Interactions of 14-3-3 proteins with Cbl

In T cells 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K). The 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell is Cbl (see Drosophila Cbl), a protooncogene product of unknown function that is a major protein-tyrosine kinase (PTK) substrate, one that interacts with several signaling molecules (including PI3-K) in T lymphocytes. The association between 14-3-3tau and Cbl is detected both in vitro and in intact T cells; in contrast to Raf-1, the association is markedly increased following T cell activation. The use of truncated 14-3-3tau fusion proteins demonstrates that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K. The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins (Liu, 1996).

Stimulation of the T cell antigen receptor (TCR).CD3 complex induces rapid tyrosine phosphorylation of Cbl, a protooncogene product that has been implicated in intracellular signaling pathways via its interaction with several signaling molecules. Cbl associates directly with a member of the 14-3-3 protein family (14-3-3tau) in T cells. The association is increased as a consequence of anti-CD3-mediated T cell activation. Phorbol ester stimulation of T cells also enhances the interaction between Cbl and two 14-3-3 isoforms (tau and zeta). Tyrosine phosphorylation of Cbl is neither sufficient nor required for this increased interaction. Thus, cotransfection of COS cells with Cbl plus Lck and/or Syk family protein-tyrosine kinases causes a marked increase in the phosphotyrosine content of Cbl without a concomitant enhancement of its association with 14-3-3. Phorbol stimulation induces serine phosphorylation of Cbl. Dephosphorylation of immunoprecipitated Cbl by a Ser/Thr phosphatase disrupts its interaction with 14-3-3. By using successive carboxyl-terminal deletion mutants of Cbl, the 14-3-3-binding domain is shown to map to a serine-rich 30-amino acid region (residues 615-644) of Cbl. Mutation of serine residues in this region further define a binding motif distinct from the consensus sequence RSXSXP, which has been identified as a 14-3-3-binding motif. These results suggest that TCR stimulation induces both tyrosine and serine phosphorylation of Cbl. These phosphorylation events allow Cbl to recruit distinct signaling elements that participate in TCR-mediated signal transduction pathways (Liu, 1997).

Interactions of 14-3-3 proteins with PKC

Potential physical and functional interactions have been examined between PKC theta (see Drosophila PKC), a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitate from Jurkat T cells; recombinant 14-3-3 tau interacts directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppresses stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This does not represent a general inhibition of activation events, since PKC-independent (but Ca[2+]-dependent) activation of an IL-4 promoter element is not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibits phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau causes increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau is more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation (Meller, 1996).

Phosphorylation of the polarity protein Par-3 by the serine/threonine kinases aPKCzeta/iota and Par-1 (EMK1/MARK2) regulates various aspects of epithelial cell polarity, but little is known about the mechanisms by which these posttranslational modifications are reversed. This study finds that the serine/threonine protein phosphatase PP1 (predominantly the alpha isoform) binds Par-3, which localizes to tight junctions in MDCKII cells. PP1alpha can associate with multiple sites on Par-3 while retaining its phosphatase activity. By using a quantitative mass spectrometry-based technique, multiple reaction monitoring, it was shown that PP1alpha specifically dephosphorylates Ser-144 and Ser-824 of mouse Par-3, as well as a peptide encompassing Ser-885. Consistent with these observations, PP1alpha regulates the binding of 14-3-3 proteins and the atypical protein kinase C (aPKC) zeta to Par-3. Furthermore, the induced expression of a catalytically inactive mutant of PP1alpha severely delays the formation of functional tight junctions in MDCKII cells. Collectively, these results show that Par-3 functions as a scaffold, coordinating both serine/threonine kinases and the PP1alpha phosphatase, thereby providing dynamic control of the phosphorylation events that regulate the Par-3/aPKC complex (Traweger, 2008).

Other 14-3-3 interactions

Regulation of cell survival is crucial to the normal physiology of multicellular organisms. Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. PI3'K may also directly interact with and be activated by the small G protein Ras (see Drosophila Ras). However, the mechanism by which Akt functions to promote survival is not understood. Growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery (Datta, 1997).

The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B (PKB; also known as c-Akt: Drosophila homolog Akt1) and phosphorylation and sequestration to protein 14-3-3 of the proapoptotic Bcl-2-family member BAD. A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases. Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3. Serum withdrawal induces a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis is largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival is blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signaling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis (Yano, 1998).

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, studies investigating the effect of phosphorylation and protein interactions on KSR function have been initiated. Five in vivo phosphorylation sites of KSR have been identified. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites does not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, it was found that the biological effect of KSR varies dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functions as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocks Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocks R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels (Cacace, 1999).

