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Purification Methods of MARCKS from Bovine Brain

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Purification Methods of MARCKS from Bovine Brain
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Chakravarty, Trina
Bubb, Michael ( Mentor )
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Gainesville, Fla.
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..Oluinie 9, issue 3 - Sprin i,. . i 200



Purification Methods of MARCKS from Bovine Brain

Trina Chakravarty



ABSTRACT


Myristoylated alanine-rich C kinase substrate (MARCKS) is a well-characterized, natively unfolded protein that

is thought to play a prominent role in the regulation of actin dynamics. MARCKS binds both calmodulin and actin

and has been implicated in processes that necessitate regulated rearrangement of the actin cytoskeleton1. MARCKS

is also known to interact with several other ligands and promote the regulation of cellular motility as well as

cellular contractile function. The MARCKS protein is redistributed from the cytoplasmic face of the plasma

membrane to the cytosol upon phosphorylation by protein kinase C following the stimulation of several cell types

due to hormones and other agonists2.



The studies conducted over the past year have focused primarily on the isolation and purification of the

MARCKS protein. Non-myristoylated MARCKS was isolated from bovine brain, and the protein was isolated

and purified using various techniques including bouncing, perchloric acid treatment (PCA), and heat, and the

samples were concentrated via dialysis. The results were analyzed via gel electrophoresis using an SDS gel

to separate the protein based on molecular weight and charge. The western blotting technique was also used

to quantitatively identify the amount of MARCKS isolated post-treatment. Using native gels, a 2-D gel analysis

was also used to verify the amount of MARCKS present in the bovine samples that were created. An anion

exchange column was finally used to identify at which salt concentration MARCKS elutes.



An understanding of MARCKS structure, of its interactions with calmodulin, actin and PKC, and of its capacity of

cycle between the membrane and the cytosol provides a conceptual framework for understanding the biological

role of the protein.



INTRODUCTION


Stimulation of membrane polyphosphoinositol turnover is one of the most commonly employed signal

transduction systems in normal physiology. The two second messengers generated by this reaction include

1,4,5-triphosphate and diacylglycerols, which can activate most isoforms of protein kinase C (PKC)3. The PKC

family members are diacylglycerol-activated, calcium-dependent protein kinases that regulate a large number

of cellular responses including synthesis of macromolecules, activation of transport systems, secretion of
5





hormones, contraction of muscles, and many others . However, the molecular steps between the activation of

PKC and the resultant biological responses are largely unknown. One approach to elucidating these

molecular pathways is to study direct cellular substrates for PKC in the hope that the knowledge gained will help

to explain downstream eventss5. Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely

distributed, specific PKC substrate whose phosphorylation has been used as a marker of PKC activation in vivo.

The MARCKS family is comprised of two groups of proteins. The members of the first group, MARCKS, are 30-35

kDa proteins encoded by the macs genes. The second group, referred to as the MARCKS related proteins (MRP)

is composed of 20 kDa proteins encoded by the mrp genes.




I 6 86 109
murie MRP Myr-GSQSSK----- ----KKXKK-FSFKKBFKLSGLSFKNRRK .........C00-
murine MARCKS A F R S F KSK
bovine MARCKS A F R S F KNK
mynsmoylatd basic domain
N-lenrinus
Figure 1. Structure of proteins of the MARCKS family. The amino acid sequence of the myristoylated

N terminus, which contains the consensus sequence GXXXS recognized by NMT, as well as of the

basic domain, which contains the series phosphorylated by protein kinase C, are shown for murine

MRP (uppersequence). Nonconserved residues (underlined in MRP sequence) are shown for murine

and bovine MARCKS (lowersequences). The amino acid residues are numbered (top) according to

the MRP sequence6.



MARCKS is an acidic protein that is unusually rich in alanine, glycine, proline, and glutamic acid. The protein is a

rod-shaped molecule containing three distinct domains: an N-terminal myristoylated domain that mediates binding

to membranes, a highly conserved MH2 domain of unknown function, and a basic effector domain containing the

PKC phosphorylation sites and the calmodulin and actin-binding sites. The effector domain (ED) is highly basic

in contrast with the rest of the highly acid protein.















