by Tkip-treated or SOCS1-KIR treated cells, compared
to control peptide-treated cells ( 11 and 33 CLM) were statistically significant as
determined by Mann-Whitney signed rank test (P < 0.03). The experiment was
carried out in triplicate and the data are representative of two independent
experiments.













z I Media
I I I Media + MBP
7000' mmTkip
6000 IIIIIIIISOCS 1-KIR
E 5000,1 I I 253-287






3.7 11 33
Peptide concentration, CIM





C Media
E 0.7- -1 I 11 1 MBP
c z mmTkip
3 "~ ~II11 1~ I IIIII~ SOCS1-KIR
4 0.5, 253 -287





0.1-


3.7 11 33
Peptide concentration, pM

Figure 4-13. Tkip and SOCS1-KIR inhibit antigen-induced B cell proliferation and antibody
production. A) Tkip and SOCS1-KIR inhibit B cell proliferation. B cells (5 x 105
cells/well) obtained from MBP sensitized SJL/J mice 2 months after last remission
were incubated with lipophilic peptides in the presence or absence of MBP and APCs.
The data presented for peptides was in the presence of MBP and APCs. The cultures
were pulse labeled with 3[H]-thymidine for 18 h and harvested. Radioactivity was
counted and is reported as cpm. The differences in inhibition of proliferation by Tkip
or SOCS1-KIR (3.7, 11, and 33 CLM), compared to the control peptide were
statistically significant as determined by Mann-Whitney test (P < 0.01). B) Tkip and
SOCS1-KIR inhibit the production of MBP-specific antibodies. B cells (5 x 105
cells/well) from MBP sensitized SJL/J mice were incubated with lipophilic peptides
in the presence of MBP (50 Cpg/mL) and APCs for 48 h. Culture supernatants were
then harvested and tested for MBP-specific antibodies by ELISA. The difference
recorded for Tkip or SOCS1-KIR (33 CLM), compared to control peptide were
statistically significant (P < 0.01) as determined by Wilcoxon matched pairs test. The
experiments were carried out in triplicate and data is representative of two
independent experiments.












10.0



7.5.1 HMLF5 Tk~p
555LF' + S0C51-VIRh



2.5-IL. IS-~i








0.1 0.5 1.0
LPS concentration, Clg/ml

Figure 4-14. Tkip inhibits LPS-induced macrophage activity. Murine macrophage cells, Raw
264.7, were incubated with varying concentrations of LPS alone or with either
lipophilic Tkip, lipophilic SOCS1-KIR or lipophilic control peptide, MulFNy(95-
106), each at 24 CLM final concentration, for 48 h. Culture supernatants were collected
and nitrite concentration determined using Greiss assay. The experiments were
carried out in triplicate and data are representative of two independent experiments.
There was statistically significant difference between Tkip and control peptide (P <
0.05), but none between SOCS1-KIR and control peptide (P > 0.05), at the
concentrations tested, as determined by Wilcoxon matched pairs test.










Figure 4-15. pJAK2(1001-1013) peptide has SOCS-1 antagonist properties. A) pJAK2(1001-
1013) enhances suboptimal IFNy-induced antiviral activity against EMC virus in
human fibroblast WISH cells as determined using a cytopathic assay. B)
pJAK2(1001-1013) reverses SOCS-1 inhibition of STAT3 phosphorylation in human
prostate cancer cells (LNCaP) transfected with a SOCS-1 plasmid DNA, pEF-FLAG-
I/mSOCS-1, provided by Dr. David Hilton. The results of an immunoblot assay with
phosphorylated STAT3 (pY705) and the corresponding densitometry readings of
band intensities are shown. Also shown are results of an immunoblot assay with
unphosphorylated STAT3 antibody as well as SOCS-1 expression in LNCaP cells.
The data are representative of two independent experiments. C) pJAK2(1001-1013)
enhances GAS promoter activity. WISH cells were transfected with a vector
expressing firefly luciferase, driven by a GAS promoter, along with a vector
expressing Renilla luciferase as a control vector. IFNy (1 U/mL) and lipo-
pJAK2(1001-1013) at 5 or 1 CIM final concentration, or a control peptide,
MulFNy(95-125) (5 CIM) were added to the cells. After 48 h incubation, the cell
extracts were assayed for relative luciferase activities using a luminometer. D)
Soluble SOCS-1 protein, similar to SOCS1-KIR inhibits the binding of biotinylated
pJAK2(1001-1013) to SOCS1-KIR. Biotinylated pJAK2(1001-1013) that had been
preincubated with varying concentrations of soluble SOCS1-KIR, SOCS-1 lysate or
control lysates, was added in triplicate to a 96 well plate coated with SOCS1-KIR.
Bound biotinylated pJAK2(1001-1013) was detected using neutravidin-HRP
conjugate as described in Chapter 2. Also shown is an immunoblot lysate showing
expression of SOCS-1 in Sf9 insect cells. Aliquots of the cell lysate were used for the
competition for binding assay.

























