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)

In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.
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PMID:Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells. 132 34

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.
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PMID:Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues. 132 62

We have performed large-scale random oligonucleotide insertion mutagenesis on a 41-kbp genomic segment derived from the unique long (UL) region of the alphaherpesvirus pseudorabies virus (PRV). This procedure has resulted in the generation of a series of PRV strains, each carrying a single gene whose termination of translation is induced by the inserted oligonucleotide. To relate the genes that were involved in the mutagenization to genes previously identified in herpes simplex virus type 1, the prototype alphaherpesvirus, we have performed cross-hybridization studies. In this way, we have mapped the location of the homolog of a gene which was described to have sequence characteristics of a eukaryotic phosphotransferase. We characterized the phenotype of a mutant PRV strain lacking this putative phosphotransferase also the phenotype of a PRV strain lacking, in addition to the UL-encoded putative phosphotransferase, the protein kinase encoded within the unique short region of the virus. To assess the enzymatic activity of the UL region-encoded phosphotransferase, we expressed the gene transiently in a eukaryotic expression system. Immunoprecipitation of the protein followed by kinase assays and phosphoamino acid analyses revealed protein-serine/threonine kinase activity. Implications of sequence divergence of this protein from classical protein-serine/threonine kinases for kinase structure and function are discussed in view of the recent resolution of the structure of the catalytic domain of cyclic AMP-dependent protein kinase.
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PMID:Herpesviruses encode an unusual protein-serine/threonine kinase which is nonessential for growth in cultured cells. 132 89

The hepatocyte nuclear factor 3 (HNF-3) gene family is composed of three proteins (alpha, beta, and gamma) that are transcription factors involved in the coordinate expression of several liver genes. All three proteins share strong homology in their DNA binding domains (region I) and are able to recognize the same DNA sequence. They also possess two similar stretches of amino acids at the carboxyl terminus (regions II and III) and a fourth segment of homology at the amino terminus (region IV). Furthermore, the HNF-3 proteins demonstrate homology with the Drosophila homeotic gene fork head in regions I, II, and III, suggesting that HNF-3 may be its mammalian homolog. In order to define HNF-3 beta protein domains involved in transcriptional activation, we have used a reporter gene, whose transcription is dependent on HNF-3 binding, for hepatoma cell cotransfection assays with expression vectors that produced different truncated HNF-3 beta proteins. A position-independent activation domain which contained conserved regions II and III was identified at the carboxyl terminus of the HNF-3 beta protein (amino acids 361 to 458). Moreover, site-directed mutations that altered the sequences within regions II and III demonstrated their importance to transactivation. The region II-III domain does not possess amino acid sequences in common with other transcription factors and may define a novel activation motif. HNF-3 beta amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-III domain. Region IV is abundant in serine amino acids and contains two putative casein kinase I phosphorylation sites, a feature similar to protein motifs described for the transcription factors Pit-1/GHF-1 and HNF-1.
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PMID:Hepatocyte nuclear factor 3 beta contains two transcriptional activation domains, one of which is novel and conserved with the Drosophila fork head protein. 132 4

Microtubule-associated protein (MAP) kinases form a group of serine/threonine kinases stimulated by various growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 kinase. To further evaluate the interactions between the different components of this cascade, we looked at the possible coprecipitation of MAP kinase activator(s) or MAP kinase substrate(s) with MAP kinase. Using antipeptides to the C terminus of the M(r) 44,000 MAP kinase, ERK1, and cell extracts from unstimulated or NGF-treated PC12 cells, we obtained in addition to MAP kinase itself coprecipitation of a protein with a M(r) in the 90,000 range. We further show that this protein is a protein kinase since it becomes phosphorylated on serine residues, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phosphorylation performed before sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates NGF-sensitive phosphorylation of this 90-kDa protein on both serine and threonine; the serine phosphorylation is likely to be due to autophosphorylation, and the threonine phosphorylation due to phosphorylation by the copurifying MAP kinase. Furthermore, immunoprecipitation of this 90-kDa protein was obtained with antibodies to S6 kinase II. Finally, using in situ chemical cross-linking, we were able to demonstrate in intact cells the occurrence of an anti-ERK1 immunoreactive species with a molecular mass of approximately 125,000 compatible with a complex between ERK1 and a 90-kDa S6 kinase. Taken together, our observations demonstrate that the 44-kDa MAP kinase is associated, in intact PC12 cells, with a protein kinase which is very likely to be S6 kinase II. In conclusion, our data represent strong evidence for a physiological role of the MAP kinase-S6 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a powerful tool to search for additional physiologically relevant substrates for MAP kinase, a key integrator enzyme for growth factors and hormones.
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PMID:Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1. 132 33

