14-3-3蛋白对植物发育的调控作用

14-3-3蛋白是高度保守并在真核生物中普遍存在的一类调节蛋白。不同的14-3-3蛋白同工型具有不同的细胞特异性, 并通过识别特异的磷酸化序列与靶蛋白相互作用, 被称为蛋白质与蛋白质相互作用的桥梁蛋白。在植物生长发育过程中, 14-3-3蛋白通过与其它蛋白的相互作用参与多种植物激素信号转导、各种代谢调控、物质运输和光信号应答等调控过程。该文主要对近年来有关14-3-3蛋白在植物生长发育中的调控作用, 特别是14-3-3蛋白参与调控植物激素信号转导等方面的研究进展进行综述。     

Structure of the p53 C-terminus bound to 14-3-3: Implications for stabilization of the p53 tetramer

The adaptor protein 14-3-3 binds to and stabilizes the tumor suppressor p53 and enhances its anti-tumour activity. In the regulatory C-terminal domain of p53 several 14-3-3 binding motifs have been identified. Here, we report the crystal structure of the extreme C-terminus (residues 385-393, p53pT387) of p53 in complex with 14-3-3σ at a resolution of 1.28 Å. p53pT387 is accommodated by 14-3-3 in a yet unrecognized fashion implying a rationale for 14-3-3 binding to the active p53 tetramer. The structure exhibits a potential binding site for small molecules that could stabilize the p53/14-3-3 protein complex suggesting the possibility for therapeutic intervention.

Structured summary

MINT-7711943: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by X-ray crystallography (MI:0114)MINT-7711931: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by isothermal titration calorimetry (MI:0065)     

植物中14-3-3蛋白的主要功能

14-3-3蛋白家族广泛存在于真核生物中,序列高度保守。主要以同源或异源二聚体形式存在,可以同时与两个靶蛋白或者与一个靶蛋白的两个结构域相互作用,通过与靶蛋白上的一小段共有序列的磷酸化丝氨酸/苏氨酸残基结合来发挥其调控功能。本文综述了植物中的14-3-3蛋白及其主要功能,并重点综述了14-3-3蛋白对植物基本碳、氮代谢的调控。     

ZmMPK6, a Novel Maize MAP Kinase that Interacts with 14-3-3 Proteins

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.     

Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen-activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)-differentiated PC12 cells, we found that constitutive and autonomous Ca2+-independent PKC activity is associated with 14-3-3 zeta. Because PKC and 14-3-3 zeta are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14-3-3 zeta in the brain and whether the PKC/14-3-3 zeta complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+-dependent classical PKC, Ca2+-independent novel PKC, and Ca2+-independent and diacylglycerol-insensitive atypical PKC, all interact with 14-3-3 zeta in the rodent brain. The pool size of 14-3-3 zeta bound form of PKC is small (1-4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14-3-3 zeta is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14-3-3 zeta could be a mechanism for the persistent PKC activation in the brain.     

The C-terminal tail of Arabidopsis 14-3-3omega functions as an autoinhibitor and may contain a tenth alpha-helix

The eukaryotic regulatory protein 14-3-3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me2+) and some polyamines interact with the loop 8 region of the 14-3-3 proteins and allow them to bind and inhibit pNR in vitro. The role of the highly variant C-terminal regions of the 14-3-3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C-terminal tail of the Arabidopsis 14-3-3omega isoform and evaluated its contributions to the inhibition of pNR. Nested C-terminal truncations of the recombinant 14-3-3omega protein revealed that the removal of the C-terminal tail renders the protein partially Mg2+-independent in both pNR binding and inhibition of activity, suggesting that the C-terminus functions as an autoinhibitor. The C-terminus of 14-3-3omega appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys-247. C-terminal truncation of 14-3-3omega at Thr-255 increased its interaction with antibodies to the C-terminus of 14-3-3omega in non-denaturing conditions, but not in denaturing conditions, suggesting that the C-terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C-terminal peptide, from Trp-234 to Lys-249, revealed that the C-terminal tail might contain a tenth alpha-helix, in agreement with the in silico predictions. The function of the putative tenth alpha-helix is not clear because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg2+. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14-3-3 protein is restricted by the C-terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.     

14-3-3 proteins act as scaffolds for GmMYB62 and GmMYB176 and regulate their intracellular localization in soybean

Isoflavonoids are plant natural compounds predominantly found in leguminous plant. They play important functions in both nitrogen fixation and stress resistance. Many clinical studies have linked dietary intake of isoflavonoids to human health benefits. Binding of 14-3-3 proteins to GmMYB176, an isoflavonoid regulator, modulates expression of key isoflavonoids gene expression and its biosynthesis. We have recently demonstrated that the interaction of 14-3-3 proteins with GmMYB176 regulates nuclear-cytoplasmic localization of GmMYB176 thereby affecting target gene expression. Here, we report GmMYB62 as a new R1 MYB client protein of soybean 14-3-3s that may function together with GmMYB176 for gene regulation in soybean.     

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。     

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.     

