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)

A protein phosphokinase (EC 2.7.1.1.37) was isolated from baker's yeast (Saccharomyces cerevisiae) after a 17,000-fold purification; the purified enzyme is homogeneous according to the criteria of gel electrophoresis and ultracentrifuge analysis. The enzyme has a high isoelectric point of ca. 9 and appears to exist as a monomer with a molecular weight of 42,000 plus or minus 1500. It is neither stimulated by cyclic 3',5'-AMP, -GMP, -CMP or -ump nor inhibited by the regulatory subunit of rabbit muscle protein kinase (Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971), J. Biol. Chem. 246, 1986). In the presence of divalent metal ions, preferably Mg-2+ or Mn-2+, the enzyme readily transfers the terminal phosphate group of ATP to phosvitin, alphaS1B- and beta a-casein and an NH2-terminal tryptic peptide derived from beta a-casein, but not to protamine, lysine, or arginine-rich histones or to yeast enzymes such as phosphorylase, phosphofructokinase, or pyruvate carboxylase; serine and polyserine were also inactive as phosphate acceptors. Km values of 0.17 mM for beta a-casein and 0.2 mMfor ATP were determined at 10 mM Mg-2+. The urified yeast protein kinase also catalyzes the reverse reaction, namely, the transfer of phosphate from fully phosphorylated beta a-casein or its NH2-terminal peptide to ADP resulting in the formation of ATP. AMP, GDP, UDP, and CDP did not serve as phosphate acceptors in this reaction. As observed by Rabinowitz and Lipmann (Rabinowitz, M., and Lipmann, F. (1960), J. Biol. Chem. 235, 1043) both reactions have different pHoptima with values of 7.5 for the forward reaction (phosphorylation of the proteins) and ca 5.2 for the formation of ATP; both are differently affected by salts. Phosphorylation of beta a-casein with [gamma-32-P]ATP followed by digestion of the labeled protein with trypsin indicated that all the radioactivity was exclusively introduced in an NH2-terminal peptide possessing the unique sequence: Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu...(Ribadeau-Dumas, B., Brignon, G., Grosclaude, F., and Mercier, J.-C. (1971), eur J. Biochem. 20, 264). By subjecting beta a-casein and its NH2-terminal peptide to the combined action of almond acid phosphatease and purified yeast protein kinase, it was determined that the phosphorylation and dephosphorylation reactions proceed randomly, i.e., all seryl phosphate residues are equally susceptible and that the rate of phosphorylation decreases drastically as the number of bound phosphate groups in the substrate diminishes.
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PMID:Purification and properties of a yeast protein kinase. 23 75

Involvement of serine protease-activation in the generation of cytoplasmic factor(s) that induced NHP-specific protein kinase activity in nuclei in anti-Ig-stimulated cells was described. DFP or PMSF with anti-Ig inhibited the induction of cytoplasmic factor(s), whereas pretreatment of cells with DFP or PMSF without anti-Ig did not show any inhibitory effect on anti-Ig-induced generation of cytoplasmic factor(s). TAME or BAME with anti-Ig inhibited the generation of cytoplasmic factor(s) and the simultaneous addition of TAME or BAME with DFP protected the generation of cytoplasmic factor(s) against the inhibitory effect of DFP, showing the involvement of trypsin-like, arginine-type serine protease in anti-Ig-induced generation of cytoplasmic factor(s). Anti-Ig-stimulated membrane preparations induced cytoplasmic factor(s) in normal cytoplasm. The m.w. of precursor proteins present in resting B cells and active cytoplasmic factor(s) were approximately 150,000 and 45,000, respectively. These results showed that anti-Ig-activated membrane-bound serine protease split precursor proteins in resting B cells into active cytoplasmic factor(s) responsible for signal transmission.
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PMID:Involvement of anti-Ig-activated serine protease in the generation of cytoplasmic factor(s) that are responsible for the transmission of Ig-receptor-mediated signals. 31 62

