Other Drosophila 14-3-3 proteins

Mutations in the gene encoding a Drosophila 14-3-3epsilon protein act as suppressors of the rough eye phenotype caused by the ectopic expression of RAS1V12. Using a simple loss-of-function 14-3-3epsilon mutation, it has been shown that 14-3-3epsilon acts to increase the efficiency of RAS1 signaling. The 14-3-3epsilon protein appears to function in multiple RTK pathways, suggesting that it is a general component of RAS1 signaling cascade. Sequence analysis of three dominant-negative alleles defines two regions of 14-3-3epsilon that participate in RAS1 signaling. 14-3-3epsilon and 14-3-3zeta, two 14-3-3 protein family members, are partially redundant for RAS1 signaling in photoreceptor formation and in animal viability. These genetic data suggest that 14-3-3epsilon functions either downstream of or parallel to RAF, but upstream of nuclear factors in RAS1 signaling (Chang, 1997).

The 14-3-3 protein family is known to interact with various proteins involved in signaling pathways. The expression pattern of the Drosophila 14-3-3 (d14-3-3epsilon) protein during embryonic development is reported. The epsilon isoform of 14-3-3 functions in the Sevenless pathway required for R7 photoreceptor formation. In syncytial blastoderm when the nuclei divide rapidly, d14-3-3epsilon localizes in the nuclei. During cellularization d14-3-3epsilon gradually becomes membrane-bound. During gastrulation, an enhanced staining in the perinuclear region is observed in various tissues. Co-labeling with dp-ERK, which recognizes the activated form of MAPK, suggests that d14-3-3epsilon is expressed prior to MAPK activation. During neuronal differentiation, the d14-3-3epsilon protein remains at a high level in the neuronal cytoplasm. The accumulation of high levels of d14-3-3epsilon may be due to continuous protein synthesis in the neurons since the mRNMA is detected specifically in the CNS and PNS during neuronal differentation (Tien, 1999).

14-3-3 proteins in yeast

The kinase Tor is the target of the immunosuppressive drug rapamycin and is a member of the phosphatidylinositol kinase (PIK)-related kinase family. It plays an essential role in progression through the G1 phase of the cell cycle. The molecular details of Tor signaling remain obscure, however. Two Saccharomyces cerevisiae genes, BMH1 and BMH2, were isolated as multicopy suppressors of the growth-inhibitory phenotype caused by rapamycin in budding yeast. BMH1 and BMH2 encode homologs of the 14-3-3 signal transduction proteins. Deletion of one or both BMH genes causes hypersensitivity to rapamycin in a manner that is dependent on gene dosage. In addition, alterations in the phosphopeptide-binding pocket of the 14-3-3 proteins have dramatically different effects on their ability to relieve the growth-arresting rapamycin phenotype. Mutations that prevented 14-3-3 from binding to a phosphoserine motif abolish its ability to confer rapamycin resistance. In contrast, substitution of two residues in 14-3-3 that surround these phosphoserine-binding sites confer a dominant rapamycin-resistant phenotype. These studies reveal 14-3-3 as an important component in rapamycin-sensitive signaling and provide significant new insights into the structure and function of 14-3-3 proteins (Bertram, 1998).

The switch from mitosis to meiosis is controlled by the Pat1(Ran1) kinase-Mei2p system in Schizosaccharomyces pombe. Mei2p promotes both premeiotic DNA synthesis and meiosis I, and its RNA binding ability is essential for these two processes. Mei2p forms a dot structure in the nucleus prior to meiosis I, aided by a specific RNA species named 'meiRNA'. Pat1 kinase phosphorylates Mei2p on two positions and downregulates its activity. Pat1 kinase undergoes inactivation under meiotic conditions, as a result of the production of a tethering pseudosubstrate Mei3p, and accumulation of the unphosphorylated form of Mei2p commits cells to meiosis. However, the mechanism of how phosphorylation of Mei2p suppresses Mei2p activity to induce meiosis remains largely unknown. S. pombe Rad24p, a 14-3-3 protein, functions as a negative factor for meiosis by antagonizing the function of meiRNA to promote the formation of a nuclear Mei2p dot. Rad24p binds preferentially to Mei2p phosphorylated by Pat1 kinase. It inhibits association of meiRNA to the phosphorylated form of Mei2p but not to the unphosphorylated form in vitro. It is speculated that Rad24p, bound tightly to the residues phosphorylated by Pat1 kinase, may mask the RNA recognition motifs on Mei2p. This model will explain, at least partly, why phosphorylation by Pat1 kinase inhibits the meiosis-inducing activity of Mei2p (Sato, 2002).

