The 14-3-3 proteins positively regulate rapamycin-sensitive signaling

BACKGROUND: The kinase Tor is the target of the immunosuppressive drug rapamycin and is a member of the phosphatidylinositol kinase (PIK)-related kinase family. It plays an essential role in progression through the G1 phase of the cell cycle. The molecular details of Tor signaling remain obscure, however. RESULTS: We isolated two Saccharomyces cerevisiae genes, BMH1 and BMH2, as multicopy suppressors of the growth-inhibitory phenotype caused by rapamycin in budding yeast. BMH1 and BMH2 encode homologs of the 14-3-3 signal transduction proteins. Deletion of one or both BMH genes caused hypersensitivity to rapamycin in a manner that was dependent on gene dosage. In addition, alterations in the phosphopeptide-binding pocket of the 14-3-3 proteins had dramatically different effects on their ability to relieve the growth-arresting rapamycin phenotype. Mutations that prevented 14-3-3 from binding to a phosphoserine motif abolished its ability to confer rapamycin resistance. In contrast, substitution of two residues in 14-3-3 that surround these phosphoserine-binding sites conferred a dominant rapamycin-resistant phenotype. CONCLUSIONS: Our studies reveal 14-3-3 as an important component in rapamycin-sensitive signaling and provide significant new insights into the structure and function of 14-3-3 proteins.     

14-3-3 proteins in neuronal development and function

The 14-3-3 proteins are small, cytosolic, evolutionarily conserved proteins expressed abundantly in the nervous system. Although they were discovered more than 30 yr ago, their function in the nervous system has remained enigmatic. Several recent studies have helped to clarify their biological function. Crystallographic investigations have revealed that 14-3-3 proteins exist as dimers and that they contain a specific region for binding to other proteins. The interacting proteins, in turn, contain a 14-3-3 binding motif; proteins that interact with 14-3-3 dimers include PKC and Raf, protein kinases with critical roles in neuronal signaling. These proteins are capable of activating Raf in vitro, and this role has been verified by in vivo studies in Drosophila. Most interestingly, mutations in the Drosophila 14-3-3 genes disrupt neuronal differentiation, synaptic plasticity, and behavioral plasticity, establishing a role for these proteins in the development and function of the nervous system.     

14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.     

Human signaling protein 14-3-3zeta interacts with platelet glycoprotein Ib subunits Ibalpha and Ibbeta

The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor and its arterial subendothelial von Willebrand factor (vWF) ligand. The intracellular signaling immediately following GPIb receptor occupancy connecting the adhesive event to platelet activation and aggregation has not been well characterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous protein family with diverse biologic roles, including functional modulation of several prominent signaling proteins. We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3zeta, and provide evidence for the potential signaling role of this protein. Two-hybrid interactions suggest that platelet 14-3-3zeta associates with the cytoplasmic domain of GPIb subunits Ibalpha and Ibbeta in transformed yeast cells. The 14-3-3 interaction with GPIbbeta may be partly mediated through the latter's phosphorylated serine 166 residue as its mutagenesis results in 20% to 40% reduced interaction. There was 51% to 59% reduced interaction between GPIb and three 14-3-3zeta deletion mutants compared with full-length 14-3-3zeta, suggesting that either the N-terminal dimerization or membrane-binding domains or more than one noncontiguous 14-3-3zeta element may be required for optimal GPIb interaction. Confocal studies of platelets and a megakaryocyte cell line provided additional evidence for interaction of 14-3-3zeta with GPIbalpha and GPIbbeta. We also found that, similar to the signaling mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3-3zeta content is increased following vWF and ristocetin stimulation. We suggest that platelet 14-3-3zeta interacts with GPIbalpha and Ibbeta, that this interaction may be partly mediated through phosphoserine recognition, and that 14-3-3zeta cytoskeletal translocation may serve as a GPIb post-receptor occupancy signaling event.     

Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.     