MKRGKED: L'./s LygLy --E S F t L ILr. .IrI -LyrLteuSerGly, � rSe# LynLysAsnrAygsLys


Figure 2. Molecular model of the ED. An atomic model of the MARCKS ED, built in agreement with

all known structural data, shows the molecule in an extended conformation. This peptide model was

built using the Insight-II/Biopolymer package (MSI) and was provided by Dr. Diana Murray of

Cornell University. The 13 basic lysine and arginine residues are colored blue, the five

aromatic phenylalanine residues are colored green and the four serine residues containing the





PKC-phosphorylated sites are colored brown. Shown as a blue mesh is the +25 mV equipotential

surface, as calculated in a 100mM univalent salt solution using GRASP. Note the strongly positive

field around the N- and C-terminal lobes of the ED. Below the primary sequences of the bovine

MARCKS and MRP EDs are aligned using the same color code. The proline residue of MRP that

replaces the second serine residue of MARCKS is underlined7.



MARCKS is known to be involved in brain development, post-natal survival, cellular migration, adhesion,

and neurosecretion. MARCKS is also reported to be involved in the etiology of diseases such as Alzheimer's,

chronic bronchitis, asthma, cystic fibrosis, and rheumatoid arthritis8. Several in vivo studies have implicated

MARCKS in an actin-regulating function, as MARCKS co-localizes with F-actin, and the dissociation of this complex

is regarded as a change in actin dynamics9.



In order to elucidate the function of MARCKS and understand its function in vitro, purification techniques that

avoid excessive denaturing steps were performed.



MATERIALS AND METHODS


Makeup of bovine brain homogenate samples (PCA treatment)


538 pL of homogenized bovine brain was combined with 12 pL diisopropylfluorophosphate (DFP),12 pL PMSF,

2000 pL 0.5M PCA and was left for five minutes on ice. The sample was then neutralized with 500 pL 2M tris base

and 30.62 pL of the detergent Triton X (10%) was added. The sample was then dounced and an additional 40 pL

of DFP was added and spun in a TL 100.4 rotor centrifuge at 65,000 rpm for 15 minutes at 40C.



Makeup of bovine brain homogenate samples (PCA and heat treatment)


In an attempt to further purify MARCKS, a combination of Triton-X, PCA, and heat is used. 538 pL of

homogenized bovine brain was combined with 12 pL diisopropylfluorophosphate (DFP),12 pL

phenylmethylfulfonyl fluoride (PMSF), 2000 pL 0.5M PCA and was left for five minutes on ice. The sample was

then neutralized with 500 pL 2M tris base and 30.62 pL of the detergent Triton X (10%) was added. The sample

was then dounced and an additional 40 pL of DFP was added and spun in a TL 100.4 rotor centrifuge at 65,000

rpm for 15 minutes at 40C. The sample was then heated for 10 minutes at 800C and spun once more in the TL

100.4 rotor at 65,000 rpm for another 15 minutes at 40C.



Testing PCA vs. PCA and heat treatment of bovine brain in the purification of MARCKS


Both treated samples of bovine brain homogenate were compared on an 8% SDS gel. A western blot analysis

was performed to determine the different techniques of purification of MARCKS.





2-D gel of heat and PCA-treated bovine brain


A 6% native gel was run using 30 pL of the PCA and heat-treated bovine brain to determine the level of purity

of MARCKS. The gel was run at 80 volts for 2 hours. The lane from the native gel containing the sample was then

run on a 10% SDS gel, which was again run at 80 volts for 2 hours.



Dialysis


The dialysis buffer used consisted of 10 mM tris-HCI, 100 pM EGTA, 5 mM p-mercaptoethanol (ME), 2 mM EDTA,

100 pM PMSF, and 0.6 mM DFP. For the dialysis, a Spectrum #1 1.0 cm dialysis tubing was used. The membrane

was soaked in deionized water for 10 minutes and 13 mL of the sample of PCA and heat-treated bovine

brain homogenate was dialyzed for 12 hours. After 12 hours, the sample was spun in a 50.2 Ti rotor at 36,000

rpm for 15 minutes at 40C.



Testing PCA vs. PCA and heat treatment of bovine brain in the purification of MARCKS


Both treated samples of bovine brain homogenate were compared on an 8% SDS gel. A western blot analysis

was performed to determine the different techniques of purification of MARCKS.



2-D gel of heat and PCA-treated bovine brain


A 6% native gel was run using 30 pL of the PCA and heat-treated bovine brain to determine the level of purity

of MARCKS. The gel was run at 80 volts for 2 hours. The lane from the native gel containing the sample was then

run on a 10% SDS gel, which was again run at 80 volts for 2 hours.