14-3-3 proteins in various species

A C. elegans homolog of a 14-3-3 protein-encoding gene has been cloned that is located within the unc-22 gene cluster on chromosome IV. Sequence analysis reveals that the cDNA-encoded product is 78% identical to both the Drosophila melanogaster and bovine 14-3-3 proteins. The cDNA hybridizes to at least three major transcripts of 1.5, 1.35 and 0.9 kb, which are all more abundant in fertile hermaphrodites than in those lacking germ cells (Wang, 1994).

A variant form of 14-3-3 zeta has been isolated from the rat hippocampal cDNA library. The cloned cDNA is 1687 bp in length and contains an entire ORF (nt = 63-797) with 245 amino acids that is characteristic to 14-3-3 zeta subtype. By comparison with reported sequences of 14-3-3 zeta, three nucleotide substitutions were found within the coding sequence in the clone: C<-->T transition at nt = 325 and G<-->C transversions at nt = 387 and 388. Both are missense mutations, leading to ACG (Thr) to ATG (Met) and CGT (Arg) to GCT (Ala) conversions at residue 88 and 109, respectively. These results show that at least three different genetic variants of 14-3-3 zeta are present in rat species, resulting in protein variations. Such mutation in the amino acid sequence is an important indication of the diverse functions of this protein and may also contribute to the recent contradictory observations regarding the role of the 14-3-3 zeta subtype (Murakami, 1997).

Par-5 is a C. elegans 14-3-3 protein involved in cell asymmetry

The establishment of anterior-posterior polarity in the C. elegans embryo requires the activity of the maternally expressed par genes. The identification and analysis of a new par gene, par-5, is reported. par-5 is required for asynchrony and asymmetry in the first embryonic cell divisions, normal pseudocleavage, normal cleavage spindle orientation at the two-cell stage, and localization of P granules and MEX-5 during the first and subsequent cell cycles. Furthermore, par-5 activity is required in the first cell cycle for the asymmetric cortical localization of PAR-1 and PAR-2 to the posterior, and PAR-3, PAR-6, and PKC-3 to the anterior. When PAR-5 is reduced by mutation or by RNA interference, these proteins spread around the cortex of the one-cell embryo and partially overlap. Sequence analysis of par-5 mutants and RNA interference show that the par-5 gene is the same as the ftt-1 gene, and encodes a 14-3-3 protein. The PAR-5 14-3-3 protein is present in gonads, oocytes, and early embryos, but is not asymmetrically distributed. This analysis indicates that the par-5 14-3-3 gene plays a crucial role in the early events leading to polarization of the C. elegans zygote (Morton, 2002).

The par-5 is one of two 14-3-3 genes in the C. elegans genome. The two 14-3-3 proteins do not appear to be acting as heterodimers in the one-cell embryo, since RNAi that specifically targets the second 14-3-3 gene, ftt-2, does not result in polarity defects. This genetic result is consistent with prior results that par-5/ftt-1 mRNA is detectable in gonads and one-cell embryos, but ftt-2 RNA is not. How does PAR-5 contribute to the polar distribution of the other PAR proteins? One possibility is that PAR-5 binds to and blocks the recruitment of one or more of the PAR proteins to the cell cortex. This could lead to an asymmetric distribution of the PAR proteins if the interaction between PAR-5 and its partners is restricted to one pole of the cell. The results rule out asymmetric distribution of PAR-5 as a means to regulate its activity. However, asymmetric binding of the proteins could be achieved by polarized phosphorylation of PAR-5 or its partners (Morton, 2002).

If PAR-5 14-3-3 acts to block recruitment of its binding partners to the cell cortex, then its ligands may include one or more of the anterior group PAR proteins, because the cortical distributions of these anterior group proteins are expanded posteriorly in par-5 mutants. The existence of a potential 14-3-3 binding motif in PAR-3 (RSKSQP) raises the possibility that PAR-5 can interact with PAR-3. Recent studies of PAR-3, PAR-6, and PKC-3 in C. elegans, and their homologs in Drosophila and mammals suggest that these three proteins are part of a multiprotein complex. It is also possible that, instead of blocking interactions among complex members, PAR-5 may bind to more than one of the proteins of the complex, facilitating stabilization of the complex in the anterior. A mammalian atypical protein kinase C, PKC-z, has been found to interact with an isotype of 14-3-3, raising the possibility that the C. elegans atypical protein kinase C, PKC-3, may be a binding partner of PAR-5 (Morton, 2002).