The subunit structure of bovine brain 14-3-2

Two aspects of the subunit structure of the bovine brain specific protein 14-3-2 have been examined. On the one hand, native 14-3-2 has been separated into two fractions by hydroxylapatite chromatography. One eluted at the same position when chromatographed on the same column, while the other redistributed into the same two fractions again. Amino acid analysis of these two forms of 14-3-2 gave results that were not significantly different under the condition of analysis. Furthermore, when each peak was subjected to cyanogen bromide cleavage, very similar elution profiles of the resultant fragments were obtained. The two pools also cross-reacted with antiserum to 14-3-2. Reaction of purified 14-3-2 with dimethylsuberimidate caused the formation of covalently bound protein units of 100,000 molecular weight when measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as opposed to the 50,000 minimal molecular weight normally detected. On the other hand, analysis of the soluble tryptic peptides of S-[14C]carboxymethyl 14-3-2 yielded only three distinct radioactive peptides, each with one residue of S-carboxymethylcysteine whereas 8 are expected on the basis of the amino acid composition of the 50,000 molecular weight polypeptide chains. Thermolysin digestion of a similarly modified 14-3-2 preparation yielded all of the radioactivity in 5 S-carboxymethylcysteine-containing peptides. The partial amino acid sequence of these peptides indicates that they represent 4 unique areas of the polypeptide chain. Since 8 such peptides were expected, that is, double the number found, the minimum structural unit of the protein must be of 25,000 molecular weight. The results of these experiments do not permit distinction between a duplication of the structure within a single polypeptide chain or the alternate possibility of two polypeptide chains bound by unusually strong non-covalent bonds. These results suggest that 14-3-2 is a covalently linked dimer of 25,000 mol.wt. units that can aggregate to form larger species of 100,000 mol.wt. and higher.     

Over-expression of plant 14-3-3 proteins in tobacco: enhancement of the plasmalemma K+ conductance of mesophyll cells

Two cDNA clones encoding 14-3-3 homologous proteins were isolated from Vicia faba. Deduced amino acid sequences share different degrees of homology with other plant 14-3-3 proteins. Both clones, under the control of the CaMV 35S promoter, were transformed into tobacco plants. Immunoblotting showed three different forms of ca. 31, 34, and 37 kDa, indicating a covalent modification of the expressed 14-3-3 proteins. These forms were mainly present in the microsomal fraction. Patch-clamp studies of mesophyll protoplasts of the transformants revealed a strongly enhanced K+ conductance compared to the wild type. This indicates the involvement of 14-3-3 proteins in ion channel regulation, presumably by modulating kinase activities or binding the channel.     

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.     

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.     

Biological significance of divalent metal ion binding to 14-3-3 proteins in relationship to nitrate reductase inactivation

In this report we address two questions regarding the regulation of phosphorylated nitrate reductase (pNR; EC 1.6.6.1) by 14-3-3 proteins. The first concerns the requirement for millimolar concentrations of a divalent cation in order to form the inactive pNR:14-3-3 complex at pH 7.5. The second concerns the reduced requirement for divalent cations at pH 6.5. In answering these questions we highlight a possible general mechanism involved in the regulation of 14-3-3 binding to target proteins. We show that divalent cations (e.g. Ca2+, Mg2+ and Mn2+) bind directly to 14-3-3s, and as a result cause a conformational change, manifested as an increase in surface hydrophobicity. A similar change is also obtained by decreasing the pH from pH 7.5 to pH 6.5, in the absence of divalent cations, and we propose that protonation of amino acid residues brings about a similar effect to metal ion binding. A possible regulatory mechanism, where the 14-3-3 protein has to be "primed" prior to binding a target protein, is discussed.     

The Arabidopsis 14-3-3 multigene family

The 14-3-3 proteins are ubiquitous eukaryotic proteins and are encoded by a gene family in many species. We examined the 14-3-3 gene family in Arabidopsis thaliana and found that it contains 10 members. Four new cDNAs, GF14 epsilon, GF14 kappa, GF14 mu, and GF14 nu, and two new genomic clones of GF14 phi and GF14 nu were isolated and characterized. Together with the six previously described 14-3-3 isoforms in Arabidopsis, they constitute a complete family of 10 distinct 14-3-3 proteins of 248 to 268 amino acids. Phylogenetic analysis revealed the presence of two ancient, distinct 14-3-3 gene classes in Arabidopsis and other plants. The epsilon forms diverged early from the other plant isoforms, and plant 14-3-3 genes displayed a different evolutionary course from that of mammals.     

