The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein–protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.     

MAP kinase cascade,but not ERKs,activated during early cleavage of mouse embryos

Mitosis in early embryos is independent of exogenous mitogens, although mitogen stimulations and subsequent activation of a mitogen-activated protein (MAP) kinase cascade are essential for the proliferation of somatic cells. The activation state of the MAP kinase cascade during early cleavage has never been reported. In the present study, factors involved in the MAP kinase cascade—Ras, Raf-1, 14–3-3, MEK, and ERKs—and their activation states were detected by immunoblotting during early cleavage of mouse embryos. We found the constant presence of these molecules in mouse early embryos and the activation of Raf-1 exclusively at the M-phase. An immunoprecipitation study revealed that active Raf-1 in the M-phase was dissociated from 14–3-3, as in somatic cells, whereas inactive Raf-1 was associated with 14–3-3. Surprisingly, the ERKs (MAP kinases) were not activated throughout early cleavage, although M-phase–specific activation of the MAP kinase kinase, MEK was observed. Myelin basic protein kinase activity was, however, significantly higher in the M-phase than in the interphase. These results indicate that the MAP kinase cascade is activated at the M-phase and that some MAP kinases other than ERKs are activated during early cleavage of mouse embryos. Mol. Reprod. Dev. 51:148–155, 1998. © 1998 Wiley-Liss, Inc.     

SIRT2 is a negative regulator of anoxia-reoxygenation tolerance via regulation of 14-3-3 zeta and BAD in H9c2 cells

Knockdown or inhibition of SIRT2 enhances biological stress-tolerance. We extend this phenotype showing that SIRT2 knockdown reduces anoxia-reoxygenation injury in H9c2 cells. Gene array analysis following SIRT2 siRNA knockdown identifies 14-3-3 zeta as the most robustly induced gene. SIRT2 knockdown evokes induction of this chaperone, facilitating cytosolic sequestration of BAD with a corresponding reduction in mitochondrial BAD localization. Concurrent siRNA against SIRT2 and 14-3-3 zeta abolishes the SIRT2-depleted cytoprotective phenotype. SIRT2 functions to moderate cellular stress-tolerance, in part, by modulating the levels of 14-3-3 zeta with the concordant control of BAD subcellular localization.     

Phosphorylation-dependent binding of 14-3-3 to Par3beta, a human Par3-related cell polarity protein

Mammalian Par3alpha and Par3beta/Par3L participate in cell polarity establishment and localize to tight junctions of epithelial cells; Par3alpha acts via binding to atypical PKC (aPKC). Here we show that Par3beta as well as Par3alpha interacts with 14-3-3 proteins in a phosphorylation-dependent manner. In the interaction, Ser-746 of Par3beta and the corresponding residue of Par3alpha (Ser-814) likely play a crucial role, since replacement of these residues by unphosphorylatable alanine results in a loss of interacting activity. The mutant Par3 proteins with the replacement are correctly recruited to tight junctions of MDCK cells and to membrane ruffles induced by an active form of the small GTPase Rac in HeLa cells. Thus, the interaction with 14-3-3 appears to be dispensable to Par3 localization. Consistent with this, the Par3alpha-14-3-3 interaction does not inhibit the Par3alpha-aPKC association required for the Par3alpha localization, although the aPKC-binding site lies close to the Ser-814-containing, 14-3-3-interacting region.     

Characterization of zetin 1/rBSPRY,a novel binding partner of 14-3-3 proteins

14-3-3 proteins are ubiquitously expressed proteins which serve as central adaptors in different signal transduction cascades. In this study, yeast two-hybrid screening of a rat brain cDNA library identified a novel gene product termed zetin 1/rBSPRY that interacts with 14-3-3 zeta. The zetin 1/rBSPRY gene is ubiquitously expressed in a variety of rat tissues, with highest expression being found in testis. In adult brain, high levels of zetin 1/rBSPRY mRNA were observed in the hippocampus, cerebral cortex, and piriform cortex. Biochemical studies confirmed zetin 1/rBSPRY to interact with 14-3-3 zeta. Transient co-transfection in COS 7 cells caused a partial redistribution of zetin 1/rBSPRY into 14-3-3 zeta enriched submembranous foci at leading edges. Our results suggest a role for zetin 1/rBSPRY-14-3-3 interactions at specialized submembrane domains.     

Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.     