Media IFNy IFNy +JAK2 IFNy+ pJAK2


I2 3 4 5 6 7 8 9


II- + +


1 1 1 1 1 '
Treatment None IFN IFN+ IFN+ IFN+
pJAK2 pJAK2 95-125
pIM 5 1 5
D


0*


0*


0*


p 15

B>
E
I 5


Lane


srcsi
Con~ol uctr
pJAK2(100-101:
11.-

pstarl

STR


SOCS-1 plasmid
Control plasmid +


SOCS-


S10.0

S7.5



S2.5


,n


MMSOCS-1 lysate
O Control lysate
mm SOCS1-KIR
C"""" 95-106


c*
60-
50

30





Peptide (pM) 100
Protein (pg/ml) 1 2


SOCS-1 Iransfctred
Control transfected+


SOCS-1


300 500
110 330


-n ,_


nn











Lane 1 2 3 4 5 6 7 8 9 1011121314


10 20 30
MVARNVA NAISPAAEPR RRSEPSSSSS
PGDTHFRTF~R SHSDYRRITIR TSALLD~ACGF
TFLVRDSRQR NCFFIALSVKM ASGPTSIRVH
LLEHYVAAPR RMLGAPLRQR RVRPLQELCR
LRDYLSSFPF QIKGNSKLRP


40 50
SSSPAAPVRP RPCPAVPABA
YWGPLSVHGA HERLRAEPVG
FEQAGRFHLDG SRETF~DCLFE
QRIVAAVGRE NIARIPLNPV


0
50
100
150
200


Figure 4-16. SOCS-1 protein was expressed in baculovirus infected Sf9 insect cells. A) The
restriction enzyme digestion pattern of pBlueBac/muSOCS 1 plasmid DNA with Lane
2 showing the correct restriction enzyme digestion product. B) Amino acid sequence
of the cloned plasmid showing full-length SOCS-1 sequence, sequences derived from
the cloning vector are underlined.











Lane 1 2 3 4 5 6 7 89101112131415


Figure 4-17. Recombinant baculovirus containing muSOCS-1 DNA. PCR amplification of
recombinant plaques (blue) with Baculovirus forward and baculovirus reverse
primers. Lanes 1, 4, 5, 6, 9, and 10 are carrying inserts of the expected size.
Constructs represented by lane 4 and 6 were chosen for further analysis.









CHAPTER 5
DISCUSSION

SOCS-1 is absolutely essential for survival of the individual. Although SOCS-1 knockout

mice appear to be normal at birth, they exhibit stunted growth and die neonately by three weeks

of age (Alexander et al. 1999). These mice exhibit a syndrome characterized by severe

lymphopenia, activation of T lymphocytes, fatty degradation and necrosis of the liver,

hematopoetic infiltration of multiple organs, and high levels of constitutive IFNy as well as

abnormal sensitivity to IFNy (Alexander et al. 1999, Reviewed in Alexander and Hilton 2004,

Yoshimura 2005). IFNy plays a central role in the pathology as SOCS-1 knockout mice that are

deficient in IFNy or IFNy receptor do not die as neonates. Similar pathology and lethality is

observed in normal neonates that are injected with IFNy. It is worth noting that SOCS-1- -, IFNy~I

double knockout mice die by 6 months of age of severe inflammatory disease (Metcalf et al.

2002) indicating that SOCS-1 regulation is not specific for IFNy.

The dynamics of induction of SOCS-1 by IFNy in cells and the activation of STATla is

illustrative of how SOCS-1 attenuates IFNy functions under physiological conditions. For

example, treatment of monocytes or astrocytes with IFNy was followed by activation of the

SOCS-1 gene at approximately 90 min (Dickensheets et al. 1999, Brysha et al. 2001). Low doses

of IFNy resulted in transient increases in SOCS-1 mRNA that returned to baseline after 4 h,

while high concentrations of IFNy resulted in increases of SOCS-1 mRNA up to 24 h. Thus, the

SOCS-1 response appears to be induced by the IFNy signal. Treatment of hepatocytes from

SOCS-1" mice with IFNy resulted in STATla activation within 15 min, which peaked by 2 h

before declining (Brysha et al. 2001). Although STATla is similarly activated in SOCS-1









deficient livers, it persists for 8 h. SOCS-1 thus appears to attenuate IFNy persistent activation of

STATla, which nonetheless allows the beneficial effects of IFNy-induced signaling.

