Protein family review
Robert J Ferl, Michael S Manak and Matthew F Reyes
Address: Program in Plant Molecular and Cellular Biology, Department of Horticultural Sciences, University of Florida, Gainesville,
FL 32611, USA.
Correspondence: Robert J Ferl. E-mail: email@example.com
Published: 27 June 2002
Genome Biology 2002, 3(7):reviews3010.1-3010.7
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2002/3/7/reviews/3010
BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)
Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far
investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s
are homo- and hetero-dimers, the biological functions of which are to interact physically with
specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved
in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis,
serving as adapters, activators, and repressors. There are currently 133 full-length sequences
available in GenBank for this highly conserved protein family. A phylogenetic tree based on the
conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family
can be divided into two clearly defined groups. The core region encodes an amphipathic groove
that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences.
The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core
region and may interact with isoform-specific client proteins and/or confer specialized subcellular
and tissue localization.
The 14-3-3 protein family is highly conserved and is repre-
sented throughout the eukaryotic branch of life. The proteins
were discovered in 1967 during a study of the soluble acidic
proteins of the mammalian brain  and were named on the
basis of fraction number during DEAE-cellulose chromato-
graphy and location after starch gel electrophoresis. For 25
years, 14-3-3s were generally thought to reside exclusively in
animal brain tissue and to be involved in the function of
neurons. During this early period of research, 14-3-3s were
characterized as a heterogeneous family of dimeric proteins
with a monomer mass of 25-32 kDa and multiple isoelectric
points. One of the first biochemical functions of the 14-3-3s
to be identified was the activation of the neurotransmitter
pathway enzymes tyrosine hydroxylase and tryptophan
hydroxylase, in a reaction requiring calcium and the cAMP-
dependent kinase or calmodulin-dependent protein kinase II
. Once 14-3-3s were found in Arabidopsis thaliana ,
maize  and other plants, and in tissues other than the
brain, however, perspectives on their presence and roles
broadened, and now 14-3-3s have been found in every
eukaryote that has been screened for their presence. The
vast number of organisms containing 14-3-3s suggests that
this family of proteins is involved in many important biologi-
cal processes .
Gene organization and evolutionary history
Each 14-3-3 protein sequence can be roughly divided into
three sections: a divergent amino terminus, the conserved
core region and a divergent carboxyl terminus. The high level
of conservation in the core region is demonstrated in
Figure la, which shows a similarity plot derived from an
alignment of selected 14-3-3s. A phylogenetic tree generated
from the full alignment of the core region is shown in
Figure 2 and demonstrates that plant 14-3-3s fall into two
groups, an epsilon (8) group and a non-epsilon group [6,7].
The plant non-epsilon group is very different from plant e iso-
forms and animal isoforms. Additional complex groupings
2 Genome Biology Vol 3 No 7 Ferl et al.
(a) H3 H7
1 50 100 150 200 250
(b) ---,.. ... .. ... .......-.. -.
Conservation of 14-3-3 proteins. (a) A graph of percentage similarity derived from (b), which shows a multiple sequence alignment of 40 selected 14-3-3
isoforms. Highly conserved helices in (a) are in red and are indicated by numbers that correspond to helices in the crystal structure of Figure 3. The
alignment in (b) was created using Clustal W . Residues in red are 100% conserved across all isoforms; residues in blue are highly conserved. These
red and blue colors correspond to the red and blue regions of the crystal structure shown in Figure 3.
isfoms iglycosrvd, elcs na)aene an aendcaebnmbrstatcorepod.oheics.n.h crystal..structure ofFgre3-h
alinmet n () as retedusig lusalW  Reidus n rd re 00 cosre_ cosalioom;rsde in ble rhghycosevd.Ths
re-n le ooscresodt h red ad-blu regions of.th crsa stutr shown in Fiur 3.I ... :
and subgroupings are apparent, but the tree must be evalu-
ated carefully because not all species are represented by a
complete genome sequence . The presence of the divergent
termini and the few amino-acid changes that do occur within
the conserved region result in multiple isoforms in most
organisms and present the potential for client-specific inter-
actions occurring in distinct cellular locations .
