Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Virus infection usually alters the host cell and shuts off the synthesis of cellular macromolecules. In order to screen the upregulated cellular transcripts during pseudorabies virus (PRV) infection, we employed the mRNA differential display technique. The screen is based on positive selection at the mRNA level for genes expressed in normal cells but increased in corresponding PRV-infected cells. Over 14000 species of mRNA, isolated from mock-infected and PRV-infected Madin-Darby bovine kidney cell at 1 h post infection, were screened, and 40 candidate clones were recovered. Southern blot analysis revealed that 17 out of 40 candidate clones, were enhanced in PRV-infected cells. Partial DNA sequences demonstrated that 17 clones were distinct cellular genes, including those encoding the modulators of signal transduction (saposin, 14-3-3, adenylate kinase, adenylyl cyclase, protein kinase C-alpha), those encoding the components of translation (fau, ribosomal proteins S11, L31, L36), other cellular genes (peptidase, cyclin E, rch1, oligo-C-rich single-stranded nucleic acid binding protein, rap, arginyl-tRNA synthetase), and two unknown genes. Thus, this study identifies successfully the transcriptionally regulated cellular genes which are associated with PRV infection. Furthermore, this study provides support for the use of mRNA differential display as a method to rapidly isolate differentially expressed genes in virus infection.
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PMID:Analysis of upregulated cellular genes in pseudorabies virus infection: use of mRNA differential display. 891 Jun 44

The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3eta protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.
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PMID:Interaction of the ligand-activated glucocorticoid receptor with the 14-3-3 eta protein. 907 30

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.
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PMID:14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain. 926 Oct 98

Protein factors playing a significant part in differentiation and development have been recently elucidated. However, low molecular factors which also seem to be essential remain still unknown, although only retinoic acid has become such a candidate. Cotylenins had been isolated as the plant-growth regulators, and have been found to affect a number of physiological processes of higher plants. Here we report that at the concentrations above 12.5 microg/ml (20 microM) cotylenin A induced the functional and morphological differentiation in murine (M1) and human myeloid leukemia (HL-60) cells. Although cotylenin A has some similarity to PMA both in carbotricyclic diterpene structure and in biological activity (i.e. differentiation-induction of HL-60 cells into macrophages), the activation of PKC and the elevation of Ca2+-levels by cotylenin A were not observed. Quite recently it has been reported that fusicoccin (closely related to cotylenin A)-targets are 14-3-3 proteins, which are at the crosspoint of a huge array of signalling and regulatory pathways. These results suggest that cotylenin A might become a useful tool for the elucidation of molecular mechanisms of differentiation and development.
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PMID:Cotylenin A, a plant-growth regulator, induces the differentiation in murine and human myeloid leukemia cells. 932 63

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.
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PMID: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. 934 31

Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.
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PMID:Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11. 936 53

14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (alpha to theta) of 14-3-3 protein are known, but the zeta subtype of this protein has been cloned only in mammals. We cloned the zeta subtype of 14-3-3 protein (14-3-3 zeta) from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3 zeta s, and 92% with human phospholipase A2 (PLA2; 14-3-3 zeta). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3 zeta mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3'-untranslated region (3'-UTR) was observed between frog and human 14-3-3 zeta cDNA. The results suggest that 14-3-3 zeta is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3'-UTR of 14-3-3 zeta cDNA may be conserved in frogs and humans.
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PMID:Molecular cloning of cDNA for the zeta isoform of the 14-3-3 protein: homologous sequences in the 3'-untranslated region of frog and human zeta isoforms. 945 Mar 88

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.
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PMID:14-3-3 proteins in neuronal development and function. 962 66

Ras-mediated signaling pathways play a critical role in cellular proliferation and differentiation. Although it has been demonstrated that Ras interacts with Raf-1 to stimulate the serine/threonine kinase activity of Raf-1, the precise mechanism by which Ras activates Raf-1 remains obscure. To address this question, we developed a cell-free system in which the activated form of H-Ras can induce Raf-1 activation. Using this system, we found the presence of a new protein factor, in cytosolic fractions of both human embryonic kidney 293 cells and rat brain tissues, that is required for Ras-dependent activation of Raf-1. The factor was purified from rat brain cytosols through successive column chromatographies on DEAE Sephacel, SP Sepharose and Sephacryl S-300. The approximate molecular weight of the activator was estimated as 400,000 by gel filtration. Its activity was sensitive to heat and trypsin treatments. The purified activator did not contain Src, 14-3-3, protein kinase C, JAK2 or Ksr-1, as judged by immunoblotting. Further characterization of the activator is underway.
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PMID:Isolation of a new protein factor required for activation of Raf-1 by Ha-Ras: partial purification from rat brain cytosols. 965 45

Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent phosphorylation of specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or induce conformational changes that alter 14-3-3 association with other kinases involved in signal transduction.
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PMID:A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein. 970 22


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