Given the critical importance of SOCS-1 in modulating the activities of IFNy and other

inflammatory cytokines that use tyrosine kinases such as JAK2 in their signaling pathways, our

laboratory developed the small tyrosine kinase inhibitor peptide, Tkip, which is a mimetic of

SOCS-1 (Flowers et al. 2004). Tkip was designed to recognize the autophosphorylation site on

JAK2 involving residues 1001 to 1013 containing the critical tyrosine at 1007 (Yasukawa et al.

1999). Flowers et al (2004) showed that Tkip blocked JAK2 autophosphorylation as well as

tyrosine phosphorylation of substrates such as STATla and IFNy receptor chain, IFNGR-1. The

authors also showed that like SOCS-1, Tkip blocked EGFR autophosphorylation, while not

affecting the tyrosine kinase function of c-Src and vascular endothelial growth factor receptor.

Additional experiments showed that Tkip blocked IL-6 induced activation of the STAT3

oncogene in LNCaP prostate cancer cells, which involved inhibition of JAK2 activation (Flowers

et al. 2005). These studies presented a proof-of-concept demonstration of a peptide mimetic of

SOCS-1 that regulates JAK2 tyrosine kinase function.

Because of its potential for regulation of inflammatory conditions where tyrosine kinases

such as JAK2 play a role in the resultant pathology, Tkip was tested in a mouse model of

multiple sclerosis called experimental allergic encephalomyelitis (EAE) (Mujtaba et al. 2005).

SJL/J mice were immunized with myelin basic protein (MBP) for induction of the

relapsing/remitting form of EAE. Tkip, 63 Clg every other day, given intraperitoneally,

completely protected the mice against relapses when compared to control groups in which

greater that 70% of the mice relapsed after primary incidence of disease. Protection of mice

correlated with lower MBP antibody titers in Tkip-treated groups as well as suppression of MBP-









induced proliferation of splenocytes taken from EAE-afflicted mice. Consistent with its JAK2

inhibition function, Tkip also inhibited the activity of inflammatory cytokine TNF-u, which uses

the STATla transcription factor. Thus, Tkip, like SOCS-1, possesses anti-inflammatory activity

that protects mice against ongoing relapsing/remitting EAE.

The design of Tkip was independent of any knowledge of the structural and functional

domains of SOCS-1. Given that the design focused on Tkip binding to the autophosphorylation

site of JAK2, I compared Tkip with regions of SOCS-1 that have been proposed to be either

directly involved in such binding or to be involved in enhancement of SOCS-1 binding to the

autophosphorylation site. Yasukawa et al. (1999) identified three regions that were directly

involved in SOCS-1 binding to JAK2, the large SH2 domain, a N-terminal 12-amino acid

sequence called extended SH2 (ESS), and an additional N-terminal 12-amino acid region called

the kinase inhibitor region (KIR). The ESS and SH2 domains were felt to bind to the

autophosphorylation or activation site of JAK2, while KIR bound to the catalytic site in this

model. The 12-amino acid ESS sequence consists of residues 68-79, while KIR consists of

residues 56-67. The SOCS1-KIR peptide sequence compared to the KIR above consists of

residues 53-68, sharing just the 168 with the ESS and containing three additional residues in its

N-terminus. In comparative binding, the SOCS1-KIR peptide bound to pJAK2(1001-1013),

while SOCS1-ESS peptide failed to bind (Figure 4-10). Close examination of N-terminal

truncated SOCS-1 expressed proteins, designated dN51 (missing residues N-terminal to 51) and

dN68, in Yasukawa et al (1999) showed that removal of the KIR region resulted in loss of

binding to JAK2 in transfected cells. Further, in direct binding to pJAK2 autophosphorylation

site peptide by truncated dN56 SOCS-1 protein, the SOCS1-KIR peptide sequence except for

residues 53-55, was present along with ESS and SH2 (Yasukawa et al. 1999). Although the









region of SOCS-1 that binds to the autophosphorylation site of JAK2 as per Yasukawa et al.