Although 14-3-3s are found throughout the eukaryotes, Ara-
bidopsis is an excellent model system for studying 14-3-3s
for two reasons: it is a higher eukaryote with a fully
sequenced genome and it has a large family of thirteen
14-3-3 isoforms ; there is also a wealth of knowledge on
key biological pathways. In Arabidopsis, the 14-3-3 epsilon
group has five members (p (mu), s, n (pi), i (iota), and
o (omicron) and the non-epsilon group has eight members
(K (kappa), X (lambda), y (psi), v (nu), v (upsilon), co (omega),
) (phi), and x (chi)). The presence and chromosomal location
of all Arabidopsis 14-3-3 isoforms is known (Table 1); there is
at least one 14-3-3 on each of the five chromosomes. Similar
diversity in chromosomal distribution occurs in other species
where multiple 14-3-3s have been found.
Characteristic structural features
The conserved middle core region of the 14-3-3s encodes an
amphipathic groove that forms the main functional domain,
a cradle for interacting with client proteins. Extensive
Figure 2 (see the figure on the next page)
Phylogenetic relationships of 14-3-3s. This tree is a rooted cladogram from a neighbor-joining analysis  of the 133 different full-length 14-3-3 isoforms
that are currently available in the GenBank database , with Dictyostelium discoideum 14-3-3 as the outgroup. Arabidopsis isoforms are highlighted in
green. The separation of plant epsilon and non-epsilon 14-3-3 proteins is clearly visible.
Figure 2 (see the legend on the previous page)
Tbcooos d Floer
Tobacco bZIP reg D75
Pop-l1s x canescens 260AAb
Soybean GF14 D
V-LC-a faba Protein B
Oryza sativa GF14d
Popuilus x canescens 261AA
Pjlsum sat-m 14331ike 260AA
Toblaccob Protein i
CPotabto p16 3k~ nonce
Tobacco Proteln A
Tomato Pt 6
Tobacco Proteln A
Tomato Proei T3
TBarley Protein C
Lll~n ongflo~n 433Like rt
Oryza sat va $94
Barley Protein A
Potato Leaf Specific
Populous y canescens 262AA P201
4 Genome Biology Vol 3 No 7 Ferl et al.
investigation of the 14-3-3 binding site of the mammalian
serine/threonine kinase Raf-1 has produced a consensus
sequence for 14-3-3-binding, RSxpSxP (in the single-letter
amino-acid code, where x denotes any amino acid and p
indicates that the next residue is phosphorylated) [io]
which has been verified through peptide library screening
. A common, but not exclusive, requirement of 14-3-3
ligands is the phosphorylation of a serine or threonine
residue in the target sequence. The phosphorylated consen-
sus sequence does not, however, fully represent every ligand
that 14-3-3s can bind: they are also known to bind other
non-phosphorylated sequences such as GHSL , and
WLDLE . A common, but not exclusive, requirement of
14-3-3 ligands is the phosphorylation of a serine residue in
the target sequence. For those client proteins whose target
sequences undergo phosphorylation, the binding of 14-3-3s
to the target is the major step of a signal-transduction event.
Despite the simplicity of the binding-site requirements, a
diverse array of proteins potentially interact with 14-3-3s;
some reports suggest that as many as 20% of Arabidopsis
proteins are clients for 14-3-3s .
Our knowledge of the three-dimensional structure of 14-3-3s
is based on the model derived from X-ray diffraction of the
crystals of the and t mammalian isoforms [15,16]. The high
level of conservation of 14-3-3 amino acid sequence in the
conserved core allows the general features of this structural
model to be applied to all known 14-3-3s. The monomer con-
sists of nine a helices organized in an antiparallel manner,
forming an L-shaped structure (Figure 3). The interior of the
L-structure is composed of four helices: H3 and H5, which
contain many charged and polar amino acids, and H7 and
H9, which contain hydrophobic amino acids. These four
helices form the concave amphipathic groove that interacts
with target peptides. An alignment of all currently known
full-length isoforms provides evidence that this groove is
over 70% conserved (Figures 1 and 3). Five of the most
highly conserved regions correspond to helices H1, H3, H5,
H7, and H9 (Figure 1). The conserved amphipathic groove is
the site for ligand binding; amino acids Lys49, Arg56, and
Arg127 in mammalian 14-3-3 sequences have been demon-
strated to interact with the phosphorylated amino acids of
ligands (serine/threonine kinases Raf-1 and Bcr) by muta-
tional [17-19] and co-crystallization [11,13] experiments; the
latter have also shown that both phosphorylated and non-
phosphorylated ligands bind in the amphipathic groove .
