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14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.  相似文献   

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Gelperin D  Horton L  DeChant A  Hensold J  Lemmon SK 《Genetics》2002,161(4):1453-1464
14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR.  相似文献   

5.
The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.  相似文献   

6.
Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The beta1,4-galactosyltransferase 1 (beta4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3beta is required to bind beta4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Ikappabeta kinase and protein kinase Cdelta is involved in the phosphorylation of 14-3-3beta on two serine residues, paralleled by release of binding of TTP and 14-3-3beta from beta4GalT1 mRNA, nuclear sequestration of TTP, and beta4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3beta.  相似文献   

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 The nitrate reductase activity from Chlamydomonas reinhardtii was not altered when extracts were incubated with yeast 14-3-3 proteins in the presence of Mg-ATP. However, the C. reinhardtii extracts contained 14-3-3 proteins capable of inhibiting the spinach nitrate reductase, raising the question of their physiological substrates. Two C. reinhardtii proteins of about 48 and 35 kDa were eluted from 14-3-3 affinity chromatography columns and bound to 14-3-3s in overlay assays. The 48-kDa protein corresponded to the cytosolic isoform of glutamine synthetase (GS1). The GS1 was phosphorylated by a Ca2+- and calmodulin-dependent protein kinase partially purified from the alga. However, neither phosphorylation nor 14-3-3 binding seemed to change GS catalytic activity. Received: 3 February 2000 / Accepted: 6 May 2000  相似文献   

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The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.  相似文献   

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Testicular protein kinase 1 (TESK1) is a serine/threonine kinase that phosphorylates cofilin and induces actin cytoskeletal reorganization. The kinase activity of TESK1 is stimulated by integrin-mediated signaling pathways, but the mechanism of regulation has remained unknown. By using the yeast two-hybrid system, we identified 14-3-3beta to be the binding protein of TESK1. Specific interaction between TESK1 and 14-3-3beta became evident in in vitro and in vivo co-precipitation assays. 14-3-3beta interacts with TESK1 through the C-terminal region of TESK1 and in a manner dependent on the phosphorylation of Ser-439 within an RXXSXP motif. Binding of 14-3-3beta inhibited the kinase activity of TESK1. During cell spreading on fibronectin, the TESK1/14-3-3beta interaction significantly decreased, in a time course that inversely correlated with increase in TESK1 kinase activity. Thus, the dissociation of 14-3-3beta from a TESK1/14-3-3beta complex is likely to be involved in the integrin-mediated TESK1 activation. In HeLa cells, TESK1, together with 14-3-3beta, accumulated at the cell periphery when cells were plated on fibronectin, whereas they were diffusely distributed in the cytoplasm in the case of non-stimulated cells. We propose that 14-3-3beta plays important roles in regulating the kinase activity of TESK1 and localizing TESK1 to cell adhesion sites following integrin stimulation.  相似文献   

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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.  相似文献   

12.
Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.  相似文献   

13.
WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.  相似文献   

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To monitor site-specific phosphorylation of spinach leaf nitrate reductase (NR) and binding of the enzyme to 14-3-3 proteins, serum antibodies were raised that select for either serine 543 phospho- or dephospho-NR. The dephospho-specific antibodies blocked NR phosphorylation on serine 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, NR inhibition by 14-3-3s, NR dephosphorylation on serine 543, and did not precipitate 14-3-3s together with NR. Together, this confirms that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543. The amounts of individual NR forms were determined in leaf extracts by immunoblotting and immunoprecipitation. The phosphorylation state of NR on serine 543 increased 2-3-fold in leaves upon a light/ dark transition. Before the transition, one-third of NR was already phosphorylated on serine 543 but was not bound to 14-3-3s. Phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves.  相似文献   

16.
14-3-3 proteins are intracellular, dimeric molecules that bind to and modify the activity of several signaling proteins. We used human 14-3-3zeta as a bait in the yeast two-hybrid system to screen a murine embryonic cDNA library. One interacting clone was found to encode the carboxyl terminus of a putative protein kinase. The coding sequence of the human form (protein kinase Ualpha, PKUalpha) of this protein kinase was found in GenBank(TM) on the basis of sequence homology. The two-hybrid clone was also highly homologous to TOUSLED, an Arabidopsis thaliana protein kinase that is required for normal flower and leaf development. PKUalpha has been found by coimmunoprecipitation to bind to 14-3-3zeta in vivo. Our confocal laser immunofluorescence microscopic experiments revealed that PKUalpha colocalizes with the cytoplasmic intermediate filament system of cultured fibroblasts in the G(1) phase of the cell cycle. PKUalpha is found in the perinuclear area of S phase cells and in the nucleus of late G(2) cells. Transfection of cells with a dominant negative form of 14-3-3eta promotes the nuclear localization of PKUalpha. These results suggest that the subcellular localization of PKUalpha is regulated, at least in part, by its association with 14-3-3.  相似文献   

17.
Proteins of the kinesin superfamily are regulated in their motor activity as well as in their ability to bind to their cargo by carboxyl-terminal associating proteins and phosphorylation. KIF1C, a recently identified member of the KIF1/Unc104 family, was shown to be involved in the retrograde vesicle transport from the Golgi-apparatus to the endoplasmic reticulum. In a yeast two-hybrid screen using the carboxyl-terminal 350 amino acids of KIF1C as a bait, we identified as binding proteins 14-3-3 beta, gamma, epsilon, and zeta. In addition, a clone encoding the carboxyl-terminal 290 amino acids of KIF1C was found, indicating a potential for KIF1C to dimerize. Subsequent transient overexpression experiments showed that KIF1C can dimerize efficiently. However, in untransfected cells, only a small portion of KIF1C was detected as a dimer. The association of 14-3-3 proteins with KIF1C could be confirmed in transient expression systems and in untransfected cells and was dependent on the phosphorylation of serine 1092 located in a consensus binding sequence for 14-3-3 ligands. Serine 1092 was a substrate for the protein kinase casein kinase II in vitro, and inhibition of casein kinase II in cells diminished the association of KIF1C with 14-3-3gamma. Our data thus suggest that KIF1C can form dimers and is associated with proteins of the 14-3-3 family.  相似文献   

18.
The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth.  相似文献   

19.
14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Forster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3zeta protein.  相似文献   

20.
Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.  相似文献   

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