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.     

Identification of FAKTS as a novel 14-3-3-associated nuclear protein

Through bioinformatics and experimental approaches, we have assigned the first biochemical property to a predicted protein product in the human genome as a new 14-3-3 binding protein. 14-3-3 client proteins represent a diverse group of regulatory molecules that often function as signaling integrators in response to various environmental cues and include proteins such as Bad and Foxo. Using 14-3-3 as a probe in a yeast two-hybrid screen, we identified a novel 14-3-3 binding protein with unknown function, initially designated as clone 546. Confocal microscopy revealed that clone 546 localized to the nucleus of mammalian cells. Additional studies show that the gene encoding clone 546 is expressed in many human tissues, including the thymus, as well as a number of cancer cell lines. The interaction of clone 546 with 14-3-3 was confirmed in mammalian cells. Interestingly, this interaction was markedly enhanced by the expression of activated Akt/PKB, suggesting a phosphorylation dependent event. Mutational analysis was carried out to identify Ser479 as the predominant residue that mediates the clone 546/14-3-3 association. Phosphorylation of Ser479 by AKT/PKB further supports a critical role for Akt/PKB in regulation of the clone 546/14-3-3 interaction. On the basis of these findings, we named this undefined protein FAKTS: Fourteen-three-three associated AKT Substrate.     

Developmental regulation of 14-3-3 ϵ isoform in rat heart

Human heart cDNA sequencing yielded a cDNA clone that is similar in DNA and amino acid sequences to that of mouse 14-3-3 ϵ isoform. The 6xHis-tagged H1433ϵ recombinant protein was expressed in Escherichia coli and its size was approximately 30 kDa. From Northern blot results with human multiple tissues, human skeletal muscle was found to have the highest level of h1433ϵ mRNA expression, whereas Northern blots of human cancer cell lines detected the highest mRNA level of h1433ϵ in colorectal adenocarcinoma SW480. The protein expression level of h1433ϵ and Raf-1 is found to be regulated coordinately during rat heart development, and their protein expression was highest from 14.5 to 16.5 days postcoitum. J. Cell. Biochem. 68:195–199, 1998. © 1998 Wiley-Liss, Inc.     

14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究

本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.     

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.     

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.     

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.     

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.     

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.     

14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.     

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.     

Deciphering the role of 14-3-3 proteins

14-3-3 Proteins are found to bind to a growing number of eukaryotic proteins and evidence is accumulating that 14-3-3 proteins serve as modulators of enzyme activity. Several 14-3-3 protein recognition motifs have been identified and an increasing number of target proteins have been found to contain more than one binding site for a 14-3-3 protein. It is thus possible that 14-3-3 dimers function as clamps that simultaneously bind to two motifs within a single binding partner. Phosphorylation of a number of binding motifs has been shown to increase the affinity for 14-3-3 proteins but other mechanisms also regulate the association. It has recently been demonstrated that fusicoccin induces a tight association between 14-3-3 proteins and the plant plasma membrane H⁺-ATPase. Phorbol esters and other hydrophobic molecules may have a similar effect on the association between 14-3-3 proteins and specific binding partners.     

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.     

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.     

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。