The peptides bound by 14-3-3s adopt an extended conforma-
tion, which is thought to reduce steric hindrance between
neighboring amino acids of the ligand .
The 14-3-3 dimerization interaction occurs between the
amino-terminal helix H1 of one monomer and helices H3
and H4 of the opposing monomer; the high conservation of
amino-acid sequence along helices H1 and H3 among
various isoforms allows 14-3-3s to heterodimerize
(Figure 3) [21,22]. Two identical or different client proteins
The crystal structure of 14-3-3s. The model shown is derived from the
human 14-3-3 isoform (PDBIQJB ) and is shown from (a) the top
and (b) one side as visualized by the 3Dmol software found in the Vector
NTI 7.0 Suite. Helix numbers are denoted from H I near the amino
terminus to H9 near the carboxyl terminus. Red and blue areas correspond
to residues of 100% identity and high conservation, respectively, and
correspond to colors on the alignment (Figure I). Yellow areas correspond
to regions of reduced similarity and green areas indicate the nuclear export
can be bound simultaneously by the dimer. This dual
interaction means that possible roles of 14-3-3s include
acting as adapters capable of bringing disparate client
proteins together or moving or rearranging two different
regions of the same protein. An example of the potential
role of 14-3-3 dimers as adapters comes from studies of
14-3-3s interacting with the plant plasma-membrane
proton ATPase and the plant toxin fusicoccin . It has
been suggested that the 14-3-3 dimer binds the carboxy-
terminal autoinhibitory (C-TA) domain of the ATPase in
the presence of magnesium, creating a binding site where
fusicoccin can then interact . Once fusicoccin is bound,
the complex of 14-3-3 and the ATPase is stabilized and the
C-TA domain is displaced, allowing the ATPase to become
Localization and function
In general, 14-3-3s are distributed widely throughout the
cell, supporting the argument that they are involved in mul-
tiple protein-protein interactions in a plethora of biological
roles. There is, however, some differential subcellular local-
ization, suggesting an element of specialization among spe-
cific isoforms. Localization data have been collected for eight
of the Arabidopsis isoforms using isoform-specific antibod-
ies and fusions of 14-3-3 to green fluorescent protein (GFP;
Table 1). These data do not necessarily exhaust all possible
locations for each isoform; instead, they support the idea
that certain isoforms are recruited to distinct subcellular
locations. The localization of 14-3-3 K and v was studied
using carboxy-terminal GFP fusions in transgenicArabidop-
sis . Fusions of K with GFP tended to localize to the
plasma membrane, whereas v-GFP fusions tended to be
found in the cytosol . Additional data collected using
microscopy and immuno-cytochemistry of total nuclear
extracts showed that at least five different forms of 14-3-3s
are found in the nucleus . Chloroplast stromal extracts
screened with isoform-specific antibodies showed that
14-3-3 p and (members of the epsilon group) and 14-3-3 v
and v (members of the non-epsilon group) were the only
14-3-3s prominently located in the chloroplast . The
presence of the two non-epsilon members 14-3-3 v and v in
the chloroplast suggests that these proteins, although
located on distinct branches of the phylogenetic tree, may
share similar roles and cellular locations . 14-3-3 e and |P
were also found in starch grains ; has also been found
Genetic, cellular and functional information on Arabidopsis 14-3-3s
at the nuclear envelope during 14-3-3-GFP studies. GFP-co
fusion studies showed that 14-3-3 nuclear localization is reg-
ulated by the cell-cycle ; generally, 14-3-3-co-GFP fusions
were excluded from the nucleus, but they accumulated in the
nucleus just after nuclear division and then relocated back
out of the nucleus just before completion of cytokinesis .