14-3-3 proteins are involved in the regulation of mammalian cell proliferation

Summary. The 14-3-3 proteins are a family of abundant, widely expressed acidic polypeptides. The seven isoforms interact with over 70 different proteins. 14-3-3 isoforms have been demonstrated to be involved in the control of positive as well as negative regulators of mammalian cell proliferation. Here we used the approach of inactivating 14-3-3 protein functions via overexpression of dominant negative mutants to analyse the role of 14-3-3 proteins in mammalian cell proliferation. We found 14-3-3 dominant negative mutants to downregulate the proliferation rates of HeLa cells. Overexpression of these dominant negative mutants triggers upregulation of the protein levels of the cyclin-dependent kinase inhibitor p27, a major negative cell cycle regulator. In addition, they downregulate the protein levels of the important cell cycle promoter cyclin D1. These data provide new insights into mammalian cell proliferation control and allow a better understanding of the functions of 14-3-3 proteins.     

Tetraspanin CD9 regulates beta 1 integrin activation and enhances cell motility to fibronectin via a PI-3 kinase-dependent pathway

Tetraspanin CD9 regulates cell motility and other adhesive processes in a variety of tissue types. Using transfected Chinese Hamster Ovary cells as our model system, we examined the cellular pathways critical for CD9 promoted cell migration. alpha 5 beta 1 integrin was directly involved as CD9 enhanced migration was abolished by the alpha 5 beta 1 blocking antibody PB1. Furthermore, the ligand mimetic peptide RGDS, significantly upregulated the expression of a beta1 ligand induced binding site (LIBS) demonstrating for the first time that CD9 expression potentiates beta1 integrin high affinity conformation states. CD9 promoted cell motility was significantly blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortmannin and LY294002, whereas inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect. PI-3K dominant/negative cDNA transfections confirmed that PI-3K was an essential component. CD9 enhanced the phosphorylation of the PI-3K substrate, Akt, in response to cell adhesion on FN. CD9 expression also upregulated p130Cas phosphorylation and total protein levels; however, p130Cas siRNA knockdown did not alter the motile phenotype. CD9 enhanced migration was also unaffected by serum deprivation suggesting that growth factors were not critical. Our studies demonstrate that CD9 upregulates beta1 LIBS, and in concert with alpha 5 beta 1, enhances cell motility to FN via a PI-3K dependent mechanism.     

日本血吸虫GST蛋白和14-3-3蛋白的研究进展

血吸虫病是严重危害人类健康的人兽共患病之一,在全球范围内,血吸虫病曾在76个国家和地区流行,有超过2亿人感染了血吸虫病。目前,世界上采取了一些人畜同步治疗等综合防治措施,虽取得了一定的成效,但仍面临着成本高、再感染率高等一系列问题。因此,寻找新的治疗药物、疫苗候选分子以及开发高度特异且敏感的标准化免疫诊断试剂是当前日本血吸虫病基础研究的重要内容。主要对WHO提出的最具潜力的疫苗候选分子血吸虫GST蛋白以及参与血吸虫许多生物学功能的14-3-3蛋白的最新研究进展进行一些阐述。     

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.     

14-3-3-Regulated Ca2+-dependent protein kinase CPK3 is required for sphingolipid-induced cell death in Arabidopsis

In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.     

Effect of 14-3-3 protein induction on cell proliferation of A549 human lung adenocarcinoma

We have previously shown that 14-3-3 protein, amultifunctional adaptor molecule involved in many aspects ofsignal transduction pathways, is a target antigen for thecancer-associated human monoclonal antibody. Although recentevidences suggest a crucial role of 14-3-3 family members inthe control of cell growth and differentiation, their actualcontribution toward tumor development is still controversial. Inthis article, we examined the effect of enforced 14-3-3overexpression on cell growth of the human lung adenocarcinomacell line, A549. To address this issue, we obtained14-3-3 protein-inducible A549 sublines by transfection with14-3-3 expression vector under the control ofdexamethasone-inducible promoter. We found that 14-3-3 proteininduction in some of these sublines promoted their cell proliferation. Microscopic observation revealed that morphologyof these cells became aggressive multilayer condition,suggesting that malignant phenotypes are also acquired uponectopic induction of 14-3-3 protein.     

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1′ insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal α-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.     

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

Akt phosphorylates Connexin43 on Ser373, a "mode-1" binding site for 14-3-3

Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques.     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.