The phosphorylation of BAD may lead to the prevention of cell death via a mechanism that involves selective association of the phosphorylated forms of BAD with 14-3-3 protein isoforms. This BAD/14-3-3 interaction can occur when BAD becomes phosphorylated at SER-112 and/or Ser-136; the induced association of BAD with 14-3-3 appears to prevent BAD association with BCL-XL or BCL-2 (Drosophila homolog: death executioner Bcl-2 homologue). Preliminary evidence suggests that consitutively active Akt can induce the association of BAD and 14-3-3zeta, and that kinase-inactive Akt does not induce this association event. Akt-induced Bad/14-3-3zeta association depends on the presence of BAD Ser-136. It has been proposed that in the absence of BAD phosphorylation, BAD may bind to BCL-XL or BCL-2 and suppress survival by inducing BAX homodimer formation. The prodeath function of BAD could also be a direct consequence of BAD's heterodimerization with BCL-XL or BCL-2, and may not involve BAX heterodimerization. It is possible that BAD brings Akt to the 14-3-3 complex, where Akt may phosphorylate additional signaling molecules to promote cell survival (Datta, 1997).

The S. cerevisiae 14-3-3 homologs BMH1 and BMH2 are not essential for viability or mating-related MAPK cascade signaling, but they are essential for pseudohyphal-development MAPK cascade signaling and other processes. Activated alleles of RAS2 and CDC42 induce pseudohyphal development and MAPK cascade signaling in Bmh+ strains, but not in ste20 (p65PAK) or bmh1 bmh2 mutant strains. Moreover, Bmh1p and Bmh2p associate with Ste20p in vivo. Three alleles of BMH1 encode proteins defective for MAPK cascade signaling and association with Ste20p, yet these alleles complement other 14-3-3 functions. Therefore, the 14-3-3 proteins are specifically required for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae. Ras2, CDC42, 14-3-3 proteins and p65PAK are required for cell elongation that takes place during pseudohyphal development, but the MAPK cascade is not required for cell elongation (Roberts, 1997).

To identify proteins that bind to mouse wee1 kinase, the yeast "two-hybrid" system was used with a mouse cDNA library. Using the carboxyl half of weel kinase, the 14-3-3 zeta protein was isolated. Recombinant 14-3-3 zeta binds to wee1 kinase in vitro. The wee1 kinase phosphorylated by cdc2 kinase also binds to 14-3-3 zeta protein. When both wee1 kinase and 14-3-3 zeta are transfected into COS-1 cells, they form a complex. The sequence of wee1 kinase necessary for the binding was tested by a two hybrid system expressing different lengths of peptides derived from wee1 kinase. Both the entire kinase domain and a sequence in the carboxyl terminus is thought to be necessary for the binding. The function of 14-3-3 zeta protein remains to be elucidated in relation to the regulation of G2 to M phase transition through wee1 kinase (Honda, 1997).

Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. Xwee1 is also regulated by association with 14-3-3 proteins. In partcular, both Xenopus 14-3-3epsilon and 14-3-3zeta are found associated with His6-GST-Xwee1 in egg extracts. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggest that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuates the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1 (Lee, 2001).

The epsilon isoform of 14-3-3 interacts with the insulin-like growth factor I receptor (IGFIR) and with insulin receptor substrate I (IRS-1), but not with the insulin receptor (IR). 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR. Specifically, peptide competition studies combined with mutational analysis suggests that the 14-3-3 interaction is dependent upon phosphorylation of IGFIR serine residues 1272 and/or 1283, a region that has been implicated in IGFIR-dependent transformation. Phosphorylation of these serines appears to be dependent upon prior IGFIR activation since no interaction of 14-3-3 is observed with a kinase-inactive IGFIR in the two-hybrid assay nor is any in vitro interaction with unstimulated IGFIR derived from mammalian cells. The interaction of 14-3-3 with IRS-1 also appears to be phosphoserine-dependent. Interestingly, 14-3-3 appears to interact with IRS-1 before and after hormonal stimulation (Craparo, 1997).

Insulin binding to its receptor induces the phosphorylation of two cytosolic substrates: insulin receptor substrate (IRS)-1 and IRS-2. These associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, a human heart cDNA library was screened with 32P-labeled recombinant IRS-1 and two isoforms (epsilon and zeta) were obtained of the 14-3-3 protein family. 14-3-3 protein associates with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, also forms a complex with 14-3-3 protein. The amount of 14-3-3 protein associated with IRS-1 is not affected by insulin stimulation but is increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 reveal that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retains the capacity to bind to 14-3-3 protein in vivo. The effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was also investigated. Phosphoamino acid analysis reveals that IRS-1 coimmunoprecipitates with anti-14-3-3 antibody and is only weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues (as compared with IRS-1, immunoprecipitated with anti-IRS-1 antibody). Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1 (Ogihara, 1997).