(1999) is referred to as SOCS-1 SH2 plus ESS, the bindings that they refer to also contained our

SOCS1-KIR sequence. Additionally, I have shown that SOCS-1 protein competed with SOCS1-

KIR for pJAK2(1001-1013), suggesting that the two recognized the JAK2 autophosphorylation

site similarly. Thus, I feel that the binding data with our SOCS1-KIR are consistent with the

binding studies of these authors. It should be noted that the peptide binding approach used here

does not involve assessment of collaboration and/or synergism among the KIR, ESS, and the

SH2 domains of SOCS-1. Thus, based on the studies reported here, along with those by others

(Yasukawa et al. 1999), KIR, ESS and SH2 may all be involved in binding to JAK2

autophosphorylation site. It remains to be determined as to the extent of their relative roles.

I have shown in this study that SOCS1-KIR, independent of other domains of SOCS-1,

can bind directly to a peptide, JAK2(1001-1013), that corresponds to the autophosphorylation

site of JAK2. Further, I showed that SOCS1-KIR competed with Tkip for binding to JAK2(1001-

1013). The competitions suggest that the peptides recognized JAK2 similarly but not exactly the

same way. Phosphorylation of tyrosine 1007 on the JAK2 peptide enhanced binding of Tkip and

SOCS1-KIR. Tkip blocked JAK2 autophosphorylation as well as JAK2 phosphorylation of

STATla, while SOCS1-KIR did not block autophosphorylation but did block phosphorylation

of STATla, similar to the pattern or profile of SOCS-1 inhibition of phosphorylation (Alexander

and Hilton 2004). The peptides were also functionally similar in inhibiting IFNy activation of

macrophages to produce NO and inhibiting antigen-specific induction of proliferation of

splenocytes, with Tkip being the more effective inhibitor. Thus, I have shown here that the KIR

region of SOCS-1 can directly bind to the autophosphorylation site of JAK2. These data are

consistent with the observation made by Babon et al. (2006) on SOCS-3, which like SOCS-1 has









KIR. These authors showed that the SH2, ESS, and the C-terminal half of KIR directly contacted

the phosphotyrosine binding loop of IL-6 receptor, gpl30, and that the N-terminal half of KIR

likely bound the JAK kinase. Hence, SOCS-1 KIR likely binds directly to JAK2

autophosphorylation site.

Tkip is relatively hydrophobic, while SOCS1-KIR is hydrophilic. However, both peptides

have hydropathic profiles that are complementary to that of pJAK2(1001-1013). ESS however,

had a hydropathic profile different from Tkip. Tkip was designed to have a hydrophathic profile

complementary to that of pJAK2(1001-1013) (Flowers et al. 2004, Weathington and Blalock

2003). Thus, Tkip would recognize primarily hydrophobic residues or groups in the pJAK2

peptide, while SOCS1-KIR would recognize primarily hydrophilic residues or groups. The

binding competition could thus be due to a steric interference, which is consistent with

differential effects of Tkip and SOCS1-KIR on JAK2 kinase activity.

It has previously been shown that Tkip has potential anti-tumor (Flowers et al. 2005) and

anti-inflammatory (Mujtaba et al. 2005) properties. Hence, Tkip may have potential as a

therapeutic agent. Here, I have shown that Tkip and SOCS1-KIR directly affect the activity of

CD4' T cells, CD8 T cells, B cells, and macrophages, which provides additional insights of the

direct effect that these peptides have on the cells of the immune system. These data further show

the probable therapeutic potential of Tkip.

The fact that the KIR region of SOCS-1 can bind directly to pJAK2(1001-1013) raises

the possibility that pJAK2(1001-1013) can function as an antagonist of SOCS-1. It has thus been

shown here under four different types of experiments that pJAK2(1001-1013) possesses SOCS-1

antagonist properties. First, pJAK2(1001-1013) enhanced suboptimal IFNy activity. Second,

prostate cancer cells transfected for constitutive production of SOCS-1 protein had reduced









activation of STAT3 by IL-6 treatment. pJAK2(1001-1013) reversed the SOCS-1 effect. Third,

pJAK2(1001-1013) enhanced IFNy activation of the luciferase reporter gene via the GAS

promoter. Fourth, pJAK2(1001-1013) enhanced antigen-specific splenocyte proliferation. As

indicated above, treatment of cells with IFNy resulted in activation of the SOCS-1 gene in

approximately 90 min and it has been proposed that it is associated with the physiological

attenuation of the IFNy response by SOCS-1 (Dickensheets et al. 1999, Brysha et al. 2001)

Consistent with this, it has recently been reported that small-interfering RNA inhibition of

SOCS-1 expression in bone marrow dendritic cells resulted in enhanced CTL activity and IFNy

production by ELISPOT, culminating in enhancement of anti-tumor immunity (Shen et al. 2004).