Dialysis


The dialysis buffer used consisted of 10 mM tris-HCI, 100 pM EGTA, 5 mM p-mercaptoethanol (ME), 2 mM EDTA,

100 pM PMSF, and 0.6 mM DFP. For the dialysis, a Spectrum #1 1.0 cm dialysis tubing was used. The membrane

was soaked in deionized water for 10 minutes and 13 mL of the sample of PCA and heat-treated bovine

brain homogenate was dialyzed for 12 hours. After 12 hours, the sample was spun in a 50.2 Ti rotor at 36,000

rpm for 15 minutes at 40C.



Anion exchange column analysis


To further purify MARCKS without denaturing the protein, an anion exchange column was performed to determine

at what salt concentration the protein elutes. The start buffer contained a 20 mM tris-HCI solution and the

finish buffer contained a mixture of 20 mM tris-HCI and 1.0 M NaCI. A HiTrap� 1 mL MonoQ column was used

to elute MARCKS.


Western blot







After the SDS gel was run, the gel, filter paper, and fiber pads were soaked in a 20% MES/MeOH transfer buffer

for 10 minutes. The PVDF membrane was equilibrated for 10 minutes as well. The gel sandwich cassette was run

at 25 volts overnight. The next day, the membrane was incubated in blocking buffer (BSA A7030 5%, 100 mM

L-lysine, diluted 1:2.5 in IX PBS) for 20 minutes. A 3-hour incubation of the membrane was performed with

a primary Abcam� goat anti-MARCKS antibody (IgG, AHP695) in a 1:1000 dilution. An hour-long incubation with

the secondary antibody (Sigma� A5420, HRP conjugate) was performed in a dilution of 1:50,000. To develop

the blot, equal parts of Supersignal West Pico Luminol Enchancer solution and peroxide solution were mixed and

the membrane was incubated in the mixture for five minutes. In the darkroom, the blot was developed for 5, 10,

and 15 seconds.



RESULTS


Western blot of PCA vs. PCA and heat treatment of bovine brain homogenate


Following a western blot assay of bovine brain treated with both PCA and heat and PCA treatment alone, it

was observed that the inclusion of heat treatment does not further degrade MARCKS (Figure 3). There was an

overall estimated 25% recovery of MARCKS following both heat and PCA and heat treatments of the bovine

brain homogenate samples.


Figure 3. Bovine brain homogenate sample treated with PCA and PCA/heat treatments. Reading from

left to right, Lane 1 is 2 pL of MARCKS control (50 pL MARCKS62 scan 1069, GFC and 450 pL

MARCKS buffer). Lane 2 is 40 pL of PCA-treated bovine brain homogenate, and Lane 3 is 40 pL of

PCA- and heat-treated bovine brain homogenate. Lanes 2 and 3 qualitatively show approximately

equal amounts of myristoylated MARCKS.




2-D gel of heat- and PCA-treated bovine brain


The gel was very light but a band which may have corresponded to MARCKS was detected.


Anion exchange column analysis

It is known that MARCKS elutes at a salt concentration of approximately 40%. According to Figure 4, it was
presumed that MARCKS eluted around fraction 8.





o v e oain m 4%t lu wa W






d n. Mane MARnCK a
- - I


U . 1i . ... $ .,



- , . '+-, 4 i.






Figure 4. Anion exchange column. Fractions 6 through 30 were collected. 100%/o elution was assumed
to have occurred by fraction 19. 40%/o elution was assumed to be at fraction 8 (not marked on
graph). Since MARCKS shows no visible absorption peak when eluted, an SDS gel was run to
determine whether MARCKS was present in anion exchange column fraction 8 (see Figure 5).


























Figure 5. SDS gel analysis of anion exchange column fractions 5-11. All lanes have 30 pL

material loaded. Reading from left to right, Lane 1 is the MARCKS control (50 pL MARCKS62 scan

1069, GFC, and 450 pL MARCKS buffer). Lane 2 is the flow through from the column. Lane 3 is the

pre-loaded anion exchange column sample. Lanes 4-10 are fractions 5, 6, 7, 8, 9, 10, and

11, respectively. There is very light visible staining at the arrowhead, indicating the possible presence

of myristoylated MARCKS. This would suggest that MARCKS eluted at fraction 9.