The yeast two-hybrid system was used to isolate cDNAs encoding proteins that interact with the human glucocorticoid receptor (GR) ligand-binding domain (LBD) in a ligand-dependent manner. One such isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induce the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, does not. Glutathione S-transferase pull-down experiments in vitro show that full-length 14-3-3eta protein also interacts with the activated GR. Transient transfection studies using COS-7 cells reveal a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. These findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems (Wakui, 1997).

PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeletal-associated proteins. PTPH1 was found to associate with 14-3-3beta using a yeast two-hybrid screen; its interaction could be reconstituted in vitro using recombinant proteins. Examination of the interaction between 14-3-3beta and various deletion mutants of PTPH1 by two-hybrid tests suggests that the integrity of the PTP is important for this binding. Although both PTPH1 and Raf-1 form complexes with 14-3-3beta, they appear to do so independently. Binding of 14-3-3beta to PTPH1 in vitro is abolished by pretreating PTPH1 with potato acid phosphatase; it is greatly enhanced by pretreating with Cdc25C-associated protein kinase. Thus the association between PTPH1 and 14-3-3beta is phosphorylation-dependent. Two novel motifs (RSLS359VE and RVDS853EP) in PTPH1 were identified as major 14-3-3beta-binding sites, both of which are distinct from the consensus binding motif RSXSXP recently found in Raf-1. Mutation of Ser359 and Ser853 to alanine significantly reduces the association between 14-3-3beta and PTPH1. The association of PTPH1 and 14-3-3beta is detected in several cell lines and is regulated in response to extracellular signals. These results raise the possibility that 14-3-3beta may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways (S. Zhang, 1997).

Serotonin N-acetyltransferase (AANAT) controls the daily rhythm in melatonin synthesis. When isolated from tissue, AANAT copurifies with isoforms epsilon and zeta of 14-3-3. The structure of AANAT bound to 14-3-3zeta, an association that is phosphorylation dependent, has been determined. AANAT is bound in the central channel of the 14-3-3zeta dimer, and is held in place by extensive interactions both with the amphipathic phosphopeptide binding groove of 14-3-3zeta and with other parts of the central channel. Thermodynamic and activity measurements, together with crystallographic analysis, indicate that binding of AANAT by 14-3-3zeta modulates AANAT's activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding (Obsil, 2001).

An affinity purification method has been used to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage (Basu, 2003).

YAP is a 65 kDa protein (sometimes termed YAP65 or YAP1) that was originally identified due to its interaction with the Src family tyrosine kinase Yes. YAP contains either one or two WW domains depending on alternative splicing and also a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain. YAP has been reported to interact with p53 binding protein-2, an important regulator of the apoptotic activity of p53. Through its carboxyl terminus, YAP binds to the PDZ-containing protein EBP50, a submembranous scaffolding protein. YAP is a transcriptional coactivator that binds and activates Runx transcription factors and the four TEAD/TEF transcription factors. YAP is homologous to TAZ (45% identity), a transcriptional coactivator that is regulated by interaction with 14-3-3 and PDZ domain-containing proteins. YAP also interacts with the p53 family member p73, resulting in an enhancement of p73's transcriptional activity. YAP phosphorylation by Akt suppresses its ability to promote p73-mediated transcription of proapoptotic genes in response to DNA damaging agents and the resulting cell death. This extends the range of mechanisms whereby Akt can promote cellular survival in the face of apoptotic stimuli (Basu, 2003).

The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) has been identified as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3 (Kanai, 2000).

Arginine-based endoplasmic reticulum (ER) localization signals are involved in the heteromultimeric assembly of membrane protein complexes like ATP-sensitive potassium channels (KATP) or GABAB G protein-coupled receptors. They constitute a trafficking checkpoint that prevents ER exit of unassembled subunits or partially assembled complexes. For KATP channels, the mechanism that leads to masking of the ER localization signals in the fully assembled octameric complex is unknown. By employing a tetrameric affinity construct of the C terminus of the KATP channel (a subunit, Kir6.2), it was found that 14-3-3 isoforms epsilon and zeta specifically recognize the arginine-based ER localization signal present in this cytosolic tail. The interaction was reconstituted by using purified 14-3-3 proteins. Competition with a nonphosphorylated 14-3-3 high-affinity binding peptide implies that the canonical substrate binding groove of 14-3-3 is involved. Comparison of monomeric CD4, dimeric CD8, and artificially tetramerized CD4 fusions correlates the copy number of the tail containing the arginine-based signal with 14-3-3 binding, resulting in the surface expression of the membrane protein. Binding experiments revealed that the COPI vesicle coat can specifically recognize the arginine-based ER localization signal and competes with 14-3-3 for the binding site. It is concluded that the COPI vesicle coat and proteins of the 14-3-3 family recognize arginine-based ER localization signals on multimeric membrane proteins. The equilibrium between these two competing reactions depends on the valency and spatial arrangement of the signal-containing tails. A mechanism in which 14-3-3 binds to the correctly assembled multimer mediates release of the complex from the ER (Yuan, 2003).

