Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.     

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.     

14-3-3ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3ζ, we demonstrate that in keratinocytes 14-3-3ζ/τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3ζ acts as a dominant negative and inhibits the interaction between 14-3-3τ and SSH1. These results implicate 14-3-3ζ/τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.     

14-3-3 蛋白

介绍了14－3－3蛋白的基本结构和功能,并简要概述了14－3－3蛋白在信号转导,细胞周期调控以及前体蛋白的折叠与运输过程中的作用机理。     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

NAMPT regulates PKM2 nuclear location through 14-3-3ζ: Conferring resistance to tamoxifen in breast cancer

The resistance against tamoxifen therapy has become one of the major obstacles in the clinical treatment of breast cancer. Nicotinamide phosphoribosyltransferase (NAMPT) is an essential enzyme catalyzing nicotinamide adenine dinucleotide biosynthesis and is important for tumor metabolism. The study here sought to explore the effect of NAMPT on breast cancer survival with tamoxifen conditioning. We found that NAMPT was highly expressed in breast cancer cells compared with normal mammary epithelial cells. Inhibition of NAMPT by FK866 inhibited cell viability and aggravated apoptosis in cancer cells treated with 4-hydroxytamoxifen. NAMPT overexpression upregulated 14-3-3ζ expression. Knockdown of 14-3-3ζ reduced cell survival and promoted apoptosis. Activation of Akt signaling, rather than ERK1/2 pathway, is responsible for 14-3-3ζ regulation by NAMPT overexpression. Furthermore, NAMPT overexpression led to PKM2 accumulation in the cell nucleus and could be dampened by 14-3-3ζ inhibition. In addition, NAMPT overexpression promoted xenografted tumor growth and apoptosis in nude mice, while 14-3-3ζ inhibition attenuated its effect. Collectively, our data demonstrate that NAMPT contributes to tamoxifen resistance through regulation of 14-3-3ζ expression and PKM2 translocation.     

Calpain regulates N-terminal interaction of GSK-3β with 14-3-3ζ, p53 and PKB but not with axin

Calpain produces a truncation of GSK3β that removes the N-terminal inhibitory domain. Here we analyze the effect of that truncation on protein-protein interaction. We pulled down GST-tagged proteins in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Commercial GSK-3β was first incubated with calpain for 2.5 min in vitro, and then with GST-tagged proteins in the presence of calpeptin, a synthetic calpain inhibitor. Western blot analyses were performed to determine if there is an interaction between these GST-tagged proteins and truncated GSK-3β. Using axin GST-tagged, we pulled down the protein in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Western blot analyses showed full length GSK-3β in the pellet as well GSK-3β cleaved by calpain. Thus axin was able to bind GSK-3β without the N-terminal end. When the same experiment was carried out with GST-tagged 14-3-3ζ, p53 and PKB, full length GSK-3β was observed in the pellet, but GSK-3β truncated by calpain was not pulled down demonstrating that GSK-3β N-terminal end is necessary to interact with these three proteins. Our data demonstrate that N-terminal end is necessary for 14-3-3ζ, p53 and PKB interaction. However, the interaction of GSK3β with axin is not altered by calpain. These data support a physiological role for GSK3β truncation mediated by calpain.     

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

Serine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.

Structured summary

MINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)MINT-7233285, MINT-7233297, MINT-7233310: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

植物14-3-3蛋白研究进展

14-3-3蛋白是真核生物中许多信号传导级联反应的主要调节分子,易于与具有磷酸化的丝氨酸和苏氨酸残基的靶蛋白互作进而调节碳氮代谢、三羧酸循环、莽草酸合成等多种生理过程中的多种酶活性。该文根据近年来国内外对14-3-3蛋白的研究进展,对植物中14-3-3蛋白的发现、基因鉴定、结构和功能以及14-3-3蛋白与其靶蛋白的互作机制进行综述,并对14-3-3蛋白的研究提出了进一步的展望。     

人类14—3—3ζ和GCH1蛋白相互作用的初步研究

目的寻找人类14-3-3ζ白的相互作用蛋白,为进一步揭示14-3-3ζ的作用机理提供线索。方法以14-3—3ζ为“诱饵”,利用酵母双杂交系统筛选人脑cDNA文库得到“猎物”蛋白,通过GSTpulldown技术和哺乳动物细胞内免疫共沉淀技术验证14-3-3ζ和“猎物”蛋白的特异性结合。结果首次利用酵母双杂交cDNA文库筛选技术发现了14-3-3ζ能够与GTP环化水解酶I（GTP cyclohydrolase1,GCHl）相互作用,并通过了体内和体外的蛋白质结合实验证实了这两个蛋白质之间的特异性相互作用。结论发现并验证了14-3-3ζ与GCH1之间的蛋白质相互作用,为进一步深入研究这两种蛋白质的功能及所引起的相关疾病的发病机理提供了新的线索。     