When vaccinia-virus-infected cells were labeled with radioactive phosphate in the absence of viral gene expression an additional phosphoprotein, containing phosphoserine, was found specifically associated with the ribosomes. The phosphoprotein was removed from the ribosomes following a 0.5 M KCl washing or after EDTA treatment. This additional phosphoprotein was found in infected cells after either a long (3-4 h) or a short (30 min) labeling period; it was detected when the infected cells were incubated in the presence or absence of an inhibitor of RNA or protein synthesis. This phosphoprotein originated from the phosphorylation of vaccinia virion structural protein VP11b (Mr 11,000) at a specific site since only a single major phosphopeptide was obtained after trypsin digestion. This phosphoprotein was also present in purified vaccinia virions labeled with radioactive phosphate. VP11b protein was phosphorylated in vitro by the protein kinase associated with the cores. When the reaction was carried out at an alkaline pH the phosphorylation in vitro occurred at different sites in the protein; at neutral pH the phosphorylation of VP11b was more specific and, as judged by tryptic peptide analysis, occurred mainly at the same site as in the phosphorylation in vivo. A role for the involvement of phosphoprotein VP11b in the establishment of the shut off of host protein synthesis by vaccinia virus is suggested.
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PMID:Phosphorylation in vivo of a vaccinia-virus structural protein found associated with the ribosomes from infected cells. 46 37

Incubation of purified skeletal muscle glycogen synthetase I with trypsin (10 mug/ml) for 15 min decreased the Stokes radius of the enzyme from 68 A to 62 A and the subunit molecular weight from 90,000 to about 73,000. No decrease in the sedimentation coefficient of 13.3 S could be detected. It was calculated that native synthetase I is a tetramer of molecular weight 360,000 to 370,000. Trypsin also catalyzed a decrease in the synthetase activity ratio (minus glucose-6-P to plus glucose-6-P) largely by reducing minus glucose-6-P activity. The magnitude of the trypsin effect on the synthetase activity ratio was very similar to that produced by phosphorylation of synthetase by the cyclic AMP-dependent protein kinase. The activity ratio could be lowered from that characteristic of synthetase I, 0.85, to 0.25 either by trypsin digestion or by incorporation of 1 mol of Pi per mol of synthetase subunit. An activity ratio of less than 0.05 could be obtained in three ways as follows, (a) phosphorylation to 2 Pi/subunit, (b) phosphorylation to 1 Pi/subunit (ratio=0.25) followed by trypsin treatment, (c) trypsin treatment (ratio=0.25) followed by phosphorylation. When trypsinized synthetase was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, 1 Pi/subunit was incorporated. Trypsin (6 mug/ml) led to a rapid release of about 50% of the radioactivity from 32P-synthetase regardless of whether the enzyme contained 1 or 2 phosphates per subunit. It was concluded that two sites on the enzyme subunit are highly susceptible to phosphorylation catalyzed by the cyclic AMP-dependent protein kinase. The data indicate that the 1st mol of Pi incorporated is distributed about equally between the two sites. A model is proposed to account for these observations. The data further indicate that a peptide containing one site is removed by trypsin. This reduces enzyme activity to the same extent as does phosphorylation of the site in the intact protein.
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PMID:Regulation of glycogen synthetase. Effects of trypsin on the structure, activity, and phosphorylation of the skeletal muscle enzyme. 81 39

The effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.
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PMID:Effect of thrombin on phosphorylation of platelet membrane proteins. 98 70