Regulators of G protein signaling (RGS) constitute a family of proteins with a conserved RGS domain of approximately 120 amino acids that accelerate the intrinsic GTP hydrolysis of activated Galpha(i) and Galpha(q) subunits. The phosphorylation-dependent interaction of 14-3-3 proteins with a subset of RGS proteins inhibits their GTPase-accelerating activity in vitro. The inhibitory interaction between 14-3-3 and RGS7 requires phosphorylation of serine 434 of RGS7. Phosphorylation of serine 434 is dynamically regulated by TNF-alpha. Cellular stimulation by TNF-alpha transiently decreases the phosphorylation of serine 434 of RGS7, abrogating the inhibitory interaction with 14-3-3. The effect of 14-3-3 was examined on RGS-mediated deactivation kinetics of G protein-coupled inwardly rectifying K(+) channels (GIRKs) in Xenopus oocytes. 14-3-3 inhibits the function of wild-type RGS7, but not that of either RSG7(P436R) or RGS4, two proteins that do not bind 14-3-3. These findings are the first evidence that extracellular signals can modulate the activity of RGS proteins by regulating their interaction with 14-3-3 (Benzing, 2002).

14-3-3epsilon isoform is associated with calmodulin. Using the voltage-clamp technique, the potential role of 14-3-3 in modulating the Ca(2+)-activated Cl(-) channel (CaCC) endogenously expressed in Xenopus oocytes has been investigated. Injection of 14-3-3epsilon antisense oligodeoxynucleotides results in potentiation of the ionomycin-induced Cl(-) current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppresses the antisense-potentiated current. The data suggest that 14-3-3epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin-dependent pathway. The potential role of 14-3-3epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed (Chan, 2000).

14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. Mass spectrometry has been used to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, a domain-based hierarchical clustering technique was developed that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, a phospho-dependent 14-3-3 binding site has been identified on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. It is concluded that 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3 (Jin, 2004).

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Cofilin-phosphatase activity of SSH1L is shown to increase approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. Evidence is provided that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induces Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta, and also that 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm (Nagata-ohashi, 2004).

The LKB1 tumour suppressor kinase (Drosophila homolog: lkb1) phosphorylates and activates a number of protein kinases belonging to the AMP-activated protein kinase (AMPK) subfamily. A modified tandem affinity purification strategy was used to identify proteins that interact with AMPKalpha, as well as the twelve AMPK-related kinases that are activated by LKB1. The AMPKbeta and AMPKgamma regulatory subunits were associated with AMPKalpha, but not with any of the AMPK-related kinases, explaining why AMP does not influence the activity of these enzymes. In addition, novel binding partners were identified that interacted with one or more of the AMPK subfamily enzymes, including fat facets/ubiquitin specific protease-9 (USP9), AAA-ATPase-p97, adenine nucleotide translocase, protein phosphatase 2A holoenzyme and isoforms of the phospho-protein binding adaptor 14-3-3. Interestingly, the 14-3-3 isoforms binds directly to the T-loop Thr residue of QSK and SIK, after these are phosphorylated by LKB1. Consistent with this, the 14-3-3 isoforms fail to interact with non-phosphorylated QSK and SIK, in LKB1 knockout muscle or in HeLa cells in which LKB1 is not expressed. Moreover, mutation of the T-loop Thr phosphorylated by LKB1, prevents QSK and SIK from interacting with 14-3-3 in vitro. Binding of 14-3-3 to QSK and SIK, enhanced catalytic activity towards the TORC2 protein and the AMARA peptide, and is required for the cytoplasmic localization of SIK and for localization of QSK to punctate structures within the cytoplasm. This study provides the first example of 14-3-3 binding directly to the T-loop of a protein kinase and influencing its catalytic activity and cellular localization (Al-Hakim, 2005).

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), affinity chromatography purification was performed using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), two Numb family proteins (Numb and Numbl) were identified from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl are efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, the phosphorylation of Numb family proteins was observed in various rat tissue extracts, and the ionomycin-induced phosphorylation of endogenous Numb was detected at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, the recruitment of 14-3-3 proteins was found to be the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein (Tokumitsu, 2005).

Myosin II phosphorylation-dependent cell motile events are regulated by myosin light chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin binding subunit of MLCP, plays a critical role in MLCP regulation. This study reports a new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3beta to MYPT1 diminishes the direct binding between MYPT1 and myosin II, and 14-3-3beta overexpression abolishes MYPT1 localization at stress fiber. Furthermore, 14-3-3beta inhibits MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3beta overexpression increased myosin II phosphorylation in cells. MYPT1 phosphorylation at Ser472 is critical for the binding to 14-3-3. EGF-stimulation increases both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreases EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase-dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity (Koga, 2007).

Hypoxia induces rapid and dramatic changes in cellular metabolism, in part through inhibition of target of rapamycin (TOR) kinase complex 1 (TORC1) activity. Genetic studies have shown the tuberous sclerosis tumor suppressors TSC1/2 and the REDD1 protein to be essential for hypoxia regulation of TORC1 activity in Drosophila and in mammalian cells. The molecular mechanism and physiologic significance of this effect of hypoxia remain unknown. This study demonstrates that hypoxia and REDD1 [also know as RTP801/Dig1/DDIT4, a member of a gene family that includes its paralog REDD2 (RTP801L, DDIT4L) and the Drosophila orthologs Scylla and Charybdis] suppress mammalian TORC1 (mTORC1) activity by releasing TSC2 from its growth factor-induced association with inhibitory 14-3-3 proteins. Endogenous REDD1 is required for both dissociation of endogenous TSC2/14-3-3 and inhibition of mTORC1 in response to hypoxia. REDD1 mutants that fail to bind 14-3-3 are defective in eliciting TSC2/14-3-3 dissociation and mTORC1 inhibition, while TSC2 mutants that do not bind 14-3-3 are inactive in hypoxia signaling to mTORC1. In vitro, loss of REDD1 signaling promotes proliferation and anchorage-independent growth under hypoxia through mTORC1 dysregulation. In vivo, REDD1 loss elicits tumorigenesis in a mouse model, and down-regulation of REDD1 is observed in a subset of human cancers. Together, these findings define a molecular mechanism of signal integration by TSC1/2 that provides insight into the ability of REDD1 to function in a hypoxia-dependent tumor suppressor pathway (DeYoung, 2008).

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Evidence is presented that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR is specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR is linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. These findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence (Kim, 2008).

Xenopus oocyte death is partly controlled by the apoptotic initiator caspase-2 (C2). Oocyte nutrient depletion activates C2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit C2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II (maintenance of NADPH levels by flux through the pentose phosphate pathway induces a suppressive phosphorylation on S135; Nutt, 2005). This study shows that C2 phosphorylated at S135 binds 14-3-3zeta, thus preventing C2 dephosphorylation. Moreover, S135 dephosphorylation is catalyzed by protein phosphatase-1 (PP1), which directly binds C2. Although C2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3zeta from C2 is controlled by metabolism and allows for C2 dephosphorylation. Accordingly, a C2 mutant unable to bind 14-3-3zeta is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, similar control of murine C2 by phosphorylation and 14-3-3 binding was found to occur in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death (Nutt, 2010).

The GRIP1/14-3-3 pathway coordinates cargo trafficking and dendrite development

Regulation of cargo transport via adaptor molecules is essential for neuronal development. However, the role of PDZ scaffolding proteins as adaptors in neuronal cargo trafficking is still poorly understood. This study shows by genetic deletion in mice that the multi-PDZ domain scaffolding protein glutamate receptor interacting protein 1 (GRIP1) is required for dendrite development. An interaction was identified between GRIP1 and 14-3-3 proteins that is essential for the function of GRIP1 as an adaptor protein in dendritic cargo transport. Mechanistically, 14-3-3 binds to the kinesin-1 binding region in GRIP1 in a phospho-dependent manner and detaches GRIP1 from the kinesin-1 motor protein complex thereby regulating cargo transport. A single point mutation in the Thr956 of GRIP1 in transgenic mice impairs dendritic development. Together, these results show a regulatory role for GRIP1 during microtubule-based transport and suggest a crucial function for 14-3-3 proteins in controlling kinesin-1 motor attachment during neuronal development (Geiger, 2014).

14-3-3 and cell polarity in mammalian epithelia

The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. This study demonstrates a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. Phosphorylated serine 144 is identified as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3ζ results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding (Hurd, 2003).

In many cases, 14-3-3 binding motifs are substrates for phosphorylation by AGC family protein kinases such as PKA, PKB, and PKC. Unlike phosphorylation of Par3 serine 827, phosphorylation of serine 144 appears to be PKC independent. Interestingly, in a recent report, the activity of the PKB-activating kinase PI3K was shown to be necessary for the correct localization of Par3 to axons of hippocampal neurons. To date, few substrates for the Par1 and Par4 homologs have been identified, and one may speculate that these kinases may directly phosphorylate other members of the Par family of polarity proteins. Indeed, recently it has been demonstrated that Drosophila 14-3-3ζ and epsilon are able to interact with Par1 via a putative 14-3-3 domain distinct from the phosphoserine binding region of the protein. It has been proposed that 14-3-3 thus acts to target Par1 to its cellular substrates. This observation would suggest that 14-3-3 may act to functionally link the Par3/Par6/aPKC complex with mammalian Par1 homologs. As such, determination of the kinases responsible for phosphorylating Par3 may provide further insight into the precise regulation of cell polarity in multiple cell types (Hurd, 2003).

aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. In contrast, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. PAR-1b is shown to be essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. In contrast, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity (Suzuki, 2004).

