The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called “14-3-3 site-finder”. We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

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。     

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。     

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.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

Monitoring 14-3-3 protein interactions with a homogeneous fluorescence polarization assay

The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)-based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 as a model system, they have achieved a simple 1-step "mix-and-measure" method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used to monitor the binding of 14-3-3 with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z' factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.     

14-3-3 and aggresome formation: Implications in neurodegenerative diseases

Protein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded proteins to dynein motors for transport to aggresomes. This molecular complex involves a dimeric binding of 14-3-3 to both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3). As 14-3-3 has been implicated in various neurodegenerative diseases, our findings may provide mechanistic insights into its role in managing misfolded protein stress during the process of neurodegeneration.     

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。     

Interaction between 14-3-3β and PrP influences the dimerization of 14-3-3 and fibrillization of PrP106–126

Proteins of the 14-3-3 family are universal participate in multiple cellular processes. However, their exact role in the pathogenesis of prion diseases remains unclear. In this study, we proposed that human PrP was able to form molecular complex with 14-3-3β. The domains responsible for the interactions between PrP and 14-3-3β were mapped at the segments of amino acid (aa) residues 106–126 within PrP and aa 1–38 within 14-3-3β. Homology modeling revealed that the key aa residues for molecular interaction were D22 and D23 in 14-3-3β as well as K110 in PrP. Mutations in these aa residues inhibited the interaction between the two proteins in vitro. Our results also showed that recombinant PrP encouraged 14-3-3β dimer formation, whereas PrP106–126 peptide inhibited it. Recombinant 14-3-3β disaggregated the mature PrP106–126 fibrils in vitro. Moreover, the PrP–14-3-3 protein complexes were observed in the brain tissues of normal and scrapie agent 263 K infected hamsters. Colocalization of PrP and 14-3-3 was seen in the cytoplasm of human neuroblastoma cell line SH-SY5Y, as well as human cervical cancer cell line HeLa transiently expressing full-length human PrP. Our current data suggest the neuroprotection of PrPC and neuron damage caused by PrPSc may be associated with their functions of 14-3-3 dimerization regulation.     

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.     

The 14-3-3s

Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization.     

Structure and Sites of Phosphorylation of 14-3-3 Protein: Role in Coordinating Signal Transduction Pathways

The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of adaptor protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX_1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.     

Purification, Properties, and Immunohistochemical Localisation of Human Brain 14-3-3 Protein

Abstract: A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition (αβ; 14-3-3-1 has the composition ββ. Peptide mapping with Stuphvlococcus aureus V8 proteinase shows that α and β subunits are unrelated but the β and β' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.     

Anoxic preconditioning up-regulates 14-3-3 protein expression in neonatal rat cardiomyocytes through extracellular signal-regulated protein kinase 1/2

Anoxic preconditioning (APC) attenuates myocardial injury caused by ischemia/reperfusion. The protective mechanisms of APC involve up-regulation of the protective proteins and inhibition of apoptosis. 14-3-3 protein, as a molecular chaperone, plays an important role in regulating cell survival and apoptosis. However, the role of 14-3-3 protein in cardioprotection of APC and the pathways determining 14-3-3 protein expression during APC are not clear. In this work, Western blotting analysis was used to detect the 14-3-3 protein expression and activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in cardiomyocytes subjected to anoxia-reoxygenation injury with and without APC and control. The cardiomyocytes from APC group were more resistant to injury induced by anoxia-reoxygenation and had much stronger phosphorylation of ERK1/2 than the control. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in cardiomyocytes induced by APC. The results indicate that APC up-regulates 14-3-3 protein expression through the ERK1/2 signaling pathways.     

SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.