An alternative model for PAR-5 function is that it plays an active role in recruiting posterior group PAR proteins to the cortex. The similarity between the par-5 and par-2 mutant phenotypes is consistent with interaction between these two gene products. The predicted PAR-2 protein sequence contains a motif similar to the RSXpSXX site: RSKSSG. Most 14-3-3 binding proteins that contain this motif have a proline in the pS + 2 position, However, glycine is also a preferred amino acid here, as determined from tests of 14-3-3 binding against degenerate peptide libraries and from successful substitution of glycine for proline in a peptide used for cocrystallization. It appears that a change in peptide direction at this position in the 14-3-3 ligand, to exit the binding cleft, is the important structural feature. If PAR-5 14-3-3 is capable of binding to either PAR-2 or PAR-3, competition for binding between the two proteins could serve as a means to mediate their mutual restriction at the cell cortex (Morton, 2002).

It is also possible that PAR-5 influences the localization of other PAR proteins by regulating the organization of the cortical cytoskeleton. Polarization of the early embryo, including P granule localization and the posterior placement of the first cleavage spindle, requires intact microfilaments. Studies of 14-3-3 function in adrenal chromaffin cells and in yeast have implicated 14-3-3 in reorganization of the actin cytoskeleton. Mutations in the C. elegansactin-binding protein gene pod-1 can also lead to mislocalization of PAR-3, and RNAi depletion of the nonmuscle myosin NMY-2 in C. elegans embryos causes a phenotype similar to par-2 and par-5 mutations. Finally, the unusual pseudocleavage of par-5 embryos may reflect a defect in the actin cytoskeleton (Morton, 2002).

Polarization of the C. elegans zygote along the anterior-posterior axis depends on cortically enriched (PAR) and cytoplasmic (MEX-5/6) proteins, which function together to localize determinants (e.g. PIE-1) in response to a polarizing cue associated with the sperm asters. Using time-lapse microscopy and GFP fusions, the localization dynamics of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 were studied in wild-type and mutant embryos. These studies reveal that polarization involves two genetically and temporally distinct phases. During the first phase (establishment), the sperm asters at one end of the embryo exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing the ring finger protein PAR-2 to accumulate in an expanding `posterior' domain. Onset of the establishment phase involves the non-muscle myosin NMY-2 and the 14-3-3 protein PAR-5. The kinase PAR-1 and the CCCH finger proteins MEX-5 and MEX-6 also function during the establishment phase in a feedback loop to regulate growth of the posterior domain. The second phase begins after pronuclear meeting, when the sperm asters begin to invade the anterior. During this phase (maintenance), PAR-2 maintains anterior-posterior polarity by excluding the PAR-3/PAR-6/PKC3 complex from the posterior. These findings provide a model for how PAR and MEX proteins convert a transient asymmetry into a stably polarized axis (Cuenca, 2003).

Previous studies have implicated the sperm-derived MTOC as the most likely source for the spatial cue that initially polarizes the zygote. Time-lapse analysis supports this view. Formation of the MTOC correlates temporally and spatially with the earliest evidences of polarity: (1) cessation of ruffling, (2) enrichment of GFP:PAR-2, and (3) loss of GFP:PAR-6 in the posterior cortex. The data also demonstrates that the primary effect of the polarizing cue is to clear the PAR-3/PAR-6/PKC-3 complex from the posterior cortex. This effect does not require PAR-2. In contrast, restriction of PAR-2 to the posterior requires PAR-6, PAR-3 and PKC-3, suggesting that PAR-2 does not sense the polarity cue directly but instead responds to local displacement of the anterior complex (Cuenca, 2003).

The establishment phase requires the class II non-muscle myosin, NMY-2: nmy-2(RNAi) prevents PAR-6 (and presumably associated PAR-3 and PKC-3) from sensing the polarity cue, causing it to remain uniformly distributed throughout the cortex. In NMY-2-depleted embryos, PAR-2 is prevented from accumulating at the cortex by PAR-6 (and/or its partners). This 'default' state of PAR-6 on/PAR-2 off is also observed in mutants lacking sperm asters and in mutants where the MTOC detaches from the cortex prematurely. These observations suggest that the initial symmetry-breaking event involves signaling between the MTOC and the actin cytoskeleton. Consistent with this view, one of the earliest signs of polarization is cessation of ruffling in the cortex nearest the MTOC. Cessation of ruffling correlates with MTOC formation, but does not appear to require PAR activity (cessation of ruffling was observed in all par mutants examined in this study). These observations suggest that modification of the actin cytoskeleton may be an obligatory step before the onset of PAR asymmetry. It is proposed that signaling from the MTOC modifies the actin cytoskeleton locally, which causes the PAR-3/PAR-6/PKC-3 complex to become destabilized, allowing PAR-2 to accumulate in its place (Cuenca, 2003).