In the C. elegans one-cell embryo as well as the Drosophila late oocyte, the complex segregate along the A-P axis: the aPKC/PAR-3/PAR-6 complex then localizes at the anterior cortex, whereas PAR-1 is at the posterior cortex. In Drosophila and mammalian epithelial cells, the complex segregates along the apicobasal axis: PAR-1 localizes at the basolateral membrane in contrast with the apical localization of the aPKC/PAR-3/PAR-6 complex. These observations raise questions whether the functional hierarchy of the aPKC/PAR-3/PAR-6 complex and PAR-1 is conserved evolutionarily. The functional relationship between aPKC, PAR-1b, and 14-3-3/PAR-5 suggested in this study is different from that suggested for Drosophila epithelial cells. In Drosophila follicle cells, PAR-1 inhibits the lateral invasion of aPKC, and the phospho-motif binding site of 14-3-3 binds to BAZ. These differences suggest the possibility that mammals and flies independently evolved similar but distinct mechanisms that regulate epithelial cell polarity using aPKC/PAR proteins. However, it is also possible that mutual regulations between PAR-1 and aPKC occur in both organisms, because most of the results in each study are not completely exclusive. For example, although the current study observed that PAR-1b depletion from MDCK cells did not induce the lateral leakage of aPKC and PAR-3, the possibility cannot be excluded that other mammalian PAR-1 proteins compensate for the PAR-1b function in these cells. To address this issue, perfectly corresponding experiments should be performed in each organism (Suzuki, 2004).

14-3-3 is involved in a switch in the function of alpha6beta4 from a mechanical adhesive device into a signaling component

Growth factors, integrins, and the extracellular matrix (ECM) are known to play key roles in epidermal wound healing, although the interplay between these proteins is not fully understood. Growth factor macrophage stimulating protein (MSP)- and its receptor Ron-mediated PI3K activation in keratinocytes induces phosphorylation of both Ron and alpha6beta4 integrin at specific 14-3-3 (see Drosophila 14-3-3zeta) binding sites. Consequently, a Ron/alpha6beta4 complex formed via 14-3-3 binding displaces alpha6beta4 from its location at hemidesmosomes (structures supporting cell adhesion) and relocalizes it to lamellipodia. Concomitant activation of alpha3beta1 and keratinocyte spreading/migration on laminin-5 occurs. Further, MSP-dependent beta4 tyrosine phosphorylation evokes p38 and NF-kappaB signaling required for keratinocyte wound closure. Based on these results, a mechanism is proposed based on MSP-Ron-dependent phosphorylation and 14-3-3 association, whereby the function of alpha6beta4 switches from a mechanical adhesive device into a signaling component, and might be critically involved in human epidermal wound healing (Santoro, 2003).

The complex interplay between the extracellular matrix (ECM), growth factors, and integrins is crucial for many biological processes, including skin wound healing. Wound healing is characterized by a number of overlapping phases including inflammation, reepithelization, granulation tissue formation, and tissue remodeling. The reepithelization process is tightly regulated by specific classes of integrin receptors and interacting extracellular matrix molecules. In particular, α3β1 and α6β4 integrins, as well as laminin-5, play a key role in keratinocyte migration. Laminin-5 is the primary ligand of adult epidermal basement membrane (BM) and is secreted by wounded keratinocytes in the provisional wound bed to promote migration. Laminin-5 is synthesized as a precursor heterotrimeric protein (α3,β3,γ2) that undergoes processing of α3 and γ2 subunits after being secreted. The domain interacting with integrin resides within the α3 C-terminal large globular domain, while the region for deposition in the BM resides in the γ2 subunit. Current models propose that laminin-5 mediates keratinocyte adhesion and migration via α6β4 and α3β1 integrins at distinct sites. α6β4 can be found in hemidesmosomes (HDs), which are structures linking ECM to keratin intermediate filaments thereby supporting cell adhesion. α3β1 is present in focal contacts and links ECM to the actin cytoskeleton, regulating cell spreading and migration. The interplay between α3β1 and α6β4 and different domains of laminin-5 is crucial at the wound edges because it regulates leading keratinocytes by (1) disassembling HDs, (2) remodeling matrix interactions with increased deposition of laminin-5 and metalloproteases (MMPs), and (3) recruiting α3β1 at focal contacts to mediate migration over the provisional matrix (Santoro, 2003 and references therein).

Macrophage stimulating protein (MSP) is the ligand of the Ron tyrosine kinase receptor. It is a biologically inactive soluble plasma factor activated at extravascular sites by specific serine proteases. The Ron receptor is selectively expressed by epithelia, including keratinocytes, and by hematopoietic cells. MSP leads to receptor trans-autophosphorylation and activation of several signaling pathways. MSP and Ron modulate keratinocyte functions such as proliferation, survival, and migration, and, recently, a role in epidermal wound healing has been suggested (Santoro, 2003 and references therein).

MSP-mediated PI3K pathway activation induces Ron serine phosphorylation at residue 1394 as well as α6β4 phosphorylation in the connecting sequence to generate 14-3-3 binding sites on both molecules. Thus, dimeric 14-3-3 proteins mediate the MSP-dependent formation of a Ron/α6β4 complex that in turn induces disassembly of HDs and α6β4 relocation at lamellipodia. Further, α3β1 integrin activation and keratinocyte spreading/migration on laminin-5 takes place. All these findings suggest a role for Ron and 14-3-3 in epidermal reepithelization processes (Santoro, 2003).

14-3-3 and cell cycle

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. Mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. DNA-responsive checkpoints also activate PP2A/B56Δ phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. These data suggest that creation of a 14-3-3 'sink,' consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56Δ as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis (Margolis, 2006).

14-3-3 coordinates microtubules, Rac, and myosin II to control cell mechanics and cytokinesis

During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, chemical genetics was used in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. 14-3-3 is enriched in the cortex, helps maintain steady-state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain, and is needed to promote myosin II bipolar thick filament remodeling. It is concluded that 14-3-3 connects microtubules, Rac, and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Furthermore, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes-cytokinesis cell shape change and cell mechanics (Zhou, 2010).

14-3-3 and DNA replication/damage checkpoint control

Human Cdc25C (see Drosophila String)is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C is phosphorylated on serine-216 and binds to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogates 14-3-3 binding. Conditional overexpression of this mutant perturbs mitotic timing and allows cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylates Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells (Peng, 1997).

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, a search was carried out for high-copy suppressors of overproducer of Cdc25p [OPcdc25(+)], which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis [sum3(+)]. This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees: cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA (Forbes, 1999).

Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. It is proposed that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. It is shown by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. It is concluded that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA (Zeng, 1998).

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2-cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3epsilon and to a lesser extent with 14-3-3zeta. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2-M transition (Kumagai, 1998).

DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage (Lopez-Girona, 1999).

Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends on preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and progesterone treatment abrogates this binding. Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation (Yang, 1999).

The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage. The 14-3-3 proteins Rad24 and Rad25 physically interact with Chk1 in fission yeast. Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage. DNA damage results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form. Genetic analysis has independently implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway. It is suggested that DNA damage-dependent association of phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to a particular substrate or to a particular location within the cell. An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site. These results suggest that in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in sharp contrast to the interaction between the 14-3-3 proteins and Chk1 (Chen, 1999).

14-3-3 protein function as a novel step in left-right patterning

To gain insight into the molecular mechanisms underlying the control of morphogenetic signals by H+ flux during embryogenesis, Fusicoccin-A (FC), a compound produced by the fungus Fusicoccum amygdali Del, was tested. In plant cells, FC complexes with 14-3-3 proteins to activate H+ pumping across the plasma membrane. It has long been thought that FC acts on higher plants only, but exposing frog embryos to FC during early development specifically results in randomization of the asymmetry of the left-right (LR) axis (heterotaxia). Biochemical and molecular-genetic evidence is presented that 14-3-3-family proteins are an obligate component of Xenopus FC receptors and that perturbation of 14-3-3 protein function results in heterotaxia. The subcellular localization of 14-3-3 mRNAs and proteins reveals novel cytoplasmic destinations, and a left-right asymmetry at the first cell division. Using gain-of-function and loss-of-function experiments, it has been shown that 14-3-3E protein is likely to be an endogenous and extremely early aspect of LR patterning. These data highlight a striking conservation of signaling pathways across kingdoms, suggest common mechanisms of polarity establishment between C. elegans and vertebrate embryos, and uncover a novel entry point into the pathway of left-right asymmetry determination (Benney, 2003).

It is thought that the most likely mechanism of 14-3-3 action involves the regulation of ion flux across cell membranes. 14-3-3 proteins are known to control a variety of H+ pumps and ion channels. Proton flux may indirectly affect permeability states of connexin-based gap junctions through changes in cytosolic pH. K+ and H+ flux is asymmetric in early embryos and controls LR asymmetry. 14-3-3 proteins (including 14-3-3E) have recently been shown to be able to modulate K+ currents in Xenopus oocytes. In light of the differential LR subcellular localization of ion pumps, such as the H+/K+-ATPase, and of the ability of 14-3-3 proteins to control the localization of their binding partner, it is proposed that 14-3-3E protein functions in the LR pathway by differentially regulating the endogenous activity and/or localization of LR-relevant ion channels or pumps on each side of the midline. Other (non-LR) roles for 14-3-3 proteins clearly exist in Xenopus (such as in mesoderm induction), and have been described in loss-of-function studies (Benney, 2003).