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.
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PMID:Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. 132 38

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.
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PMID:The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase. 132 41

The phosphorylated state of the vesicular stomatitis virus phosphoprotein (P), an essential component of the virion-associated RNA polymerase complex, has been shown to be important for the transcriptional activity of the complex. Recent studies indicate that phosphorylation within the acidic domain of the P protein by cellular casein kinase II is necessary for its activity. In an attempt to identify the exact location of the cell kinase-mediated phosphorylation, we altered specific serine and threonine residues within the acidic domain of the New Jersey serotype of P protein by site-directed mutagenesis. The altered P proteins were then tested to determine what effect these mutations had on the phosphorylated state of the protein in vivo as well as its transcriptional activity in vitro. We report that serine residues 59 and 61 within the acidic domain of the P protein must be phosphorylated for it to be functionally active in a reconstituted transcription assay. These results demonstrate the importance of site-specific phosphorylation in the transcriptional activity of a negative-strand RNA viral phosphoprotein and the crucial role played by a cell protein kinase in this process.
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PMID:Phosphorylation of specific serine residues within the acidic domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro. 132 45

Mitogen-activated protein (MAP) kinases are 42- and 44-kD serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation in cells stimulated with mitogens and growth factors. MAP kinase and the protein kinase that activates it (MAP kinase kinase) were constitutively activated in NIH 3T3 cells infected with viruses containing either of two oncogenic forms (p35EC12, p3722W) of the c-Raf-1 protein kinase. The v-Raf proteins purified from cells infected with EC12 or 22W viruses activated MAP kinase kinase from skeletal muscle in vitro. Furthermore, a bacterially expressed v-Raf fusion protein (glutathione S-transferase-p3722W) also activated MAP kinase kinase in vitro. These findings suggest that one function of c-Raf-1 in mitogenic signaling is to phosphorylate and activate MAP kinase kinase.
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PMID:Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. 138 11

The DNA sequence of the short (S) genomic component of the equine herpesvirus type 1 (EHV-1)KyA strain has been determined recently in our laboratory. Analysis of a 1353-bp BamHI/PvuII clone mapping at the unique short/terminal inverted repeat (Us/TR) junction revealed 507 bp of Us and 846 bp of TR sequences as well as an open reading frame (ORF) that is contained entirely within the Us. This ORF encodes a potential polypeptide of 219 amino acids that shows significant homology to the US9 proteins of herpes simplex virus type 1 (HSV-1), EHV-4, pseudorabies virus (PRV), and varicella zoster virus (VZV). The US9 polypeptides of the two equine herpesviruses exhibit 50% identity but are twice as large as their counterparts in HSV-1, PRV, and VZV. All five US9 proteins are enriched for serine and threonine residues and share a conserved domain of highly basic residues followed by a region of nonpolar amino acids. DNA sequence and Southern blot hybridization analyses revealed that the Us of EHV-1 KyA differs from the Us of EHV-1 KyD and AB1 in that the ORFs encoding glycoproteins I and E and a unique 10-kDa polypeptide are deleted from the KyA genome. These data demonstrate that the predicted 10-kDa protein unique to EHV-1 is nonessential for replication in vitro and that EHV-1 glycoproteins I and E, like their equivalents in HSV-1 and PRV, are also nonessential. These findings and those reported previously by this laboratory and others reveal that the Us segment of EHV-1 comprises nine ORFs, two of which, US4 and 10-kDa ORF, are unique to EHV-1. The gene order of the Us is US2, protein kinase, gG, US4, gD, gI, gE, 10 kDa, and US9.
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PMID:The equine herpesvirus type 1 (EHV-1) homolog of herpes simplex virus type 1 US9 and the nature of a major deletion within the unique short segment of the EHV-1 KyA strain genome. 132 5


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