Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phosphoprotein P gene of measles virus (Edmonston strain) has been cloned in the Escherichia coli expression vector pET-3a with a histidine tag at the C-terminal end. The expressed protein was soluble, unphosphorylated, and constituted 10 to 20% of total cellular protein. Recombinant P protein purified by Ni-affinity chromatography was found to be efficiently phosphorylated in vitro by recombinant casein kinase II (CKII) or by the CKII activity present in the uninfected cell extract. A comparison of phosphopeptide analyses between the in vivo- and the in vitro-32P-labeled P proteins revealed that both proteins share common phosphorylation sites. In an attempt to identify the exact site of the CKII-mediated phosphorylation, we altered specific serine residues located within the CKII consensus motif to alanine by site-directed mutagenesis. The results indicate that Ser 86, Ser 151, and Ser 180 located within the N-terminal half of the P protein are involved in the CKII-mediated phosphorylation of the P protein.
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PMID:Involvement of cellular casein kinase II in the phosphorylation of measles virus P protein: identification of phosphorylation sites. 764 14

Casein kinase-II (CK-II) is a widely distributed protein kinase, which plays numerous roles in the regulation of transcription through modification of transacting transcription factors. Phosphorylation of vesicular stomatitis virus (VSV) P protein by CK-II was found to be both necessary and sufficient for transcriptional activation. Upon treatment of P by CK-II, activity was acquired faster (t1/2 = 3.7 min) than were total phosphates (t1/2 = 7.4 min). Stoichiometry was 2 mol phosphate/mol P, indicating activation by phosphorylation at either one or both of two independent sites. The sites were identified by substituting aspartate (D) residues at either S60 or T62, producing proteins that were partly active without phosphorylation, but were fully active at higher concentrations; CK-II added only a single phosphate group to each of these, and conferred full activity. P protein doubly substituted with D at S60 and T62 was fully active without phosphorylation, and was not a substrate for CK-II. Active P protein, whether CK-II treated or doubly substituted, was shown by gel filtration and crosslinking to exist as a discretely multimeric, probably tetrameric, structure. The singly substituted mutants were partly multimeric, becoming fully so after CK-II treatment. Phosphorylation by CK-II thus mediates the self-association of P into the multimeric, transcriptionally active form.
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PMID:Multimerization and transcriptional activation of the phosphoprotein (P) of vesicular stomatitis virus by casein kinase-II. 772 Jul 14

Specific in vivo interaction between the phosphoprotein (P) and the large polymerase protein (L) from the Indiana serotype of vesicular stomatitis virus was studied using a two-hybrid system. Transfection of CHO cells with plasmids encoding GALPIND and VPLIND fusion proteins resulted in an easily detectable level of CAT activity, indicating that PIND and LIND associate in vivo in the absence of other viral proteins. Mutational studies of PIND demonstrated that both domains I and II of PIND are important for PIND-LIND association. In addition, casein kinase II (CKII)-mediated phosphorylation within domain I of PIND was necessary for efficient association with LIND. We have also used the two-hybrid system to show PIND interaction with NIND in vivo. PIND and NIND associated more strongly than PIND and LIND. A similar strong association was observed in heterologous interaction studies between Indiana and New Jersey serotype P and N proteins. Mutational studies of PIND demonstrated that, unlike what was found for PNJ-NNJ association, only the C-terminal region of the P protein was important for efficient association with NIND. Like PNJ, CKII-mediated phosphorylation within domain I of PIND was not required for P-N association and, like NNJ, the C-terminal five amino acids of the NIND protein were critical for P association with N. These results demonstrate the importance of phosphorylation and specific domains of the P protein in its interaction with the L and N proteins, which are necessary for viral transcription and replication, respectively.
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PMID:Efficient interaction of the vesicular stomatitis virus P protein with the L protein or the N protein in cells expressing the recombinant proteins. 774 58

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.
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PMID:Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication. 776 74

Chandipura (CHP) virus, a member of the vesiculovirus genus within the Rhabdoviridae family, was first isolated from human patients in India. The full length phosphoprotein P gene of CHP have been cloned and expressed in Escherichia coli using the T7 polymerase-based pET-3 series of expression vectors. Under optimal conditions of induction with IPTG, the recombinant P protein constituted 35% of the total bacterial protein. The bacterially expressed protein was found to be phosphate-free. Deletion analysis suggested that the anomalous mobility of the P protein was due to its high acidity. The expressed protein can be phosphorylated in vitro by the extracts prepared from baby hamster kidney cells or rabbit reticulocytes. The cellular kinase involved in phosphorylation appears to be casein kinase II.
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PMID:Cloning of the chandipura virus phosphoprotein encoding gene and its expression in Escherichia coli. 778 87