Recently, it was reported that in Caenorhabditis elegans, a 14-3-3 protein (PAR-5) is required for cellular asymmetry in the early embryo. PAR5 likewise functions in axial asymmetry in Drosophila. Analogous to the suggested role of the FC/14-3-3 receptor in Xenopus, PAR-5 in C. elegans acts at an early step in establishing polarity, but its precise role is unclear. It was suggested that PAR-5 binds to and blocks recruitment of one or more PAR proteins (notably PAR-2 and PAR-3) to the cell cortex. The involvement of 14-3-3 proteins in cellular asymmetry in early cleavages of both C. elegans and Xenopus is further evidence of a deep and fundamental underlying similarity in the mechanisms by which asymmetry and polarity, whether on the cellular level, or on the scale of the organism, is established. The finding that elements of FC signaling are conserved from plants to animals presents a new perspective from which to investigate novel aspects of large-scale morphogenetic control in vertebrates, and is likely to make manipulation of 14-3-3 protein signaling a powerful tool for addressing aspects of asymmetry at every scale (Benney, 2003).

14-3-3 proteins and apoptosis

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. A Bcl-2 interacting protein (BAG-1) enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1 (H. G. Wang, 1996).

Extracellular survival factors alter a cell's susceptibility to apoptosis, often through posttranslational mechanisms. However, no consistent relationship has been established between such survival signals and the BCL-2 family, where the balance of death agonists versus antagonists determines susceptibility. One distant member, BAD, heterodimerizes with BCL-X(L) or BCL-2, neutralizing their protective effect and promoting cell death. In the presence of survival factor IL-3, cells phosphorylate BAD on two serine residues embedded in 14-3-3 consensus binding sites. Only the nonphosphorylated BAD heterodimerizes with BCL-X(L) at membrane sites to promote cell death. Phosphorylated BAD is sequestered in the cytosol bound to 14-3-3. Substitution of serine phosphorylation sites further enhance BAD's death-promoting activity. The rapid phosphorylation of BAD following IL-3 connects a proximal survival signal with the BCL-2 family, modulating this checkpoint for apoptosis (Zha, 1996).

A20, a novel zinc finger protein, is an inhibitor of tumor necrosis factor-induced apoptosis. The mechanism by which A20 exerts its protective effect is currently unknown. Several isoforms of the 14-3-3 proteins have been found to interact with A20 in a yeast two-hybrid screen. A20 binds several 14-3-3 isoforms in vitro. Transfected A20 preferentially binds the endogenous eta14-3-3 isoform, whereas the beta/zeta isoforms co-immunoprecipitate much less efficiently, and epsilon14-3-3 has an intermediate affinity. Importantly, c-Raf, a 14-3-3-interacting protein, also preferentially binds the eta isoform. The cellular localization and subcellular fractionation of A20 is dramatically altered by co-transfected 14-3-3, providing the first experimental evidence for the notion that 14-3-3 can function as a chaperone. Furthermore, c-Raf and A20 co-immunoprecipitate in a 14-3-3-dependent manner, suggesting that 14-3-3 can function as a bridging or adapter molecule (Vincenz, 1996).

Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component of a signaling pathway induced by many death stimuli, including tumor necrosis factor alpha, Fas, and the anticancer drugs cisplatin and paclitaxel. ASK1 proapoptotic activity is antagonized by association with 14-3-3 proteins. ASK1 specifically binds 14-3-3 proteins via a site involving Ser-967 of ASK1. Interestingly, overexpression of 14-3-3 in HeLa cells blocks ASK1-induced apoptosis whereas disruption of the ASK1/14-3-3 interaction dramatically accelerates ASK1-induced cell death. Targeting of ASK1 by a 14-3-3-mediated survival pathway may provide a novel mechanism for the suppression of apoptosis (Zhang, 1999).

14-3-3 family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control pathways. In this work, dominant-negative mutant forms of 14-3-3 were used to disrupt 14-3-3 function in cultured cells and in transgenic animals. Transfection of cultured fibroblasts with the R56A and R60A double mutant form of 14-3-3zeta (DN-14-3-3zeta) inhibits serum-stimulated ERK MAPK activation, but increases the basal activation of JNK1 and p38 MAPK. Fibroblasts transfected with DN-14-3-3zeta exhibit markedly increased apoptosis in response to UVC irradiation that is blocked by pre-treatment with a p38 MAPK inhibitor, SB202190. Targeted expression of DN-14-3-3eta to murine postnatal cardiac tissue increases the basal activation of JNK1 and p38 MAPK, and affects the ability of mice to compensate for pressure overload, which results in increased mortality, dilated cardiomyopathy and massive cardiomyocyte apoptosis. These results demonstrate that a primary function of mammalian 14-3-3 proteins is to inhibit apoptosis (Xing, 2000).

Back to leonardo Evolutionary homologs part1/2

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.