Structural view of a fungal toxin acting on a 14-3-3 regulatory complex

The fungal phytotoxin fusicoccin stabilizes the interaction between the C-terminus of the plant plasma membrane H(+)-ATPase and 14-3-3 proteins, thus leading to permanent activation of the proton pump. This results in an irreversible opening of the stomatal pore, followed by wilting of plants. Here, we report the crystal structure of the ternary complex between a plant 14-3-3 protein, fusicoccin and a phosphopeptide derived from the C-terminus of the H(+)-ATPase. Comparison with the corresponding binary 14-3-3 complexes indicates no major conformational change induced by fusicoccin. The compound rather fills a cavity in the protein-phosphopeptide interaction surface. Isothermal titration calorimetry indicates that the toxin alone binds only weakly to 14-3-3 and that peptide and toxin mutually increase each others' binding affinity approximately 90-fold. These results are important for herbicide development but might have general implications for drug development, since rather than inhibiting protein-protein interactions, which is difficult to accomplish, it might be easier to reverse the strategy and stabilize protein-protein complexes. As the fusicoccin interaction shows, only low-affinity interactions would be required for this strategy.     

Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-d-aspartate (NMDA) Receptor Trafficking

The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, β, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission.     

Post-translational modification of 14-3-3 isoforms and regulation of cellular function

14-3-3 is now well established as a family of dimeric proteins that can modulate interaction between proteins involved in a wide range of functions. In many cases, these proteins show a distinct preference for a particular isoform(s) of 14-3-3 and in many cases a specific repertoire of dimer formation influences the particular proteins that 14-3-3 interact. Well over 200 proteins have been shown to interact with 14-3-3. The purpose of this review is to give an overview of the recently identified post-translational modifications of 14-3-3 isoforms and how this regulates function, interaction, specificity of dimerisation between isoforms and cellular location of target proteins. The association between 14-3-3 and its targets usually involves phosphorylation of the interacting protein which has been the subject of many reviews and discussion of this is included in other reviews in this series. However, it is now realised that in some cases the phosphorylation and a number of other, novel covalent modifications of 14-3-3 isoforms may modulate interaction and dimerisation of 14-3-3. Since this aspect is now emerging to be of major importance in the mechanism of regulation by 14-3-3 isoforms and has not been the focus of previous reviews, this will be detailed here.     

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.     

Binding of 14-3-3 proteins to a single stranded oligodeoxynucleotide aptamer

A synthetic library of ca. 10¹³ single stranded oligodeoxynucleotides, each comprising a randomized 40mer sequence and homogeneous 10mer flanking regions, was screened for binding to recombinant human 14-3-3γ. A single aptamer, which showed similar affinities (K_D ∼ 10⁻⁸ M) for six isoforms of the protein, has been shown to bind to undenatured 14-3-3 protein in the cerebral spinal fluid of scrapie infected sheep.     

14-3-3 epsilon modulates the stimulated secretion of endopeptidase 24.15

Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine-scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser(644) by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 microm). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 microm) from 10%[1.4 +/- 0.24 AFU/(min 10(6) cells)] to 19%[2.54 +/- 0.24 AFU/(min 10(6) cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.     

A multimeric membrane protein reveals 14-3-3 isoform specificity in forward transport in yeast

Arginine (Arg)-based endoplasmic reticulum (ER) localization signals are sorting motifs involved in the quality control of multimeric membrane proteins. They are distinct from other ER localization signals like the C-terminal di-lysine [-K(X)KXX] signal. The Pmp2p isoproteolipid, a type I yeast membrane protein, reports faithfully on the activity of sorting signals when fused to a tail containing either an Arg-based motif or a -KKXX signal. This reporter reveals that the Arg-based ER localization signals from mammalian Kir6.2 and GB1 proteins are functional in yeast. Thus, the machinery involved in recognition of Arg-based signals is evolutionarily conserved. Multimeric presentation of the Arg-based signal from Kir6.2 on Pmp2p results in forward transport, which requires 14-3-3 proteins encoded in yeast by BMH1 and BMH2 in two isoforms. Comparison of a strain without any 14-3-3 proteins (Deltabmh2) and the individual Deltabmh1 or Deltabmh2 shows that the role of 14-3-3 in the trafficking of this multimeric Pmp2p reporter is isoform-specific. Efficient forward transport requires the presence of Bmh1p. The specific role of Bmh1p is not due to differences in abundance or affinity between the isoforms. Our results imply that 14-3-3 proteins mediate forward transport by a mechanism distinct from simple masking of the Arg-based signal.     

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.     

水稻14-3-3蛋白家族的生物信息学分析

通过隐马尔柯夫模型(Hidden Markov Model,HMM),对粳稻(Oryza sativa L.ssp．japonica)基因组的蛋白质数据库进行搜索,结果获得8个14—3—3蛋白的同源序列,其中发现4个新基因。通过对所有粳稻的14—3—3蛋白的DNA序列与各种表达序列标签(Expression Sequence Tags,ESTs)进一步比对,为14-3-3蛋白找到了ESTs的证据。结果说明这些基因在水稻不同的处理和不同的部位都有所表达,而且不同成员之间的表达模式存在较大的差异。蛋白质多序列联配分析结果表明,存在可能的功能多态位点。通过基因结构和染色体定位的分析,确认了水稻基因组中存在E样和非E样两类14-3-3蛋白。此外,对目前植物中的14—3—3家族作了初步的进化分析。