The establishment phase also requires the 14-3-3 protein PAR-5. In its absence, PAR-6 responds only weakly, if at all, to the polarity cue and PAR-2 is no longer excluded from the cortex by the PAR-3/PAR-6/PKC-3 complex. Cessation of ruffling in the posterior, however, still occurs in par-5(RNAi) embryos, suggesting that PAR-5 is not required for the initial MTOC/actin cytoskeleton interaction. Although this interpretation is complicated by the fact that residual PAR-5 activity may persist in par-5(RNAi) embryos, it is proposed that PAR-5 functions primarily by regulating the ability of the PAR-3/PAR-6/PKC-3 complex to (1) exclude PAR-2 and (2) respond to changes in the cytoskeleton. The presence of a potential 14-3-3 binding motif in PAR-3 is consistent with the possibility that PAR-5 regulates the PAR-3/PAR-6/PKC-3 complex by binding to it directly (Cuenca, 2003).

Surprisingly, it was found that the predominantly cytoplasmic MEX-5 and MEX-6 also play a role during the establishment phase. In the absence of MEX-5 and MEX-6, the posterior domain occasionally does not form (15%-30% of embryos), and frequently (50% or more of embryos) is slow to reach its final configuration. These observations indicate that, although MEX-5 and MEX-6 are not absolutely required for PAR localization in the zygote, they do play a role in ensuring a robust response by the PAR-3/PAR-6/PKC-3 complex to the MTOC/actin cytoskeleton signal (Cuenca, 2003).

This aspect of MEX-5/6 function is negatively regulated by PAR-1. In par-1 mutants, MEX-5 and MEX-6 cause the posterior domain to extend further towards the anterior during the establishment phase. Since PAR-1 itself becomes enriched in the posterior domain, one attractive possibility is that PAR-1 and MEX-5/6 participate in a feedback loop that limits expansion of the posterior domain. It is proposed that at the beginning of the establishment phase, MEX-5 and MEX-6 levels are high throughout the zygote and help clear the PAR-3/PAR-6/PKC-3 complex from the region nearest the sperm asters. This clearing allows PAR-2 and PAR-1 to accumulate on the cortex, which in turn reduces MEX-5/6 activity and/or levels in the surrounding cytoplasm. Eventually, MEX-5/6 levels become too low to fuel further expansion of the posterior domain. It is not yet known whether the partial penetrance of the mex-5(-);mex-6(-) phenotype is due to redundancy with other factors, or is indicative of a minor role for the feedback loop in regulating PAR asymmetry (Cuenca, 2003).

The finding that the sperm-derived MTOCs play a role in initiating polarity raised the question of how polarity is maintained after pronuclear meeting, when the pronuclei/centrosome complex rotates and microtubules invade the anterior end of the embryo. In the absence of PAR-2, PAR-3 and PAR-6 and PKC-3 can become asymmetric before pronuclear meeting, but return into the posterior domain afterwards. This finding demonstrates two points: (1) the PAR-6/PAR-3/PKC-3 complex no longer responds to the MTOC-dependent cue after pronuclear meeting, and (2) PAR-2 is required after pronuclear meeting, but not earlier, to exclude the PAR-6/PAR-3/PKC-3 complex from the posterior. It is proposed that pronuclear meeting (and/or the end of prophase) triggers a change in the cytoskeleton, or in the PAR-6/PAR-3/PKC-3 complex, that turns off the MTOC-dependent polarity signal, or the ability to respond to it. From that point on, PAR-2 becomes essential to keep PAR-6/PAR-3/PKC-3 out of the posterior cortex. It is intriguing that PAR-6 briefly localizes to nuclei at pronuclear meeting, raising the possibility that it becomes modified at that time (Cuenca, 2003).

