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)

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism.
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PMID:Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes. 184 60

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.
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PMID:Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells. 184 70

Homogenates prepared from the temporal cortex and hippocampus of individuals who had histopathologically confirmed Alzheimer's disease exhibited reduced in vitro cyclic AMP-dependent phosphorylation of synapsin I, neuronal phosphoprotein. One specific phosphorylation site (site 1) was affected while two other sites, which are phosphorylated by calcium/calmodulin kinase II, exhibited no such differences. Other phosphoproteins such as pyruvate dehydrogenase, did not show these differences. The reductions were not observed in either cerebellum or thalamus of Alzheimer's disease brain. Analysis by immunoblots indicated that the reductions were not caused by a decrease in absolute amounts of the protein. The reduced AD synapsin I phosphorylation was not overcome by the addition of purified cyclic AMP-dependent protein kinase. No differences were detected in total cyclic AMP-dependent protein kinase activity between the control and Alzheimer samples. However, dephosphorylation of the synapsin I prior to the in vitro phosphorylation reversed the differences observed between the control and AD homogenates. Thus, the reduced in vitro phosphorylation of the synapsin I in the Alzheimer homogenate reflects a reduced phosphorylatability of the protein due to either an increased phosphate content or some other alteration of the phosphorylation site.
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PMID:Reduced in vitro phosphorylation of synapsin I (site 1) in Alzheimer's disease postmortem tissues. 185 67

The aim of this study was to demonstrate the presence of calmodulin-stimulated protein kinase II, protein kinase C, and cyclic AMP-stimulated protein kinase in isolated myenteric ganglia and to characterize the major ganglia phosphoproteins using biochemical and immunochemical techniques. Ganglia from the small intestine of guinea-pigs were isolated, disrupted by sonication in Triton X-100, and phosphorylated. The phosphoprotein patterns obtained were compared with those of synaptosomes from guinea-pig and rat cerebral cortex. Myenteric ganglia were as rich in protein kinase C and cyclic AMP-stimulated protein kinase as brain tissue, but the level of calmodulin-stimulated protein kinase II was relatively lower. The alpha subunit of calmodulin-stimulated protein kinase II was detected by immunoblotting and the beta subunit by autophosphorylation. The ratio of beta to alpha subunit was considerably higher in ganglia than in brain and ganglia beta subunit had a lower apparent molecular weight than the brain enzyme. A number of neuronal phosphoproteins were found in ganglia including the 87,000 mol. wt phosphoprotein, synapsins 1a and 1b, and proteins IIIa and IIIb. A phosphoprotein of 48,000 mol. wt had many of the characteristics of the B-50 protein but was not the same. In addition, a number of other phosphoproteins not previously identified in neurons were found in ganglia including those with apparent molecular weights of 60,000 and 58,000 that were the major calmodulin kinase substrates. The guinea-pig enteric nervous system has been extensively studied but, unlike other parts of the mammalian nervous system, little is known about the intracellular mechanisms underlying its functions. A technique for isolating myenteric ganglia is now available and we have used this preparation to characterize the major protein kinase and phosphoproteins present in this tissue. The results obtained will allow the phosphorylation of the various proteins to be investigated after physiological or pharmacological manipulation of myenteric ganglia in situ and in vivo.
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PMID:Protein phosphorylation in guinea-pig myenteric ganglia and brain: presence of calmodulin kinase II. protein kinase C and cyclic AMP kinase and characterization of major phosphoproteins. 185 Dec 58

The Drosophila developmental antigen recognized by the monoclonal antibody F7D6 is expressed in dividing embryonic and imaginal cells but is lost from all differentiating tissues except electrogenic cells of the nervous system and spontaneously contracting muscles. The 63 kDa antigen is associated with the inner surface of plasma membranes and is expressed in several classes of tumorous mutants of Drosophila. The monoclonal antibody was used for immunoprecipitating the antigen for biochemical characterization and for screening expression vector cDNA libraries. Here we report that this oncodevelopmental antigen is a phosphoprotein and a serine-threonine specific protein kinase. A 1.6 kb cDNA isolated by immunological screening of an ovarian library hybridized to a single band on polytene chromosomes, localizing the gene to 72F on the left arm of the third chromosome. Immunofluorescence assays of deficiency stocks in the region confirmed the location of the gene and identity of the cDNA clone, and mapped the gene between the left breakpoints of Df(3L) st100.62 and Df(3L) stj7, i.e., between 72F3-7 and 73A1-2. The biochemical and genetic properties indicate that this is a novel growth-related kinase of Drosophila.
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PMID:Kinase activity and genetic characterization of a growth related antigen of Drosophila. 186 24

Skeletal-muscle sarcoplasmic reticulum (SR) comprises two distinct domains, corresponding to the free membrane of longitudinal SR (LSR) and the junctional membrane region of the terminal cisternae (TC), respectively. The junctional membrane contains the ryanodine receptor (RyR)/Ca(2+)-release channel and additional minor protein components that still require biochemical investigation, in relation to excitation-contraction coupling. Recent findings suggested the involvement in this process of a 170 kDa protein [Kim, Caswell, Talvenheimo & Brandt (1990) Biochemistry 29, 9281-9289], also characterized as a phosphoprotein in junctional TC in independent studies [Chu, Submilla, Inesi, Jay & Campbell (1990) Biochemistry 29, 5899-5905]. We show that this protein is a specific substrate of exogenous cyclic AMP-dependent protein kinase, that it is exposed to the outer surface of intact TC vesicles, and that it co-localizes with the RyR to the junctional membrane. Comparative analysis of LSR and TC subfractions for the 160 kDa glycoprotein sarcalumenin, using Western-blot techniques and specific monoclonal antibodies or concanavalin A as a ligand, revealed that the distribution of this protein within the SR corresponds inversely to both that of the RyR and of the 170 kDa protein. The 170 kDa protein, like sarcalumenin, stains blue with the cationic dye Stains-All and binds 45Ca2+ on blots, but it is uniquely distinguished by its ability to bind 125I-labelled low-density lipoprotein. The similarity of these properties, as well as the pI and solubility properties, to those described for the SR protein, recently purified and cloned and named histidine-rich Ca(2+)-binding protein [HCP; Hofmann, Brown, Lee, Pathak, Anderson & Goldstein (1989) J. Biol. Chem. 264, 8260-8270], makes it very likely that our protein and HCP may indeed be identical. The protein described in the present study differs from sarcalumenin because its migration in SDS/PAGE is accelerated in the presence of Ca2+, a previously reported property of other Ca(2+)-binding proteins [leMaire, Lund, Viel, Champeil & Moller (1989) J. Biol. Chem. 265, 1111-1123], arguing for Ca(2+)-induced protein-conformational changes. Kinase-dependent phosphorylation of our protein is another distinguishing feature, which, although not previously reported for HCP, is consistent with the presence of potential serine/threonine phosphorylation sites in the middle portion of the cloned HCP molecule. The finding that HCP, contrary to early views, selectively binds to the cytoplasmic side of the junctional membrane, together with its newly characterized properties, seem to provide new clues as to a possible role in electromechanical coupling and/or Ca2+ release.
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PMID:Subcellular fractionation to junctional sarcoplasmic reticulum and biochemical characterization of 170 kDa Ca(2+)- and low-density-lipoprotein-binding protein in rabbit skeletal muscle. 187 15

The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.
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PMID:Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. 190 79

In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987], the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.
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PMID:Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress. 196 22

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate), a phosphoprotein substrate for cAMP-dependent protein kinase, is unevenly distributed in adult rat brain. Using immunoblotting and phosphorylation in vitro followed by immunoprecipitation, ARPP-21 was found to be enriched in caudate-putamen, substantia nigra, nucleus accumbens and olfactory tubercle. Intermediate levels were found in cerebral cortex and hippocampus. ARPP-21 was very low in most other brain areas and was not detected in any of the peripheral tissues studied. Following unilateral lesion of the caudate-putamen with quinolinic acid, a marked decrease in the levels of ARPP-21 was observed in both the lesioned caudate-putamen (-75%) and the ipsilateral substantia nigra (-70%) compared with the unlesioned side. This result demonstrates the enrichment of ARPP-21 in striatonigral neurons. In slices of caudate-putamen, substantia nigra or cerebral cortex incubated in vitro, the phosphorylation of ARPP-21 was enhanced by 8-Br-cAMP, a stable analog of cAMP. In striatal slices, forskolin, a compound which stimulates adenylate cyclase directly, enhanced the phosphorylation of ARPP-21 with an EC50 of 0.5 microM. In conclusion, ARPP-21 is a neuron-specific phosphoprotein enriched in specific brain areas which are known to receive a rich dopaminergic innervation and to contain high levels of D1 dopamine receptors. The phosphorylation of ARPP-21 is likely to mediate some of the intracellular effects of neurotransmitters which stimulate adenylate cyclase in these regions, in particular dopamine and vasoactive intestinal peptide.
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PMID:ARPP-21, a cAMP-regulated phosphoprotein enriched in dopamine-innervated brain regions: tissue distribution and regulation of phosphorylation in rat brain. 196 23

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.
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PMID:Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein. 196 66


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