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 catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6-8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (less than 5 micrograms/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 microM and Vmax of 20 mumol X min-1 X mg-1, corresponding values with mixed histones were 12 microM and 1.2 mumol X min-1 X mg-1. With both protein substrates the apparent Km for ATP was 11 microM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7-2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [gamma-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.
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PMID:Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue. 715 5

A rice (Oryza sativa) seed plasma-membrane calcium-dependent serine/threonine protein kinase (CDPK) has been partially purified. Comparing results in seeds that were treated with and without the plant hormone gibberellin (GA) for 10 min showed that rice CDPK was highly induced by GA. After separating solubilized membrane proteins by sodium dodecyl sulfate-gel electrophoresis, followed by renaturation, a radiolabeled phosphoprotein band of approximately 58 kD was detected, and it was apparently produced by autophosphorylation. There are five aspects of the rice CDPK that show similarity to mammalian protein kinase C (PKC) and to other plant CDPKs: (a) Histone IIIS and PKC peptide-ser25 (19-31) are phosphorylated by rice CDPK. (b) The phosphorylation reaction is strictly dependent on calcium. (c) The activity of the rice CDPK is inhibited by either staurosporine or the PKC inhibitory peptide (19-36). (d) Addition of calmodulin has no effect on the activity of the enzyme; however, the CDPK is inhibited by the calmodulin antagonists trifluoperazine and W-7. (e) The rice CDPK reacts with a mammalian anti-PKC antibody in immunoblotting analysis. However, there is one major difference between the rice CDPK and other CDPKs: the rice CDPK is induced by GA, whereas no mammalian PKC or other plant CDPKs are known to be induced by any hormone.
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PMID:A rice membrane calcium-dependent protein kinase is induced by gibberellin. 761 Jan 67

Mitogen-activated protein kinase (MAP kinase) plays a role in the cascade of protein kinase activation in cultured cells. To investigate the involvement of MAP kinase in meiotic maturation, we measured MAP kinase activity, using myelin basic protein as a substrate, with histone H1 kinase activity, in mouse oocytes. MAP kinase activity was low 1 h after isolation from follicles (when oocytes lost their germinal vesicle), increased abruptly at 2 h, and remained high until the second metaphase (13 h after isolation from follicles). Histone H1 kinase activity increased gradually from 2 to 7 h after isolation. When immature oocytes were treated with puromycin, MAP kinase activity did not increase after isolation from follicles. In the presence of 3-isobutyl-1-methylxanthine, the treatment of immature oocytes with okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, induced germinal vesicle breakdown and activation of MAP kinase. These results suggest that MAP kinase is involved in the regulation of meiotic maturation, and that the activation of MAP kinase requires protein synthesis and is inhibited by the protein phosphatase during meiotic maturation in mouse oocytes.
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PMID:Activation of mitogen-activated protein kinase during meiotic maturation in mouse oocytes. 768 86

A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.
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PMID:Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation. 782 30

The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57 and the whole triplet Glu59-Asp-Glu61. The activity of CK2 holoenzyme, either native or reconstituted, toward calmodulin can be elicited by a variety of polybasic effectors, including polylysine, polyarginine, salmine, and histones H4, H3, and, to a lesser extent, H2a and H2b. Histone H1 and polyamines are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate. 815 51

Maturation promoting factor (MPF) is universally recognized as the biological entity responsible for driving the cell cycle from G2- to M-phase. Histone H1 kinase activity is widely accepted as a biochemical indicator of p34cdc2 protein kinase complex activity and therefore MPF activity. In this paper we present results which indicate that during the G2- to M-phase transition in mouse oocytes the dynamic of p34cdc2 related histone H1 kinase activity differs markedly from the biological activity of MPF as measured by classical cell fusion procedures. MPF is activated just before germinal vesicle breakdown (GVBD) whereas histone H1 kinase is activated 5-7 h later coincident with the formation of the definitive first metaphase plate. The biological activity of MPF is merely reduced to about 50% of control levels by a short period of protein synthesis inhibition (1-2 h) and completely suppressed after a prolonged period of inhibition (4-5 h). By contrast, inhibition of protein synthesis in mouse oocytes results in a rapid and complete suppression of histone H1 kinase activity. Therefore, biological MPF and histone H1 kinase activity should not be used in an interchangeable manner during the G2- to M-phase transition in mouse oocytes.
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PMID:Kinetics of MPF and histone H1 kinase activity differ during the G2- to M-phase transition in mouse oocytes. 818 3