A third functional isoform enriched in mushroom body neurons is encoded by the Drosophila 14-3-3ζ gene

14-3-3 Proteins are highly conserved across eukaryotes, typically encoded by multiple genes in most species. Drosophila has only two such genes, 14-3-3ζ (leo), encoding two isoforms LEOI and LEOII, and 14-3-3ε. We report a bona fide third functional isoform encoded by leo divergent from the other two in structurally and functionally significant areas, thus increasing 14-3-3 diversity in Drosophila. Furthermore, we used a novel approach of spatially restricted leo abrogation by RNA-interference and revealed differential LEO distribution in adult heads, with LEOIII enrichment in neurons essential for learning and memory in Drosophila.     

14-3-3:保护性信号转导调节蛋白

14 3 3蛋白家族是真核细胞中高度保守的可溶性蛋白。在哺乳动物 ,14 3 3蛋白主要存在于脑。 14 3 3蛋白与许多蛋白结合 ,在细胞凋亡、生长、增殖的信号转导过程中发挥关键的调节作用 ,是细胞内重要的保护性蛋白。 14 3 3蛋白还是一些脑疾病的诊断标志。 14 3 3蛋白有可能成为治疗一些疾病的靶点     

14-3-3蛋白与植物细胞信号转导

14-3-3蛋白通过直接蛋白质-蛋白质相互作用对植物代谢关键酶、质膜H^＋ -ATP酶等发挥广泛调节作用。越来越多证据显示14-3-3蛋白通过与转录因子和其他信号分子结合参与调控植物细胞信号转导。对植物细胞中14-3-3蛋白调控信号转导途径,尤其是植物细胞对胁迫响应的调控机制进行了综述。     

14-3-3蛋白和神经退行性疾病

14-3-3蛋白以二聚体形式存在,识别磷酸丝氨酸／苏氨酸连接的信号分子,通过与其配体蛋白质相互作用,参与细胞信号转导、细胞周期调控和细胞问运输。14-3-3蛋自在脑组织中含量丰富,所有神经元、星形胶质细胞、少突肢质细胞、小胶质细胞的胞液与胞核中都有表述。14-3-3蛋白与神经退行性疾病阿尔茨海默病和帕金森病的发生过程关系密切。     

14-3-3? and 14-3-3σ Inhibit Toll-like Receptor (TLR)-mediated Proinflammatory Cytokine Induction

Toll-like receptors (TLRs) are a group of pattern recognition receptors that play a crucial role in the induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral double-stranded RNA. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Limited studies have applied proteomics toward understanding the dynamics of TLR signaling. Herein, a proteomics approach identified 14-3-3ϵ and 14-3-3σ proteins as new members of the TLR signaling complex. Toward the functional characterization of 14-3-3ϵ and 14-3-3σ in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligand-induced IL-6, TNFα, and IFN-β production. We also show that 14-3-3ϵ and 14-3-3σ impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-κB and IFN-β reporter gene activity. Interestingly, although the 14-3-3 proteins inhibit poly(I:C)-mediated RANTES production, 14-3-3 proteins augment Pam₃CSK₄, LPS, R848, and CpG-mediated production of RANTES (regulated on activation normal T cell expressed and secreted) in a Mal (MyD88 adaptor-like)/MyD88-dependent manner. 14-3-3ϵ and 14-3-3σ also bind to the TLR adaptors and to both TRAF3 and TRAF6. Our study conclusively shows that 14-3-3ϵ and 14-3-3σ play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections through modulation of the TLR signaling pathway. Thus, manipulation of 14-3-3 proteins may represent novel therapeutic targets for inflammatory conditions and infections.     

Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin β Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation

Integrins are hetero-dimeric (α and β subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the β CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of β CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3ζ binds to phosphorylated β CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated β CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3ζ, Dok1, and phosphorylated integrin β2 and β3 CTs. We show that the scaffold protein 14-3-3ζ interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin β CTs. The interactions were mapped onto the β-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3ζ/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin β CTs. We demonstrate that Thr phosphorylated pTTT β2 CT or pTST β3 CT can bind to 14-3-3ζ that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated β2 CT or Tyr phosphorylated β3 CT interacted with Dok1 in 14-3-3ζ/Dok1 complex. Based on these data, we propose that 14-3-3ζ/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation.