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)

Voltage-dependent sodium channels from a variety of tissues are known to be phosphorylated by the cAMP-dependent protein kinase, protein kinase A. However, the functional significance of sodium channel phosphorylation is not clearly understood. Using whole-cell voltage-clamp techniques, we show that sodium currents (INas) in rabbit cardiac myocytes are enhanced by isoproterenol (ISO). This enhancement of INa by ISO 1) is holding potential dependent, 2) can be mimicked by forskolin and dibutyryl cAMP, and 3) is accompanied by an increase in the rate of Na+ channel inactivation. In single-channel, inside-out patch experiments, the catalytic subunit of protein kinase A also enhances INa and increases the rate of inactivation, suggesting that cardiac Na+ channel phosphorylation may be physiologically important. Addition of the protein kinase A inhibitor to the pipette solution in whole-cell experiments blocks the stimulatory effect of forskolin without blocking the effect of ISO, suggesting that ISO also enhances INa through a cAMP-independent pathway. To determine if ISO may stimulate INa through a direct G protein pathway, single channels were recorded in the presence of the Gs-activating GTP analogue, GTP gamma S, and the stimulatory G protein subunit, Gs alpha. Both of these agents enhanced INa without affecting the rate of Na+ channel inactivation. These results suggest that ISO enhances rabbit cardiac INa through a dual (direct and indirect) G protein regulatory pathway.
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PMID:Enhancement of rabbit cardiac sodium channels by beta-adrenergic stimulation. 130 15

A photoaffinity analogue of ATP, 8-azido-adenosine 5'-triphosphate (8-N3ATP), was used to probe ATP-binding sites in native transcription complexes of vesicular stomatitis virus (VSV) (New Jersey serotype). The analogue was found to be a substrate for a serine/threonine protein kinase that phosphorylated both the NS and L proteins of native complexes. The analogue failed to interact with the RNA polymerase, another ATP-utilizing activity associated with the transcription complex. Kinetic analyses of both ATP and 8-N3ATP utilization by the protein kinase yielded biphasic saturation curves. Photolysis of 8-N3ATP in the presence of VSV transcription complexes resulted in selective labelling of the L protein. The photolabelling of L was saturable and apparently biphasic. Photolabelling of the L protein was significantly reduced by competition with ATP whereas other nucleoside triphosphates (GTP, UTP and CTP) were ineffective competitors. The stoichiometry of photolabelling was 0.2 at 10 microM-8N3ATP and 1.3 at 100 microM-ATP. These data provide chemical evidence for a virus-encoded serine/threonine protein kinase which resides on the L protein.
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PMID:Identification and characterization of serine/threonine protein kinase activity intrinsic to the L protein of vesicular stomatitis virus New Jersey. 130 63

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 micrograms/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159-166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca(2+)-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca2+ concentrations tested. Adenylate cyclase at pCa 6.2 was synergistically activated either by GTP and CaM or by CaM and beta-adrenergic agonist, (+/-)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca(2+)-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca2+. The former had bigger molecular size (greater than or equal to 49 kDa) than the latter (less than or equal to 34 kDa). Yield of Ca(2+)-dependent CaMBPs was not affected by Ca2+ concentration during preparation of the SM while that of Ca(2+)-independent CaMBPs was reduced by exposure to 100 microM Ca2+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.
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PMID:Characterization of EGTA-washed synaptosomal membrane with emphasis on its calmodulin-binding proteins. Demonstration of possible reconstitution with added calcium/calmodulin. 131 53

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.
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PMID:Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes. 131 May 90

Herpes simplex virus type 1 (HSV-1) induces a protein kinase (PK) activity in infected cell nuclei. In vitro, the enzyme is able to phosphorylate exogenous casein (albeit inefficiently) but not protamine, can use ATP or GTP as a phosphate donor, is stimulated by high salt concentrations and is insensitive to inhibition by heparin. On the basis of these properties, the PK appears to be distinct from previously described cellular enzymes and from the cytoplasmic PK encoded by the viral US3 gene. A major substrate of the enzyme in vitro is a virus-induced protein with an Mr of 57000 (Vmw57). The gene encoding Vmw57 was mapped using recombinants between HSV-1 and HSV-2 to a region of the virus genome containing genes UL9 to UL15. Use of a monospecific rabbit antiserum showed that Vmw57 is a virion structural protein encoded by gene UL13. These results, in conjunction with previous reports that the UL13 protein contains PK sequence motifs, support the notions that the nuclear PK and Vmw57 are identical, and that the observed reactivity is due to autophosphorylation.
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PMID:The UL13 virion protein of herpes simplex virus type 1 is phosphorylated by a novel virus-induced protein kinase. 131 59

Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.
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PMID:Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes. 131 71

Adenylyl cyclase in rat adipose cells is stimulated by ligands for Rs receptors (e.g. isoproterenol) and inhibited by ligands for Ri receptors (e.g. adenosine). In contrast, Rs receptors mediate inhibition and Ri receptors mediate augmentation of insulin-stimulated glucose transport activity by a process independent of changes in cellular cAMP-dependent protein kinase activity [Kuroda M., Honnor R. C., Cushman S. W., Londos C. and Simpson I. A. (1987) J. biol. Chem. 262, 245-253]. The present study examines the possible role of G-proteins in the regulation of insulin-stimulated glucose transport activity by Rs and Ri receptors. First, conditions were established that permit intoxication of isolated rat adipocytes by cholera and pertussis toxins without compromising cell integrity. Effectiveness of toxin treatment was monitored by examining adenylyl cyclase activity in isolated plasma membranes. Secondly, neither toxin interfered with the ability of a maximal concentration insulin to initiate the glucose transport response. Thirdly, pertussis toxin eliminated the augmenting effects of adenosine on insulin-stimulated glucose transport activity, but enhanced the inhibitory effects of isoproterenol. Findings with ligands for other Ri receptors (nicotinic acid and prostaglandin E2) mirrored those with adenosine. Finally, cholera toxin elicited a modest depression of transport activity, and only in the absence of an Ri ligand (e.g. adenosine). Furthermore, in contrast to the enhanced stimulation of adenylyl cyclase by isoproterenol and GTP, cholera toxin eliminated the inhibitory effect of isoproterenol on transport activity. The augmentative effects of adenosine on transport activity were unchanged. Measurements of (-/+cAMP) cAMP-dependent protein kinase activity ratios reinforce the notion that modulation of glucose transport activity is independent of changes in cAMP. We conclude that regulation of glucose transport activity by Rs and Ri receptors is mediated by the G-proteins, Gs and Gi (or other toxin substrates), respectively. Inasmuch as such regulation occurs at the plasma membrane and appears to be cAMP-independent, it is suggested that glucose transporters may be direct targets for receptor: G-protein interactions.
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PMID:Cholera and pertussis toxins modify regulation of glucose transport activity in rat adipose cells: evidence for mediation of a cAMP-independent process by G-proteins. 131 47

Studies were performed to examine the regulation of atrial natriuretic peptide- (ANP) stimulated guanylate cyclase in the the inner medulla. Primary cultures of rat inner medullary collecting tubular cells exposed to 10(-7) M ANP increased cGMP formation to 31.2 +/- 1.8 compared to the basal production of 2.1 +/- 0.6 fm/micrograms protein. This response did not appear to be transduced via a Gi protein, as preincubation with pertussis toxin did not alter the response to 10(-7) M ANP, and saponized cells exposed to 10 microM GTP gamma S did not enhance the response to ANP (77.3 +/- 5.9 vs. 86.7 +/- 6.3 g/micrograms). Likewise, changes in extracellular Ca2+ from 0.5 to 3.0 mM, decrements in intracellular Ca2+ with EGTA or increments in intracellular Ca2+ with ionomycin (5 microM) did not significantly alter the response to ANP. Neither activation of protein kinase A with forskolin (36.5 +/- 5.1) nor of protein kinase C with s,n-1,2-dioctanoylglycerol (33.2 +/- 2.5) altered the response to 10(-7) M ANP (32.2 +/- 3.3, NS). As the inner medullary environment was hypertonic, the effect of altering tonicity was studied. Cells grown for 48 hours in hypertonic media (600 mOsm/kg H2O) displayed enhanced response to 10(-8) and 10(-7) M ANP when osmolality was raised by either Na+ alone or in combination with urea, but not by urea alone. Our studies demonstrate that ANP-stimulated guanylate cyclase is insensitive to alterations in either intra- or extracellular Ca2+, is not subject to inhibition by protein kinase, and does not involve a pertussis-sensitive G protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of atrial natriuretic peptide-stimulated cGMP production in the inner medulla. 131 78