Phosphorylase kinase was activated 5--10-fold in vivo by an intravenous injection of adrenalin. Sodium fluoride an inhibitor of phosphorylase kinase phosphatase, was required to prevent the reversal of this process; the activated and non-activated forms of the enzyme were indistinguishable by dodecylsulphate gel electrophoresis. This suggested that the activation had resulted from a phosphorylation of the enzyme, and that it was not a consequence of the well known activation by proteolytic cleavage that can be demonstrated in vitro. Phosphorylase kinase activated in vivo was purified and digested with trypsin, and the two tryptic peptides which contain the serine residues which are phosphorylated in vitro by the action of cyclic-AMP (adenosine 3':5'-monophosphate) dependent protein kinase, were isolated. It was found that the same nine-amino-acid segment of the beta chain and the same seven-amino-acid segment of the alpha chain had become phosphorylated in vivo in response to adrenalin, as were phosphorylated in vitro. The degree of phosphorylation of each of the two sites was at least 50%. The data provide direct proof that the activation of phosphorylase kinase which occurs in vivo in response to adrenalin results from a phosphorylation of the enzyme. They also indicate that the novel form of regulation associated with the phosphorylation of the alpha subunit, the stimulation of protein dephosphorylation by "second site phosphorylation", can now be regarded as a new form of enzyme control mechanism which operates in vivo. The regulation of phosphorylase kinase activity was studied in the protein - glycogen complex from skeletal muscle. The enzyme could be rapidly converted to a phosphorylated form in a cyclic-AMP-stimulated reaction upon addition of magnesium ions and ATP, but the conversion of phosphorylase b to phosphorylase a in the complex still showed an absolute requirement for calcium ions. The implications of these findings and major problems in the hormonal control of skeletal muscle glycogenolysis which are not yet resolved, are discussed.
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PMID:The hormonal control of activity of skeletal muscle phosphorylase kinase. Phosphorylation of the enzyme at two sites in vivo in response to adrenalin. 112 18

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.
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PMID:The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles. 130 52

The ATP.Mg-dependent type-1 protein phosphatase and its activating factor (protein kinase FA) were identified to exist in brain synaptosome. The inactive protein phosphatase was found to exist in the synaptosomal cytosol whereas its activating factor (protein kinase FA) was present in the synaptosomal membrane, indicating that the inactive protein phosphatase and its activating factor FA are localized in two separate subcellular compartments. The membrane-bound FA was found to exist in two forms; approximately 75% of FA is inactive and trypsin-resistant, whereas 25% of FA is active and trypsin-labile. When membranes were incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be activated and sequestered to become the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, the results provide initial evidence that the activation-sequestration of membrane-bound protein kinase FA may represent one mode of control modulating the activity of protein kinase FA and thereby to activate protein phosphatase in brain synaptosome, representing an efficient regulatory mechanism for regulating neurotransmission in the central nervous system.
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PMID:The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes. 131 12

We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99-126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition (approximately 45%) was observed in the presence of 10-30 microM GTP gamma S, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na+ enhanced the degree of inhibition by about 60%, whereas K+ and Li+ suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn2+ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 mM, and at 2 mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A2 treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca(2+)-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.
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PMID:Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase. 132 94

Resealed human red cell ghosts were loaded with Fura-2, ATP, Mg2+, and either calmodulin (CaM) or, to prevent CaM activation of the Ca2+ pump, a synthetic peptide that antagonized endogenous CaM (an analogue of the CaM binding domain of protein kinase II, referred to as 'antiCaM'). The ghosts reduced the cytosolic concentration of ionized calcium ([Ca2+]i) to 193 +/- 60 nM (SD, n = 15) in a medium containing 1 mM Ca2+ and to 30 +/- 27 nM (SD, n = 62) in a medium without Ca2+ addition. Without ATP, i.e. no fuelling of the Ca2+ pump, the [Ca2+]i remained high (approx. 5 microM or higher). The simultaneous addition of the ionophore A23187 and Ca2+ rapidly increased the Ca2+ influx, which in the CaM loaded ghosts caused a solitary spike of [Ca2+]i, reaching maximum around 2 microM within 24 +/- 6 s (SD, n = 40). On the contrary, in the ghosts loaded with antiCaM, the addition of A23187 with Ca2+ raised [Ca2+]i during the first 2 min to a high level (2-4 microM) with no preceding spike. Pre-incubation of CaM-ghosts with Ca2+ diminished the height of the Ca2+ spike, and treatment with trypsin even removed the Ca2+ spike. The trypsin treatment activated the Ca2+ pump prior to the rise of [Ca2+]i, making the time-consuming CaM activation unnecessary. In conclusion, the Ca2+ spiking is dependent on a delayed CaM activation of the plasma membrane Ca2+ pump in response to a rapid increase of Ca2+ influx.
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PMID:Solitary calcium spike dependent on calmodulin and plasma membrane Ca2+ pump. 133 11


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