Protein kinase activities associated with a highly purified transcriptionally active ribonucleoprotein complex from the virions of vesicular stomatitis virus (VSV) were isolated and characterized. Based upon several biochemical and immunological criteria, the protein kinase activity, which phosphorylated the bacterially expressed unphosphorylated (Po) protein, was shown to be cellular casein kinase II (CKII). These studies included inhibition of the protein kinase by specific inhibitors, phosphorylation of mutant phosphoproteins (P), immunoprecipitation by CKII antibody and Western blot analyses, and finally its ability to activate Po to synthesize RNA in a transcription-reconstitution reaction. The P protein is phosphorylated intracellularly by cellular CKII. The present study demonstrates that VSV specifically packages CKII which remains strongly associated with the ribonucleoprotein complex during morphogenesis.
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PMID:Casein kinase II is the P protein phosphorylating cellular kinase associated with the ribonucleoprotein complex of purified vesicular stomatitis virus. 784 56

To determine which human respiratory syncytial virus (HRSV) P protein serine residues are modified by cellular protein kinase(s), several mutated versions of P protein were expressed in the absence of other viral proteins. Mutations at serines 232 or 232 and 237 drastically reduced the extent of phosphorylation P protein in vivo. Serine 232 is the main site of modification and is also essential for in vitro phosphorylation by casein kinase II. Additional in vivo phosphorylation was also detected in the region containing serines 116, 117 and 119.
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PMID:C-terminal phosphorylation of human respiratory syncytial virus P protein occurs mainly at serine residue 232. 784 63

The transcription complex of the human respiratory syncytial virus was biochemically dissected and reconstituted in vitro with purified viral macromolecules. The minimal complex consisted of the viral N-RNA template, viral phosphoprotein (P), and the large protein (L) along with host cellular factor(s), possibly actin. Active transcription could also be reconstituted using bacterially synthesized recombinant P protein provided the P protein was phosphorylated by cellular casein kinase II. Elimination of phosphorylation by inhibition of CKII or by mutation of the Ser residue at position 237 of the P protein also abrogated RSV transcription. In addition, the phosphorylation-defective P mutants exhibited a trans-dominant negative phenotype, consistent with the finding that the mutant proteins bound to the N-RNA template as efficiently as the wild type. Once engaged in transcription, however, the wild-type P protein became refractory to trans-inhibition by the mutant.
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PMID:Requirement of casein kinase II-mediated phosphorylation for the transcriptional activity of human respiratory syncytial viral phosphoprotein P: transdominant negative phenotype of phosphorylation-defective P mutants. 797 5

The phosphoprotein P gene of human respiratory syncytial virus has been cloned and the protein expressed in Escherichia coli. The expressed protein was soluble, unphosphorylated, and constituted approximately 10% of the total bacterial protein. Electrophoretic and antigenic analyses demonstrated the identity of the recombinant protein with viral P protein and P protein synthesized in reticulocyte lysates. Purified recombinant P protein was efficiently phosphorylated in vitro by purified native as well as recombinant casein kinase II (CKII) or by the CKII activity in uninfected cell extracts. Through deletions and site-directed mutagenesis, the site of CKII phosphorylation was mapped to a single serine residue (Ser237) near the C-terminal end of the P protein.
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PMID:Bacterial expression of human respiratory syncytial viral phosphoprotein P and identification of Ser237 as the site of phosphorylation by cellular casein kinase II. 797 41

The immunodominant epitope recognized by lupus antiribosomal P protein antibodies (anti-P antibodies) is located within the 11 C-terminal residues common to the three P proteins. This epitope contains a potential phosphorylation site for casein kinase II and clusters of acidic and hydrophobic amino acids. To determine the role of each of these features in antigen recognition, lupus anti-P sera were tested for binding to phospho- and dephospho- forms of the P proteins and to synthetic peptide antigens in which site-specific modifications had been introduced. Immunoblot analysis revealed that anti-P antibodies specific for the phospho- form of the P proteins represented only a minor population of anti-P antibodies and, in many cases, were absent altogether. In contrast, when charged substitutions were introduced into either the acidic or hydrophobic clusters and tested by ELISA, striking reductions of 64-86% were observed. Conservative Gly-->Pro substitutions also produced a 73% average reduction in anti-P binding whereas substitution of either Ser-105 or the C-terminal Asp-115 resulted in a < 35% reduction in binding. These findings suggest that phosphorylation of the P proteins does not play a role in antibody recognition but that anti-P antibodies require both the acidic and hydrophobic clusters for optimal binding to synthetic peptide antigens. The remarkable degree of specificity demonstrated by these antibodies supports the view that anti-P autoantibodies result from a highly specific (at the B cell level) immune response to self antigen.
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PMID:The effect of phosphorylation and site-specific mutations in the immunodominant epitope of the human ribosomal P proteins. 805 Feb 1


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