14-3-3 proteins and the response to abiotic and biotic stress

14-3-3 proteins function as regulators of a wide range of target proteins in all eukaryotes by effecting direct protein-protein interactions. Primarily, interactions between 14-3-3 proteins and their targets are mediated by phosphorylation at specific sites on the target protein. Hence, interactions with 14-3-3s are subject to environmental control through signalling pathways which impact on 14-3-3 binding sites. Because 14-3-3 proteins regulate the activities of many proteins involved in signal transduction, there are multiple levels at which 14-3-3 proteins may play roles in stress responses in higher plants. In this article, we review evidence which implicates 14-3-3 proteins in responses to environmental, metabolic and nutritional stresses, as well as in defence responses to wounding and pathogen attack. This evidence includes stress-inducible changes in 14-3-3 gene expression, interactions between 14-3-3 proteins and signalling proteins and interactions between 14-3-3 proteins and proteins with defensive functions.     

Investigation on the induction of 14-3-3 in white spruce

 A cDNA encoding a 14-3-3 protein was isolated from white spruce. The corresponding polypeptide contains several motifs that are conserved in this type of protein and is predicted to be 260 amino acids in length. Multiple banding in Southern blot analysis suggests that the gene encoding this cDNA is, in fact, part of a small family of genes. Wounding and chitosan treatment of spruce plants followed by Northern blot analysis indicates that these stimuli caused the accumulation of 14-3-3 mRNA. In addition, cell suspension cultures treated with methyl jasmonate showed up-regulation of 14-3-3-encoding mRNA. Chitosan and methyl jasmonate are both signalling molecules in the activation of plant defense response genes. Therefore, our results suggest a possible role for this 14-3-3 protein in the pathogen defense response of coniferous trees. Received: 13 December 1999 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Five new 14-3-3 isoforms from Nicotiana tabacum L.: implications for the phylogeny of plant 14-3-3 proteins

About thirty years after the initial identification of 14-3-3 proteins in mammalian brain, they are now thought to be ubiquitous among eukaryotes. We identified five cDNAs encoding 14-3-3 proteins of Nicotiana tabacum L. using a polymerase chain reaction (PCR)-based screening strategy. A phylogenetic analysis was carried out with 14-3-3 amino-acid sequences from twelve plant species. The results showed that 14-3-3 proteins of plants can be divided into at least five different subgroups. Four of these subgroups resulted from early gene duplication events that happened prior to the speciation of most of the plant species considered. Interestingly, 14-3-3 epsilon isoforms from mammals and insects form one subgroup together with epsilon-like isoforms from plants. The 14-3-3 genes known from monocots descend from the same ancestor, forming the fifth subgroup. Received: 30 June 1997 / Accepted: 29 August 1997     

FTICR-MS analysis of 14-3-3 isoform substrate selection

The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client.     

Molecular evolution of the 14-3-3 protein family

Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene fromCaenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2–5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins fromEntamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/ HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed. Correspondence to: D.C. Shakes     

14-3-3蛋白家族的调控机制和生物学功能

14-3-3蛋白家族在真核细胞中广泛表达并高度保守,它们主要以同源／异源二聚体形式存在,可以同时与两个靶蛋白或一个靶蛋白的两个结构域相互作用。14-3-3蛋白通过磷酸化丝氨酸／苏氨酸介导和靶蛋白结合,从而发挥其调控功能。现对14-3-3蛋白的识别序列、与配体相互作用的特点,及其在细胞周期、凋亡、信号转导、线粒体／叶绿体前体蛋白跨膜转运中的调控机制和发挥的生物学功能进行综述。     

Multifunctional 14-3-3 proteins of plant cell

The 14-3-3 proteins are a family of highly conserved proteins found in all eukaryotes - from the yeasts to mammals. They regulate several cellular processes recognizing unique conservative, mostly phosphorylated motif of partner proteins. Binding of the 14-3-3 proteins regulates their partners through a variety of mechanisms, such as altering their catalytic activity, subcellular localization, stability or altering their interactions with other protein molecules. The native 14-3-3 proteins are present in form of homo- and hetero-dimers. The most structurally variable N-and C-termini are responsible for isoform specific protein-protein interactions, and cellular localization. In plant cell, 14-3-3 proteins appear to play an important role in regulation of key enzymes of carbon and nitrogen metabolism, modulation ion pumps and channels. They are also involved in signal transduction pathways and even in gene expression.     

14-3-3蛋白参与植物应答非生物胁迫的研究进展

14-3-3蛋白是一种在真核生物细胞中普遍存在且高度保守的蛋白。该蛋白在大多数物种中由一个基因家族编码,并以同源或异源二聚体的形式存在。不同的14-3-3蛋白同工型具有不同的细胞特异性,可通过识别特异的磷酸化或非磷酸化序列与靶蛋白相互作用。14-3-3蛋白在植物生长和发育的各个方面都起重要作用。本文主要围绕植物14-3-3蛋白的种类、结构、磷酸化或非磷酸化识别序列及其响应干旱、冷冻、盐碱、营养和机械胁迫等的分子机制研究进展进行综述。     

Structural organisation,expression, and promoter analysis of a 16R isoform of 14-3-3 protein gene from potato

Six full-length cDNAs encoding 14-3-3 proteins from potato (Solanum tuberosum L. cv. Desiree) plants have been recently isolated and sequenced. Screening of a potato genomic library with the 16R cDNA encoding 14-3-3 protein isoform resulted in the identification and isolation of the respective genomic clone. The gene contains four exons and three introns. Inspection of the promoter sequence of the 16R gene revealed several boxes important for the regulation of the gene expression. The induction of the promoter activity by sucrose, IAA, ABA and salicylic acid has been shown. Dof protein-binding sequences, E-boxes and sequences responsible for developmental regulation are most frequently represented. Northern blot and fluorometric analyses, as well as the microscopic examination of transgenic potato plants transformed with GUS reporter under 14-3-3 protein promoter, provide evidence for tissue-specific expression and age-dependent promoter activity. Significant GUS expression was observed in young organs or organ portions, as well as in minor vascular bundles of mature organs.     

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。     

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.     

A proteomic analysis of 14-3-3 binding proteins from developing barley grains

14-3-3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare L.) 14-3-3A was over-expressed, immobilised and used to affinity purify 14-3-3 binding proteins from developing barley grains. Binding was shown to be phosphorylation-dependent. These proteins were fractionated by PAGE and identified by MALDI-TOF MS. In total, 54 14-3-3 binding proteins were identified, 49 of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14-3-3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co-ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14-3-3 in a dosage-dependent manner. The second-largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14-3-3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence.     

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.     

The role of 14-3-3 proteins in cell signalling pathways and virus infection

14-3-3 proteins are highly conserved in species ranging from yeast to mammals and regulate numerous signalling pathways via direct interactions with proteins carrying phosphorylated 14-3-3–binding motifs. Recent studies have shown that 14-3-3 proteins can also play a role in viral infections. This review summarizes the biological functions of 14-3-3 proteins in protein trafficking, cell-cycle control, apoptosis, autophagy and other cell signal transduction pathways, as well as the associated mechanisms. Recent findings regarding the role of 14-3-3 proteins in viral infection and innate immunity are also reviewed.     

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.