In addition, a nuclear export signal was identified in the
14-3-3s of the fission yeast Schizosaccharomyces pombe
that is required (in concert with the Crmi nuclear export
machinery) for the shuttling of the mitosis-inducing protein
Cdc25 out of the nucleus following DNA damage .
Nuclear shuttling has emerged as an important biological
role for 14-3-3s , and the nuclear export signal
(I/LxxxLxxxLxL) is highly conserved in the 133 full-length
14-3-3 sequences currently available (Figures 1 and 3).
The 14-3-3s are also differentially expressed among tissues
and organs (Table 1). Arabidopsis ly, X, p, and are found in
the leaves; y and X are also expressed in the stems and
flowers [30o], and X is expressed in pollen grains and stigma
papillar cells . The fact that 14-3-3s are differentially
expressed in various tissues and differentially localized in
subcellular compartments adds a layer of complexity to the
examination and determination of biological roles for
14-3-3s, a complexity that must be reconciled with the highly
conserved nature of 14-3-3-target interactions.
The identification of mutants and the use of transgenic
organisms have provided some insight into some of the
Gene name Isoform
Locus* Cellular localizationt Tissue distribution
Nm, Pm, Ct
Nm, Pm, Ct
N, Pm, Ct
Nm, Pm, Ct
N, Pm/Cw, Ct
Ne, Pm, Ct, Sg
Gene accession number 
Pollen, stigma papillar cells
?Stems, leaves, flowers
Stems, leaves, flowers
Stems, leaves, flowers
Stems, roots, flowers
*The number after 'At' denotes the chromosome. tN, nucleus; Nm, nuclear membrane; Pm, plasma membrane; Ct, cytoplasm; Cw, cell wall; Sg, leaf
starch grain; Ne, nuclear envelope [7,14,30]. For further information on 14-3-3s, please see the Ferl lab website .
6 Genome Biology Vol 3 No 7 Ferl et al.
biological roles and locations of some 14-3-3s. For instance,
a mutation in the RAD24 protein, one of the two 14-3-3s in
S. pombe, reduces the yeast's ability to keep DNA damage in
check . In Saccharomyces cerevisiae, disruption of the
14-3-3 genes BMH1 and BMH2 creates a lethal phenotype
that can be rescued by introducing 14-3-3 isoforms from Ara-
bidopsis, Dictyostelium discoideum, or Homo sapiens .
The Leonardo (14-3-3 t) protein of Drosophila melanogaster
is known to regulate presynaptic function, and its mutation
results in the death of mature embryos . Two transgenic
Arabidopsis lines, one carrying an antisense construct
against 14-3-3 p and another against 14-3-3 e show dramatic
increases in starch production in leaves .
Various 14-3-3s in a variety of species have been found to
interact with proteins involved in signal transduction (such
as Raf-1 ), apoptosis (such as the Bcl2-related protein
Bad ), cell-cycle control (such as Cdc25 ), starch syn-
thesis , nitrogen metabolism , and ATP regulation
(reviewed in ).
Initially, discoveries of 14-3-3s were almost coincidental in
nature; in many cases their identification was serendipitous
after investigating other biochemical questions. Once it
became established that these proteins were ubiquitous,
research was directed toward identifying the number and
sequences of isoforms present in different species as well as
determining their functional diversity. As more genomes are
sequenced, experimental tasks will move towards elucida-
tion of general roles as well as investigation of isoform-spe-
cific roles in order to address the implications of 14-3-3
family diversity within organisms. Such studies are key to
understanding the current conundrum: the conservation of
14-3-3s throughout eukaryotes suggests that some central
biological roles might be served by any 14-3-3 protein, yet
the diversity of 14-3-3 isoforms argues for a multitude of
specific roles. Indeed, some combination of both concepts
might be the case, with some roles being served by any
isoform, and other roles requiring isoform-specific interac-
tions. In any case, all the current data suggest that interac-
tions involving 14-3-3 proteins are critical for the correct
function of higher-order biological systems.
The presence of 14-3-3 proteins in most, if not all, eukaryotic
cells, but not in any prokaryotic cells, offers an interesting
opportunity to study the early evolutionary history of this
protein family and the concomitant development of eukary-
otic regulatory processes.