The existence of distinct establishment and maintenance phases is also supported by the observation that cdc-42 is required after prophase, but not earlier, for PAR-3, PAR-6 and PKC-3 asymmetry. Analysis of GFP:PAR-6 dynamics in par-1(RNAi) embryos suggests that PAR-1 also contributes to maintenance of PAR asymmetry after pronuclear meeting. How PAR-2, CDC-42 and PAR-1 function together to maintain the balance between anterior and posterior PAR domains remains to be determined (Cuenca, 2003).

A novel role for 14-3-3sigma in regulating epithelial cell polarity

The loss of epithelial polarity is thought to play an important role during mammary tumor progression. Using a unique transgenic mouse model of ErbB2-induced mammary tumorigenesis, it has been demonstrated that amplification of ErbB2 is frequently accompanied by loss of the 14-3-3sigma gene. This study demonstrates that ectopic expression of 14-3-3sigma results in restoration of epithelial polarity in ErbB2-transformed mammary tumor cells. Targeted deletion of 14-3-3sigma within primary mammary epithelial cells increases their proliferative capacity and adversely affects their ability to form polarized structures. Finally, it was shown that 14-3-3sigma can specifically form complexes with Par3, a protein that is essential for the maintenance of a polarized epithelial state. Taken together, these observations suggest that 14-3-3sigma plays a critical role in retaining epithelial polarity (Ling, 2010).

The concept that 14-3-3sigma plays an instrumental role in the regulation of epithelial polarity in vivo is supported by the mammary epithelial disruption of this key tumor suppressor. Although mammary epithelial-specific disruption of 14-3-3sigma had little impact on the initial stages of ductal outgrowth, histological analysis of 14-3-3sigma-deficient epithelium revealed an increase in the number of luminal epithelial cells that correlated with an increase in the proliferative capacity of these cells. Furthermore, in contrast to well-organized single-layer epithelial organoids derived from wild-type mammary epithelium, the 14-3-3sigma-deficient organoids were filled structures that bore a remarkable similarity to mammary organoids that ectopically express ErbB2. Consistent with the importance of 14-3-3sigma in controlling polarity of luminal mammary epithelium, the mammary glands derived from the hypomorphic conditional 14-3-3sigma strain exhibited an identical luminal hyperproliferation phenotype, suggesting that a critical threshold of 14-3-3sigma is required to restrict luminal epithelial proliferation (Ling, 2010).

While the precise mechanisms that modulate epithelial polarity are unclear, proteomic analyses have revealed that 14-3-3sigma can interact with several proteins that play important roles in regulating cell contacts. Indeed, this study showed that 14-3-3sigma interacts with the Par3 component of the polarity complex, although the association with Par3 might not be direct. Loss of 14-3-3sigma leads to dislocation of Par3 from cell membranes, while it has little impact on Par3 protein levels in TM15, mouse primary mammary epithelium, or MDCK cells. Previous studies in Drosophila have indicated that 14-3-3 protein plays a crucial role in establishing epithelial polarity through its action on Par3 function (Benton, 2003). There is also recent evidence to suggest that Par3 plays a crucial function in regulating mammary epithelial biology. Disruption of Par3 in the mammary epithelium results in mammary epithelial hyperplasia with luminal filling (McCaffrey, 2009). Further evidence supporting the role of 14-3-3sigma in epithelial polarity derives from studies of a mouse mutant bearing a truncated 14-3-3sigma protein. Mice heterozygous for this truncated 14-3-3sigma gene have disrupted epithelial stratification in the skin, and homozygous fetuses die shortly after birth with severe skin abnormality. In addition, primary corneal epithelial cells expressing this dominant-negative protein failed to differentiate and form tight junctions (Xin, 2010). Taken together with the current observations, these data suggest that 14-3-3sigma plays a critical role in regulating epithelial polarity. In contrast to the positive effects of 14-3-3sigma on epithelial polarity, ectopic expression of 14-3-3zeta results in disruption of epithelial polarity of MDCK cells through binding Par3. Moreover, elevated expression of 14-3-3zeta can enhance the invasiveness of mammary tumor cell lines. It is conceivable that the opposing effects on epithelial polarity of these two closely related 14-3-3 proteins is due to their ability to localize Par3 to distinct apical and basal compartments within the cell (Ling, 2010).

The observation that ErbB2 tumors are associated with loss of 14-3-3sigma has important implications in understanding the genetic events involved in ErbB2-induced tumor progression. The fact that 14-3-3sigma is a major positive regulator of epithelial polarity suggests that loss of polarity may be an important step in tumorigenesis and metastasis. Consistent with this concept, it has been demonstrated recently that loss of the Scribble polarity regulator plays an important role in c-Myc-induced mammary tumors. Whether 14-3-3sigma plays a comparable role in ErbB2 mammary tumor progression remains to be addressed (Ling, 2010).