DISCUSSION


The findings in this study support the hypothesis that PCA and heat treatments of bovine brain homogenate do

not detrimentally reduce the recovery of MARCKS. In agreement with previous studies, MARCKS is a heat-

stable protein and will elute only in the absence of calcium.10 While excessively stringent detergent usage

can severely alter the amount of MARCKS in a bovine brain homogenate sample, the techniques listed above aided

in proposing reasonable purification methods of MARCKS.



However, since MARCKS function is greatly regulated via posttranslational modifications including

phosphorylation and myristoylation, it is vital to further investigate these posttranslational modifications in detail

to understand their physiological functions11. More importantly, the mechanism of PKC-dependent cellular

responses could be understood to a greater extent. Although the MARCKS PSD peptide is a substrate for PKC as

well as conventional species, almost nothing is known about its comparative behavior as a substrate for the 10

or more PKC isoenzymes described to date, and thus a study in this direction would be vital in understanding

its interactions with other isoenzymes.



Another area of further interest is MARCKS myristoylation. MARCKS was first found to be myristoylated by Aderem

et a112 in mouse macrophages. Initial suggestions that the protein might be myristoylated posttranslationally in

an agonist-dependent manner were not supported by James and Olson13, who demonstrated in other cells that

the protein was myristoylated co-translationally. Another recent study14 showed, however, that there exists a pool

of non-myristoylated MARCKS in rat brain, which conceivably could be myristoylated posttranslationally. The






question of possible posttranslational myristoylation and demyristoylation, perhaps stimulated by agonists,

has therefore not been completely resolved and is another interesting field to explore.






REFERENCES



1. Aderem A. The MARCKS family of protein kinase-C substrates. Biochem. Soc. trans 1995;587-591.

2. Manenti S, Sorokin 0 et. al. Protein Kinase C-mediated Phosphorylation and Calmodulin Binding of

Recombinant Myristoylated Alanine-rich C Kinase Substrate (MARCKS) and MARCKS related protein. Biochem

J. 1993;269:9361-9367.

3. Wang J, Walaas S, Sihra T, Aderem A, Greengard P. Phosphorylation and associated translocation of the 87-

kDa protein, a major protein kinase C substrate, in isolated nerve terminals. Proc. Nat/. Acad. Sci. USA.

1989;86: 2253-2256.

4. Ramsden, J. MARCKS: a case of molecular exaptation? IJBCB 2000;32: 475-479.

5. Blackshear P. The MARCKS Family of Cellular Protein Kinase C Substrates. J. Biol. Chem. 268: 1501-1504.

6. Vergeres G, Manenti S, Weber T, St?rzinger C. The Myristoyl Moiety of Myristoylated Alanine-rich C Kinase

Substrate (MARCKS) and MARCKS-related Protein Is Embedded in the Membrane. The American Society

for Biochemistry and Molecular Biology, Inc. 1995;270: 19879-19887.

7. Arbuzova A, Arndt AP, Vergeres G. Cross-Talk Unfolded: MARCKS proteins. Biochem J 2002;362: 1-12.

8. lioka H, Ueno N, Kinoshita N. Essential role of MARCKS in cortical actin dynamics during gastrulation movements.

J. cell boil. 2004:164;169-174.

9. Zhao Y, Neltner B, Davis H. Role of MARCKS in regulating endothelial cell proliferation. Am J. Physiol. Cell

Physiol. 2000;279: C1611-C1621.

10. Manenti S, Sorokine 0 et al. Isolation of the Non-myristoylated form of a major substrate of protein kinase

C (MARCKS) from Bovine Brain. J Biol. Chem. 1993; 268:6878.

11. Taniguichi et al.. Myristoylated Alanine-rich C Kinase Substrate (IMARCKS), a Major Protein Kinase C Substrate, is

an in Vivo Substrate of Proline-directed Protein Kinase(s). J. Biol. Chem . 1994; 269: 18299.

12. Aderem, A. A,, Albert, K. A,, Keum, M. M., Wang, J. K. T., Greengard, P. and Cohn, Z. A. (1988) Nature 332,362-364

13. James, G., and Olson, E. N. (1989) J. Biol. Chem. 264,20928-20933

14. McIllhinney, R. A. J., and McGlone, K. (1990) Biochem. J. 271,681-685


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