A single Arabidopsis GF14 isoform possesses biochemical characteristics of diverse 14-3-3 homologues

Arabidopsis cDNA clones of GF14 proteins originally were isolated on the basis of their association with the G-box DNA/protein complex by a monoclonal antibody screening approach. GF14 proteins are homologous to the 14-3-3 family of mammalian proteins. Here we demonstrate that recombinant GF14 , one member of the Arabidopsis GF14 protein family, is a dimeric protein that possesses many of the attributes of diverse mammalian 14-3-3 homologues. GF14 activates rat brain tryptophan hydroxylase and protein kinase C in a manner similar to the bovine 14-3-3 protein. It also activates exoenzyme S of Pseudomonas aeruginosa as does bovine brain factor activating exoenzyme S (FAS), which is itself a member of 14-3-3 proteins. In addition, GF14 binds calcium, as does the human 14-3-3 homologue reported to be a phospholipase A2. These results indicate that a single isoform of this plant protein family can have multiple functions and that individual GF14 isoforms may have multiple roles in mediating signal transductions in plants. However, GF14 does not regulate growth in an in vivo test for functional similarity to the yeast 14-3-3 homologue, BMH1. Thus, while a single plant GF14 isoform can exhibit many of the biochemical attributes of diverse mammalian 14-3-3 homologues, open questions remain regarding the physiological functions of GF14/14-3-3 proteins.     

Exploring the binding diversity of intrinsically disordered proteins involved in one‐to‐many binding

Molecular recognition features (MoRFs) are intrinsically disordered protein regions that bind to partners via disorder‐to‐order transitions. In one‐to‐many binding, a single MoRF binds to two or more different partners individually. MoRF‐based one‐to‐many protein–protein interaction (PPI) examples were collected from the Protein Data Bank, yielding 23 MoRFs bound to 2–9 partners, with all pairs of same‐MoRF partners having less than 25% sequence identity. Of these, 8 MoRFs were bound to 2–9 partners having completely different folds, whereas 15 MoRFs were bound to 2–5 partners having the same folds but with low sequence identities. For both types of partner variation, backbone and side chain torsion angle rotations were used to bring about the conformational changes needed to enable close fits between a single MoRF and distinct partners. Alternative splicing events (ASEs) and posttranslational modifications (PTMs) were also found to contribute to distinct partner binding. Because ASEs and PTMs both commonly occur in disordered regions, and because both ASEs and PTMs are often tissue‐specific, these data suggest that MoRFs, ASEs, and PTMs may collaborate to alter PPI networks in different cell types. These data enlarge the set of carefully studied MoRFs that use inherent flexibility and that also use ASE‐based and/or PTM‐based surface modifications to enable the same disordered segment to selectively associate with two or more partners. The small number of residues involved in MoRFs and in their modifications by ASEs or PTMs may simplify the evolvability of signaling network diversity.     

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。     

14-3-3 proteins: regulation of subcellular localization by molecular interference

14-3-3 family of proteins plays a key regulatory role in signal transduction, checkpoint control, apoptotic, and nutrient-sensing pathways. 14-3-3 proteins act by binding to partner proteins, and this binding often leads to the altered subcellular localization of the partner. 14-3-3 proteins promote the cytoplasmic localization of many binding partners, including the pro-apoptotic protein BAD and the cell cycle regulatory phosphatase Cdc25C, but they can also promote the nuclear localization of other partners, such as the catalytic subunit of telomerase (TERT). In some cases, 14-3-3 binding has no effect on the subcellular localization of a partner. 14-3-3 may affect the localization of a protein by interfering with the function of a nearby targeting sequence, such as a nuclear localization sequence (NLS) or a nuclear export sequence (NES), on the binding partner.     

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.     

The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions and undergoes constant remodeling through formation and loss of the three-way junctions. Transmembrane and coiled-coil domain family 3 (TMCC3), an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N terminus and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wildtype TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.     

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.     

Tyrosine phosphorylation inhibits the interaction of 14-3-3 proteins with the plant plasma membrane H+-ATPase

Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.     

Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.     

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.     

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。     

Using collections of structural models to predict changes of binding affinity caused by mutations in protein–protein interactions

Protein–protein interactions (PPIs) in all the molecular aspects that take place both inside and outside cells. However, determining experimentally the structure and affinity of PPIs is expensive and time consuming. Therefore, the development of computational tools, as a complement to experimental methods, is fundamental. Here, we present a computational suite: MODPIN, to model and predict the changes of binding affinity of PPIs. In this approach we use homology modeling to derive the structures of PPIs and score them using state‐of‐the‐art scoring functions. We explore the conformational space of PPIs by generating not a single structural model but a collection of structural models with different conformations based on several templates. We apply the approach to predict the changes in free energy upon mutations and splicing variants of large datasets of PPIs to statistically quantify the quality and accuracy of the predictions. As an example, we use MODPIN to study the effect of mutations in the interaction between colicin endonuclease 9 and colicin endonuclease 2 immune protein from Escherichia coli. Finally, we have compared our results with other state‐of‐art methods.     

Binding of phosphorylated peptides and inhibition of their interaction with disease‐relevant human proteins by synthetic metal‐chelate receptors

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal‐chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal‐chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon‐γ receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Discovery of binding proteins for a protein target using protein–protein docking‐based virtual screening

Target structure‐based virtual screening, which employs protein‐small molecule docking to identify potential ligands, has been widely used in small‐molecule drug discovery. In the present study, we used a protein–protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all‐to‐all protein–protein docking run on a large dataset was performed. The three‐dimensional rigid docking program SDOCK was used to examine protein–protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z‐score, and convergency of the low‐score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all‐to‐all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor‐α (TNFα), which is a well‐known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top‐ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein–protein docking for the discovery of novel binding proteins for specific protein targets. Proteins 2014; 82:2472–2482. © 2014 Wiley Periodicals, Inc.     

Great interactions: How binding incorrect partners can teach us about protein recognition and function

Protein–protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross‐docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross‐docking predictions using the area under the specificity‐sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding‐site predictions resulting from the cross‐docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408–1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

14-3-3 proteins: regulators of numerous eukaryotic proteins

14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.     

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.