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.
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PMID:cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization. 834 7

Histone H1 is highly phosphorylated in mitotic HeLa cells, but is quickly dephosphorylated in vivo at the end of mitosis and in vitro following cell lysis. We show here that okadaic acid and microcystin-LR block the in vitro dephosphorylation of H1 and that they do so directly by inhibiting the histone H1 phosphatase rather than by some indirect mechanism. The concentrations of microcystin and okadaic acid required for inhibition strongly suggest that the histone H1 phosphatase is either PP1 or an unknown protein phosphatase with okadaic acid-sensitivity similar to PP1. The histone H1 phosphatase is predominantly located in chromosomes with at most one copy for every 86 nucleosomes. This tends to support its identification as PP1, since localization in mitotic chromosomes is a characteristic of PP1 but not of the other known okadaic acid-sensitive protein phosphatases. We also show that treatment of metaphase-arrested HeLa cells with staurosporine and olomoucine, inhibitors of p34cdc2 and other protein kinases, rapidly induces reassembly of interphase nuclei and dephosphorylation of histone H1 without chromosome segregation. This result indicates that protein kinase activity must remain elevated to maintain a mitotic block. Using this as a model system for the M- to G1-phase transition, we present evidence from inhibitor studies suggesting that the in vivo histone H1 phosphatase may be either PP1 or another phosphatase with similar okadaic acid-sensitivity, but not PP2A.
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PMID:Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A. 879 31

Phosphorylation of buffalo sperm chromatin proteins under optimum conditions (8 mM Mg2+, pH 8.0, and at 30 degrees C) using [gamma-32P]ATP and endogenous protein kinase activity was linear for 15 min incubation time and up to 330 micrograms protein. The 32P transferred from [gamma-32P]ATP was located in protein as a phosphoester bond. Fractionation with 1.2 M NaCl-4 M urea-0.2 M 2-mercaptoethanol-1 mM PMSF followed by acid treatment solubilized 87% of the total chromatin proteins termed "sperm histones." The remaining 21% nonhistone protein was tightly bound to DNA. Follow-up of the label showed 91% of the 32P in sperm histone and 9% with DNA-associated proteins. Histone kinase activity was solubilized with 0.35 M NaCl, which extracted 70% of the initial enzyme activity associated with chromatin. Of the different histones tested as substrates, histone kinase phosphorylated only histone H3 and, therefore, is highly specific for arginine rich histone. It also phosphorylates the acidic protein, casein. Cyclic AMP at concentrations up to 50 microM had no effect on the phosphorylation of histone H3. Phosphoamino acid analysis using histone H3 as the substrate showed serine to be the acceptor amino acid for phosphoester link.
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PMID:Histone kinase activity of buffalo sperm chromatin. 914 Jun 15

Cyclin-dependent kinase 5 (CDK5) is the 34 kDa catalytic subunit of a recently characterized neuronal cdc2-like protein kinase which appears to be involved in regulation of the neurocytoskeleton. Using the rat postdecapitative model, the effect of brain ischemia on histone H1 and tau protein CDK5 phosphorylating activity was examined. Histone H1 kinase activity increased in both cytosolic and particulate fractions of the hippocampus and neocortex after 5 min and 15 min of ischemia, then declined to control levels. CDK5 tau protein phosphorylating activity increased after 15 min ischemia; however, no electrophoretic shifts or changes in radiodensity of the tau bands were observed autoradiographically. On Western blot analysis, the CDK5 protein band did not change after 25 min ischemia, despite the increase and subsequent decline in enzyme activity. These data demonstrate a postischemic increase in CDK5 activity, an associated increase in CDK5 tau phosphorylating activity and a decline in activity in the absence of massive proteolysis. CDK5 appears to play a role in the events associated with neuronal response to ischemic injury.
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PMID:Cyclin-dependent protein kinase 5 activity increases in rat brain following ischemia. 930 12


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