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In Physiological and Biochemical Aspects of Nervous Integration. Edited by
Carlson FD. Englewood Cliffs, NJ: Prentice-Hall; 1967: 343-359.
The first identification of 14-3-3 proteins in mammal brains.
2. Ichimura T, Isobe T, Okuyama T, Yamauchi T, Fujisawa H: Brain
14-3-3 protein is an activator protein that activates trypto-
phan 5-monooxygenase and tyrosine 3-monooxygenase in
the presence of Ca2+,calmodulin-dependent protein kinase
II. FEBS Lett 1987, 219:79-82.
This study was the first to assign a role to 14-3-3 proteins. It was
shown that 14-3-3s activate tyrosine and tryptophan hydroxylases in a
calcium- and kinase-dependent manner.
3. Lu G, DeLisle AJ, de Vetten NC, Ferl RJ: Brain proteins in plants:
an Arabidopsis homolog to neurotransmitter pathway acti-
vators is part of a DNA binding complex. Proc Natl Acad Sci
USA 1992, 89:1 1490-1 1494.
The first identification of 14-3-3 proteins in Arabidopsis thalana. They
were found to be associated with G-box-binding factors.
4. de Vetten NC, Lu G, Ferl RJ: A maize protein associated with
the G-box binding complex has homology to brain regula-
tory proteins. Plant Cell 1992, 4:1295-1307.
The first identification of 14-3-3 proteins in maize.
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A review discussing the roles of 14-3-3s in cell-cycle control, signal
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A review of the participation of 14-3-3s in plant regulatory events,
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A review of Arabidopsis 14-3-3 gene structure and phylogeny.
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forms in multicellular organisms reflect functional speci-
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This study describes the binding affinity of Arabidopsis 14-3-3 isoforms
to a unique peptide and discusses the potential for functional specificity
for these proteins across all multicellular species. A substantial family
tree is also presented.
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A comprehensive review of the history, functions, cell-specific expres-
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This study was the first to show that 14-3-3 is a specific phosphoserine-
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Two different binding motifs were identified after screening mammalian
and yeast peptide libraries with 14-3-3 proteins. Additionally, the crystal
structure of 14-3-3 ( bound to a phosphoserine target peptide was
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14-3-3 crystal structures in complex with phosphorylated and unphos-
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Crystal structure of the mammalian 14-3-3 ( isoform.
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Crystal structure of the mammalian 14-3-3 t isoform.
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Serine phosphorylation-dependent association of the band
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Mutation of serine to alanine severely reduced the level of 14-3-3 and
PTPH I binding.
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hydrophobic surface of an amphipathic groove of 14-3-3zeta
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Mutation of hydrophobic residues on the 14-3-3 amphipathic groove
shows that these residues are involved with binding the client protein.
The mutated residues are conserved among isoforms, suggesting a
general importance for ligand binding.
19. Thorson JA, Yu LW, Hsu AL, Shih NY, Graves PR, Tanner JW, Allen
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Evidence that 14-3-3 proteins form both hetero- and homodimers.
22. Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ,
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share topology with other signals recognized by the CrmlI nuclear
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Scanning confocal microscopy and immunocytochemistry with mono-
clonal antibodies against plant 14-3-3 proteins were used for localiza-
24. Sehnke PC, Henry R, Cline K, Ferl RJ: Interaction of a plant
14-3-3 protein with the signal peptide of a thylakoid-tar-
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14-3-3 isoforms in the chloroplast stroma. Plant Physiol 2000,
Immuno-electron microscopy of leaf tissue and western-blotting analy-
sis of chloroplast fractions with monoclonal anti-14-3-3 antibodies
localized 14-3-3 proteins to the chloroplast stroma and the stromal
side of thylakoid membranes.
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In situ hybridization showed that expression of X (chi) mRNA was
prominent in epidermal tissue of roots, petals, and sepals of flower
buds, papillae cells of flowers, siliques, and endosperm of immature
seeds. These results show plant 14-3-3 gene expression exhibits cell-
and tissue-specific localization rivaling that observed for 14-3-3 proteins
within the mammalian brain.
