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)

To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Control of glycogenolysis and effects of exercise on phosphorylase kinase and cAMP-dependent protein kinase in rainbow trout organs. 819 87

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.
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PMID:Insulin does not induce the hydrolysis of a glycosyl phosphatidylinositol in rat fetal hepatocytes. 834 37

We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.
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PMID:Identification of a serine/threonine-specific protein phosphatase from the archaebacterium Sulfolobus solfataricus. 838 14

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.
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PMID:Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases. 840 Apr 54

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
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PMID:Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A. 855 May 70

To better understand the physical interaction between glycogen phosphorylase-b (P-b) and its only known kinase, phosphorylase kinase (PbK) and the relationship of this interaction to the activation of PbK, direct binding studies are necessary. By utilizing an enzyme-linked immunosorbent assay, a method was developed for measuring the binding of PbK to immobilized P-b under a variety of experimental conditions. A monoclonal antibody specific for the alpha subunit of PbK that had no effect on the phosphorylation of P-b by PbK or on the interaction of PbK with known effectors was used to detect PbK bound to plated P-b. Hyperbolic binding curves were obtained regardless of whether the concentration of Pbk or P-b was varied, and the assay detected changes in relative affinity caused by certain effectors of the kinase. The allosteric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant changes in its relative affinity for P-b; however, Ca2+ and Mg2+ ions, which also stimulate PbK, increased its affinity for P-b, with Mg2+ being more effective. Mn2+, which inhibits the P-b conversion activity of PbK, was found to be the most potent enhancer of its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with Ca2+ and Mg2+ to stimulate catalytic assay conditions did not further affect the apparent affinity for P-b, which is consistent with the previously reported rapid equilibrium random bi-bi kinetic mechanism for P-b conversion.
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PMID:Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase. 866 94

The specificity of the catalytic subunit of protein phosphatase-1 (PP1c) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1c-binding domains on GL and GM, the subunits that target PP1c to hepatic and muscle glycogen, respectively, and on M110, the subunit that targets PP1c to smooth muscle myosin. GM-(G63-T93) interacted with PP1c and prevented GL from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate GL from PP1c or affect other characteristic properties of the PP1GL complex. These results indicate that GL contains two PP1c-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, GM-(G63-N75) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates GM from PP1c because phosphate is inserted into the PP1c-binding domain of GM. M110-(M1-E309) and M110-(M1-F38), but not M110-(D39-E309), mimicked the M110 subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M110 subunit and M110-(M1-E309), neither M110-(M1-F38) nor M110-(D39-E309) suppressed the PP1c-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M110 subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M110-(M1-F38) displaced GL from PP1c, while GM-(G63-T93) displaced M110 from PP1c in vitro. These observations indicate that the region(s) of PP1c that interact with GM/GL and M110 overlap, explaining why different forms of PP1c contain just a single targetting subunit.
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PMID:Identification of protein-phosphatase-1-binding domains on the glycogen and myofibrillar targetting subunits. 870 35

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.
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PMID:Characterization of protein kinase and phosphatase systems in chick ciliary ganglion. 884 61

Multiple endocrine neoplasia type 1 (MEN1) is tightly linked to the muscle-type glycogen phosphorylase (PYGM) gene in 11q13. This region of the human genome contains additional disease-related loci implicated in the development of insulin-dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome and translocation t(11;17) described in B-cell non-Hodgkin's lymphoma. We approached cloning of candidate disease genes from 11q13 by large-scale genomic sequencing. We obtained > 106 kb of sequence around the PYGM gene and established a transcriptional map that includes: (i) two genes previously localized to 11q13, PYGM and a zinc-finger protein (ZFM1) gene; (ii) the germinal center kinase (GCK, human B-lymphocyte serine/threonine protein kinase) gene; (iii) a novel human CDC25-like (HCDC25L) gene; (iv) a dystrophia myotonica protein kinase-like (DMPKL) gene; and (v) a novel ubiquitously expressed gene of unknown function (germinal center kinase- neighboring gene, GCKNG).
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PMID:The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13. 934 81

The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.
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PMID:Relationship of glycogen synthase and glycogen phosphorylase to protein phosphatase 2C and cAMP-dependent protein kinase in liver of obese rhesus monkeys. 944 47


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