14-3-3 inhibits the Dictyostelium myosin II heavy-chain-specific protein kinase C activity by a direct interaction: identification of the 14-3-3 binding domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14-3-3 protein (Dd14-3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14-3-3 zeta isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14-3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14-3-3. This suggests that Dd14-3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14-3-3 as well as 14-3-3 zeta through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14-3-3 family and demonstrate that MHC-PKC interacts directly with Dd14-3-3 and 14-3-3 zeta through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.     

Association between ligand-induced conformational changes of integrin IIbbeta3 and IIbbeta3-mediated intracellular Ca2+ signaling

Platelet IIbbeta3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbbeta3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbbeta3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbbeta3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbbeta3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and beta3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the beta3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbbeta3. Neither group I nor group II antagonist increased intracellular Ca2+ concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbbeta3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbbeta3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbbeta3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between beta3 LIBS expression and IIbbeta3-mediated intracellular Ca2+ signaling in platelets.     

Ser-534 in the hinge 1 region of Arabidopsis nitrate reductase is conditionally required for binding of 14-3-3 proteins and in vitro inhibition

14-3-3 proteins bind to the hinge 1 region of nitrate reductase (NR) and inhibit its activity. To determine which residues of NR are required for 14-3-3-inhibitory interactions, wild-type and mutant forms of Arabidopsis NR were examined in the yeast two-hybrid system and in vitro inhibition assays. NR fragments with or without hinge 1 were introduced into yeast with one of seven Arabidopsis 14-3-3 isoforms (called GF14s). NR fragments (residues 1-562 or 487-562) containing hinge 1 interacted with all GF-14s tested; an NR fragment (residues 1-487) lacking hinge 1 did not. GF14 binding to NR fragments was dependent on Ser-534, since Asp or Ala substitutions at this site blocked the interaction. Revertants with second site substitutions restoring interaction between GF14omega and the Ala- or Asp-substituted NR fragments were identified. One isolate had a Lys to Glu substitution at position 531, which is in hinge 1, and six isolates had Ile to Leu or Phe substitutions at 561 in the heme binding region. Double mutant forms of holo-NR (S534D plus K531E, I561F, or I561L) were constructed and found to be partially inhibited by protein extracts from Arabidopsis containing 14-3-3 proteins. Wild-type NR is phosphorylated and inhibited by these extracts, but S534D single mutant forms are not. These results show that inhibitory NR/14-3-3 interactions are dependent on Ser-534 but only in the context of the wild-type sequence, since substitutions at second sites render 14-3-3 binding and in vitro NR inhibition independent of Ser-534.     

Human cruciform binding protein belongs to the 14-3-3 family

Cruciform DNA has been implicated in the initiation of DNA replication. Recently, we identified and purified from human (HeLa) cells a protein, CBP, with binding specificity for cruciform DNA. We have reported previously that the CBP activity sediments at approximately 66 kDa in a glycerol gradient. Here, photochemical cross-linking studies and Southwestern analyses confirm that a 70 kDa polypeptide interacts specifically with cruciform DNA. Microsequence analysis of tryptic peptides of the 70 kDa CBP reveals that it is 100% homologous to the 14-3-3 family of proteins and shows that CBP contains the epsilon, beta, gamma, and zeta isoforms of the 14-3-3 family. In addition to polypeptides with the characteristic molecular mass of 14-3-3 proteins (30 and 33 kDa), CBP also contains a polypeptide of 35 kDa which is recognized by an antibody specific for the epsilon isoform of 14-3-3. Cruciform-specific binding activity is also detected in 14-3-3 proteins purified from sheep brain. Immunofluorescene studies confirm the presence of the epsilon, beta, and zeta isoforms of 14-3-3 proteins in the nuclei of HeLa cells. The 14-3-3 family of proteins has been implicated in cell cycle control, and members of this family have been shown to interact with various signaling proteins. Cruciform binding is a new activity associated with the 14-3-3 family.