14-3-3 proteins, nuclear export, and ligand binding

The 14-3-3 family of proteins interact with many key signaling molecules to regulate intracellular signal transduction events. These ubiquitous ~30 kDa proteins (which include nine highly homologous members in mammals [alpha, beta, gamma, delta, epsilon,eta, sigma, tau, and zeta] and two to five members in plants, yeast, and fungi) form homo- and hetero-dimeric cup-shaped structures. 14-3-3 proteins bind to discrete phosphoserine-containing motifs present in many signaling molecules to control the onset of cell division, apoptosis, or differentiation. 14-3-3 molecules have been shown to bind to, and to regulate the activities of, Raf, Cdc25, and BAD, presumably by acting as molecular scaffolds or chaperones. More recently, a novel function for 14-3-3 has emerged from observations that 14-3-3, together with the nuclear export protein Crm1, may act as a ligand-dependent nuclear export machine retaining bound protein such as Cdc25 within the cytoplasmic subcellular compartment (Rittinger, 1999 and references).

To develop a general understanding of 14-3-3's function in regulating signaling events, a combinatorial peptide library and structural approach were pursued with respect to 14-3-3 binding to ligand. Oriented peptide library screening has been used to identify two distinct phosphoserine motifs with consensus sequences that are recognized by all 14-3-3 isoforms and the structure of a 14-3-3:peptide complex containing one of these motifs was solved at medium resolution by X-ray crystallography. The 2 Å structures of 14-3-3 bound to each of these phosphoserine motifs is described and the ligand and protein structural components necessary for binding were examined. A nuclear export signal (NES) has been identified in the S. pombe 14-3-3 homolog that appears essential for shuttling Cdc25 out of the nucleus in a phosphorylation- and Crm1-dependent manner following DNA damage. Similarly, 14-3-3 binding to a Forkhead transcription factor in response to phosphorylation by AKT is responsible for its cytoplasmic localization. Intriguingly, many of the contacts between 14-3-3 and each of the bound peptides in 14-3-3:peptide complexes arise from direct interaction with residues in an amphipathic alpha helix containing the NES sequence of 14-3-3, suggesting a dual role for this helix in ligand-dependent nuclear transport. Comparison of the 14-3-3 NES with other NES-containing proteins defines a conserved structural topology that is likely to be the recognition signal for Crm1-dependent export. All critical 14-3-3 residues involved in peptide binding are evolutionarily conserved within all 14-3-3 isotypes, and it is further demonstrated that all 14-3-3 isotypes are capable of binding to A-raf, B-raf, c-Raf-1, BAD, and IRS-1 in cell lysates. Finally, the effects of several 14-3-3 mutations and deletions reported by others are rationalized on the basis of 14-3-3:ligand structures and it has been demonstrated that a dominant-negative mutation in 14-3-3epsiolon that disrupts Raf-dependent MAP kinase signaling in Drosophila functions, not by eliminating a particularly critical interaction in the wild-type 14-3-3, but by altering the preferred phosphoserine motif and substrates recognized by the mutant 14-3-3 protein (Rittinger, 1999).

Chang and Rubin (1997) identified several mutations in 14-3-3epsilon that disrupte D-raf signaling in Drosophila photoreceptor cells in the presence of an activated Ras allele. One of these, a charge reversal mutation exchanging Glu-180 for Lys, was intriguing because this residue is located in the peptide-binding groove of 14-3-3 and makes direct contact with the phosphopeptide ligands. GST fusion constructs containing this mutation in 14-3-3epsilon fail to bind A-raf, B-raf, c-Raf-1, or BAD in PC12 cell lysates. This mutation does not universally disrupt 14-3-3 function because both wild-type and mutant 14-3-3epsilon bind to IRS-1, though binding to the mutant epsilon is slightly diminished. The contribution of Glu-180 to mode 1 and mode 2 peptide binding is quite modest -- in the mode 1 cocrystals, Glu-180 forms a hydrogen bond to the peptide's Ser hydroxyl in the pSer -2 position, whereas in the mode 2 structure, Glu-180 forms a salt bridge to the Arg residue in the pSer -4 position. Mutation of this Glu to Lys, however, significantly alters the electrostatic potential along the peptide-binding surface of 14-3-3, suggesting that the E180K mutant binds a slightly different sequence motif. Phosphoserine-oriented peptide library screening of 14-3-3epsilon E180K reveals a different motif than either the mode 1 or mode 2 consensus sequences. A GST fusion protein of the 14-3-3epsilon E180K mutant either fails to bind or else weakly binds many proteins capable of interacting with wild-type 14-3-3epsilon in 35-labeled Sf9 insect cells. Instead, several proteins, particularly one migrating at ~135 kDa, were found to selectively interact with the mutant but not with wild-type 14-3-3, in a competitive manner with the peptide containing the mutant peptide–binding motif. The dominant-negative phenotype seen with this 14-3-3epsilon E180K mutant in vivo was unexpected, since these cells contain normal amounts of the alternate 14-3-3zeta isotype, which is fully able to bind to Raf. These data suggests that this phenotype results either from a diminished pool size of functional 14-3-3, below some critical value required to activate Raf, or from the inadvertent binding of the 14-3-3epsilon mutant to other proteins that do not ordinarily interact with wild-type 14-3-3 (Rittinger, 1999).