I have thus shown here that SOCS1-KIR binds directly to the autophosphorylation site of

JAK2, similar to the binding of Tkip SOCS-1 mimetic, which results in inhibition of JAK2

phosphorylation of substrate. This directly identifies a region of SOCS-1 that possesses intrinsic

anti-kinase function. Related to this, the autophosphorylation site peptide, pJAK2(1001-1013),

functioned as an antagonist of SOCS-1. These findings with SOCS-1 mimetics and antagonists

have implications for novel therapeutic approaches to mimicking SOCS-1 for treatment of

inflammatory diseases and for suppressing SOCS-1 in order to enhance the immune response

against cancer and infectious diseases.









CHAPTER 6
FUTURE WORK

Future work will involve expressing and purifying large quantities of murine SOCS-1 and

using the purified protein to characterize the functional regions of SOCS-1 protein. I have also

designed additional experiments to determine specifically the role of the SOCS-1 KIR and ESS

regions. This would provide additional insight on the minimum domain essential for SOCS-1

activity .

Mouse studies are currently underway attempting to determine whether Tkip can be used

for treatment of ongoing relapsing/remitting form of EAE, with implications for treatment of

multiple sclerosis. Further, additional experiments are being designed to determine whether Tkip

binds to the other JAK kinases, JAKl, JAK3 and TYK2, implication of which Tkip would be

used to regulate signaling by these kinases.

For SOCS-1 antagonist studies, mouse studies are being carried out in which attempts are

being made to determine whether pJAK2(1001-1013) can enhance protection against ongoing

infectious disease.





APPENDIX: VECTOR MAP OF THE TRANSFER (CLONING) VECTOR


~I c~4~y~i r=IF;~EI


L~IW~


Cosmments fo~r pBlueBac4.5NB-His
5027 nucleotide


Polyhedrin promoter (pp,J bases 7-95 y
Multiple cloning site: bases 132-189
V5 epitope: bases 196-237
8xH is tag: bases 247-284
SV40 polya8denylation sequence: 289-416
F en d of Ac-PH: bases 420-521
S end of ORF1829: 1429-551 (complemenrtary)
Sequence homologous to ORF1829 sequence in BeeN-Blue DNA: bases 833-1431 (complemaentary)
Arupicill; n resistance gene: 1817-2674
pUC origin: 2822-3495
EE tecZ fragmenrt: 4725-381 3 (complementary)
Sequence homologous to incZ sequence in Bee-N-Blue DBNA: bases 4498-3613 (corplernentary)
Early to late promoter: bases 5027-4728 (corplemnentary)


Figure A-1. A map of the pBlueBac4.5/V5-His vector. Adapted from pBlueBac4.5/V5-His
TOPO Expression Kit manual (Invitrogen).


s~ ~ib I











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BIOGRAPHICAL SKETCH

Lilian Wangechi Waiboci was born on October 5, 1971, in Nyeri, Kenya to Mr. Francis

Waiboci Matu and Mrs. Esther K. Waiboci. She grew up in Nyeri, graduating from Bishop

Gatimu Ngandu Girls High School in 1989. She earned her B.S. in Biochemistry and Zoology

and her M.S. in Biochemistry from the University of Nairobi, Kenya in 1995 and 2001,

respectively.

Upon graduating in 1995, Lilian worked as a high school teacher, teaching Chemistry and

Biology. During her M.S. program, she was a research assistant at the International Livestock

Research Institute (ILRI) and upon completion of her degree requirements worked as a part-time

lecturer at Jomo Kenyatta University of Agriculture and Technology (JKUAT), and later for the

Walter Reed Army Medical Research Proj ect, HIV Laboratory in Kericho, Kenya as a

Laboratory Manager and Research Technician.

Lilian earned her Ph.D. from the Department of Microbiology and Cell Science,

University of Florida in May 2007. She will pursue a carrier that includes both aspects of

research and teaching in a University or a Research Institute. Lilian is married to Dr. George

Muhia Kariuki and they have a son, Victor Kariuki Muhia.