26. Sehnke PC, Chung HJ, Wu K, Ferl RJ: Regulation of starch accu-
mulation by granule-associated plant 14-3-3 proteins. Proc
Natl Acad Sci USA 2001, 98:765-770.
14-3-3 proteins in Arabidopsis were shown to regulate starch synthesis
through the use of two antisense constructs of 14-3-3 e and 14-3-3 P.
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microscopy and showed localization to nuclei undergoing cytokinesis.
28. Ford JC, al-Khodairy F, Fotou E, Sheldrick KS, Griffiths DJ, Carr AM:
14-3-3 protein homologs required for the DNA damage
checkpoint in fission yeast. Science 1994, 265:533-535.
This investigation identified two 14-3-3 proteins, Rad24 and Rad25,
that are involved in the DNA-damage checkpoint during mitosis.
29. Lopez-Girona A, Furnari B, Mondesert 0, Russell P: Nuclear local-
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protein. Nature 1999, 397:172-175.
The Rad24 14-3-3 protein controls the intracellular distribution of Cdc25.
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An entry in the Plant Gene Register. Sequences were obtained from
stem and flower tissues.
31. van Heusden GP, van der Zanden AL, Ferl RJ, Steensma HY: Four
Arabidopsis thaliana 14-3-3 protein isoforms can comple-
ment the lethal yeast bmhl bmh2 double disruption. FEBS Lett
In this exquisite study, the genes encoding two indigenous 14-3-3 iso-
forms in bakers' yeast were deleted to produce a lethal phenotype and
the phenotypes were rescued by replacement of the genes with those
of four of six Arabidopsis isoforms.
32. Broadie K, Rushton E, Skoulakis EM, Davis RL: Leonardo, a
Drosophila 14-3-3 protein involved in learning, regulates
presynaptic function. Neuron 1997, 19:391-402.
Immunolocalization studies showed that the Leonardo 14-3-3 protein
was expressed at synaptic connections enriched in presynaptic boutons
of the neuromuscular junction. Null leonardo mutants die as mature
33. Chang HC, Rubin GM: 14-3-3 epsilon positively regulates Ras-
mediated signaling in Drosophila. Genes Dev 1997, 11:1132-
This study discusses how 14-3-3 e protein appears to function in multi-
ple receptor tyrosine kinase signal transduction pathways.
34. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ: Serine phospho-
rylation of death agonist BAD in response to survival factor
results in binding to 14-3-3 not BCL-X(L). Cell 1996, 87:619-
An example of how a 14-3-3 is involved with apoptosis. In the pres-
ence of a survival factor, Bad was phosphorylated on two serine
residues embedded in 14-3-3 consensus binding sites. Only the non-
phosphorylated Bad promotes cell death.
35. Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms H:
Mitotic and G2 checkpoint control: regulation of 14-3-3
protein binding by phosphorylation of Cdc25C on serine-
216. Science 1997, 277:1501 -1505.
An example of 14-3-3s involved in cell-cycle control. A mutation in
human Cdc25c prevented the phosphorylation of Ser216, thus pre-
venting 14-3-3 binding. Conditional overexpression of this mutant per-
turbed mitotic timing and allowed cells to escape the G2 checkpoint
arrest induced by either unreplicated DNA or radiation-induced
36. Bachmann M, Huber JL, Athwal GS, Wu K, Ferl RJ, Huber SC:
14-3-3 proteins associate with the regulatory phosphoryla-
tion site of spinach leaf nitrate reductase in an isoform-spe-
cific manner and reduce dephosphorylation of Ser-543 by
endogenous protein phosphatases. FEBS Lett 1996, 398:26-30.
14-3-3 proteins were shown to regulate nitrate reductase by binding a
synthetic phosphonitrate reductase peptide.
37. Entrez nucleotide view
Access to the GenBank sequence database.
38. The laboratory of Robert J. Ferl [http://www.hos.ufl.edu/ferllab/]
Our lab's website contains additional information on 14-3-3s and
39. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties
and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680.
CLUSTAL W is a common method used to generate multiple
40. Saitou N, Nei M: The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 1987,
The neighbor-joining method is a statistical method used to generate
possible phylogenetic trees.