The two 14-3-3:peptide structures presented in this study suggest an intriguing model for how 14-3-3 might function to spatially regulate signaling in a phosphoserine-dependent manner, particularly given the dimeric nature of 14-3-3:ligand binding. In the absence of bound ligands, helix alpha I should be freely available to interact with Crm1, shuttling 14-3-3 out of the nuclear compartment. Upon ligand binding, the NES within the bound monomer is prevented from interacting with Crm1, leaving the compartmental fate of the complex dependent upon the availability of the NES from the other 14-3-3 subunit in the dimer, or from additional NES/NLS sequences within the bound ligand. For example, 14-3-3 binding to Xenopus Cdc25 sequesters an NLS sequence within the frog protein, restricting Cdc25 to the cytoplasm in G2-arrested oocytes by impairing nuclear import. Similar cytoplasmic restriction is seen for 14-3-3-bound human Cdc25C during interphase, and for 14-3-3-bound Forkhead transcription factors in response to AKT phosphorylation. Another group of 14-3-3 ligands could conceivably be targeted to the nucleus through 14-3-3 binding, particularly if two proteins simultaneously bind a single 14-3-3 dimer, with each molecule blocking Crm1:NES interaction while exposing one or more NLS sequences. These complexes would then remain in the nucleus until a dephosphorylation event and/or release of one ligand from 14-3-3 reexposes one of the NES sites. In this manner, a single 14-3-3:ligand complex could be directed to different subcellular compartments, depending upon the identity of its neighbor in the adjacent monomer. 14-3-3 molecules would function simultaneously as both scaffolding molecules and localizing anchors, similar to what has been described for AKAPs. Studies directed toward the cell biology of distinct 14-3-3 ligand complexes will be essential to examine these possibilities (Rittinger, 1999 and references).

In C. elegans embryos, a Wnt/MAPK signaling pathway downregulates the TCF/LEF transcription factor POP-1, resulting in a lower nuclear level in signal-responsive cells compared to their sisters. Although the ß-catenin WRM-1 is required for POP-1 downregulation, a direct interaction between these two proteins does not seem to be required, since the ß-catenin-interacting domain of POP-1 is dispensable for both POP-1 downregulation and function in early embryos. WRM-1 downregulates POP-1 by promoting its phosphorylation by the MAP kinase LIT-1 and subsequent nuclear export via a 14-3-3 protein, PAR-5. In signal-responsive cells, a concurrent upregulation of nuclear LIT-1 that is dependent on Wnt/MAPK signaling is also detected. These results suggest a model whereby Wnt/MAPK signaling downregulates POP-1 levels in responsive cells, in part by increasing nuclear LIT-1 levels, thereby increasing POP-1 phosphorylation and PAR-5-mediated nuclear export (Lo, 2004).

Is the Wnt/MAPK-induced nuclear export of a TCF protein described in this study a C. elegans-specific phenomenon? C. elegans POP-1 is the only TCF protein known to undergo nucleocytoplasmic redistribution upon Wnt signaling. TCF/LEF proteins appear to be constitutive nuclear proteins in all other organisms examined so far. In addition, the canonical Wnt signaling pathway results in the activation of Wnt-responsive genes via a TCF/ß-catenin complex. It would seem counterintuitive to lower the level of nuclear TCF/LEF proteins in order to activate transcription in this model. Therefore, it is possible that the Wnt-induced nuclear export of TCF proteins only occurs in C. elegans embryos where POP-1 functions mainly as a repressor. However, two results suggest that Wnt signaling-induced nuclear export of TCF proteins may not be limited to C. elegans embryos. (1) It has been shown in flies that reduction of dTcf (Pangolin) partially suppresses, whereas its overexpression enhances, the wingless mutant phenotype. This is consistent with a model where Wnt signaling lowers the level of TCF proteins. (2) In the development of C. elegans male tail, Wnt signaling lowers the nuclear level of POP-1 in the cell T.p, whose fate is specified by POP-1. LIT-1 homologs have been shown to regulate the activity of TCF/LEF proteins in a variety of organisms, and 14-3-3 proteins are highly conserved among eukaryotes. Therefore, it is an intriguing possibility that LIT-1 homologs and 14-3-3 proteins may also regulate nuclear export of TCF/LEF in other organisms (Lo, 2004).

Interactions of 14-3-3 proteins with Raf and KSR

In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus, as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly affected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT, either phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. These data indicate that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by the binding of activated Ras to c-Raf-1 (Rommel, 1996).

14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, the 14-3-3 binding motif has been identified. Most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif can disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. These findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes (Muslin, 1996).

The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands The contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove have been investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decrease the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E shows only subtle effects on the binding. Interestingly, these mutations exhibit similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increase by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta (L. Zhang, 1997).

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains unclear. Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. 14-3-3 zeta binds directly to the isolated Raf-CRD. Mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding result in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1 (Clark, 1997).

KSR (see Drosophila Kinase suppressor of ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates C. elegans and Drosophila. The function of vertebrate KSR is not well characterized biochemically or biologically. The physiological role of KSR was examined in vertebrate signal transduction using Xenopus oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induces Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation is blocked by co-expression of dominant-negative Raf-1. KSR contains multiple potential binding sites for 14-3-3, and the yeast two-hybrid system and co-immunoprecipitation experiments were used to show that KSR can bind to 14-3-3. KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies reveal that KSR forms a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only forms a complex with Raf-1 in the membrane fraction. These findings suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms (Xing, 1997).

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. 14-3-3 is shown to bind to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol (Roy, 1998).

A model is proposed that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation. The Ras interaction with Raf's Ras binding domain (RBD) brings the Raf-14-3-3 complex to the membrane and sets in train subsequent activation events: Raf cystine rich doman (CRD)-Ras interactions then act in concert with CRD-phosphatidylserine interactions and lead to (1) partial uncovering and activation of the Raf kinase domain, (2) displacement of 14-3-3 from the Raf N terminus, and (3) more favorable presentation of Raf Y340 and Y341 for phosphorylation. At some early point in the activation process, 14-3-3 is also displaced from the Raf-1 C terminus; displacement of 14-3-3 allows for dephosphorylation of S259 and S621. Successful completion of all these events is required for full Raf-1 activation. A continuing interaction between activated Raf-1 and 14-3-3 is not required to maintain the activity of Raf-1 at the plasma membrane, because such an interaction cannot be demonstrated in vivo. Following rephosphorylation of S621 and/or S259, 14-3-3 rebinds to inactive Raf-1 and sequesters it to the cytosol. This model explains why 14-3-3 functions as a negative regulator in some assays (because it must be displaced from Raf-1 for activation and may be involved in removing Raf-1 from the plasma membrane) but appears to be essential for Ras-to-Raf-1 signaling in genetic assays (because it is permissive for Ras-dependent membrane recruitment and activation) (Roy, 1998).

cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to three-fold increase in overall phosphorylation of Raf on serine/threonine residues; treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity has been uncertain. Active Raf is shown to be almost completely deactivated in vitro as a consequence of the displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by the addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. It is proposed that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo (Tzivion, 1998).

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. However, the exact mechanism of their action is still largely unknown. A requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation has been demonstrated in this study. Expression of dominant negative forms of 14-3-3 results in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. However, phosphorylation of Raf-1 is insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogates kinase activity, whereas addition of 14-3-3 restores it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions (Thorson, 1998).

Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. Using mass spectrometry to analyze the KSR1-scaffolding complex, the serine/threonine protein phosphatase PP2A has been identified as a KSR1-associated protein; PP2A is a critical regulator of KSR1 activity. The enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites (Ory, 2003).

Interactions of 14-3-3 proteins with Cbl

Continued: see leonardo Evolutionary homologs part 2/2

14-3-3zeta/leonardo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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