Modulation of inositol phospholipid (InsPL) hydrolysis in response to increasing intracellular concentrations of cyclic AMP (cAMP) was studied in a murine T helper type II (Th2) lymphocyte clone, 8-5-5. Intact 8-5-5 cells produced maximal amounts of cAMP in response to prostaglandin E2 (PGE2), cholera toxin (CTx) or 7 beta-deacetyl-7 beta-(gamma-N-methylpiperazino)butyryl forskolin (dmpb-forskolin). cAMP generation reached a plateau after 5 min of treatment with dmpb-forskolin (300 microM) or PGE2 (1 microM), but required 60 min of treatment with CTx (1 microgram/ml). Preincubation of 8-5-5 cells with 1 microM-PGE2 or 300 microM-dmpb-forskolin (10 min at 37 degrees C) or with 1 microgram of CTx/ml (60 min at 37 degrees C) completely inhibited InsPL hydrolysis induced by perturbation of the T cell receptor (TCR)/CD3 complex with the monoclonal antibody 145.2C11. Preincubation with the cAMP analogue 8-bromo-cyclic AMP (8-Br-cAMP) also inhibited InsPL hydrolysis. Tetanolysin-permeabilized 8-5-5 cells produced cAMP in response to PGE2, dmpb-forskolin and guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-cell-permeating, non-hydrolysable analogue of GTP that directly activates G-proteins. No inhibition of TCR/CD3-induced InsPL hydrolysis was observed under these conditions. InsPL hydrolysis was also unaffected when permeabilized cells were incubated with up to 10 mM-8-Br-cAMP, suggesting that permeabilized cells lost (a) soluble effector molecule(s) involved in mediating the inhibitory effect observed in intact cells. Treatment of 8-5-5 cells with dmpb-forskolin or CTx prior to permeabilization resulted in inhibition of TCR/CD3-induced InsPL hydrolysis, but did not affect InsPL hydrolysis induced via G-protein stimulation with GTP[S]. Treatment of permeabilized 8-5-5 cells with purified cAMP-dependent protein kinase (PKA) resulted in inhibition of TCR/CD3- but not GTP[S]-induced InsPL hydrolysis. This effect was associated with phosphorylation of phospholipase (PLC)-gamma 1 in the absence of phosphorylation of components of the TCR/CD3 complex. These results suggest that PKA-mediated phosphorylation of PLC may regulate TCR/CD3-induced InsPL hydrolysis.
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PMID:Increased intracellular cyclic AMP inhibits inositol phospholipid hydrolysis induced by perturbation of the T cell receptor/CD3 complex but not by G-protein stimulation. Association with protein kinase A-mediated phosphorylation of phospholipase C-gamma 1. 131 20

G-proteins are heterotrimeric proteins involved in many transmembrane signaling events. Both the renal basolateral membrane and the renal brush border membrane contain large quantities of these proteins. G-proteins appear related to hormonal signaling in the basolateral membrane and presumably affect ion gating in the brush border. We investigated the influence of G-proteins on the amiloride-sensitive Na/H exchanger, the activity of which is regulated at least in part by cAMP-dependent protein kinase, by measuring the amiloride-sensitive component of [22Na+] uptake in rat renal brush border membrane vesicles (BBMV) in the presence of a pH gradient. Incubation of vesicles with AlF4- (10 microM Al3+, 10 mM F-) resulted in significant inhibition of amiloride-sensitive [22Na+] uptake at both 20 seconds and 5 minutes of incubation. Incorporation of GTP gamma S into BBMV by transient hypotonic lysis also resulted in significantly reduced amiloride-sensitive [22Na+] uptake compared to controls at both time points. This inhibition could be reversed by GDP beta S. Similar lysis in the presence of 10 microM GDP beta S alone had no significant effect. When Na(+)-dependent [14C]-D-glucose uptake into BBMV was studied no significant effect of these G-protein modulating agents was observed. Adenylate cyclase activity could not be stimulated in these BBMV preparations using standard techniques. Furthermore, cAMP-dependent protein kinase activity, strongly stimulated in these BBMV by exogenously added cAMP, was not stimulated by 10 microM GTP gamma S alone. These findings suggest that the amiloride-sensitive Na/H exchanger can be regulated by G-proteins independently of adenylate cyclase and cAMP-dependent protein kinase.
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PMID:G-protein stimulation inhibits amiloride-sensitive Na/H exchange independently of cyclic AMP. 132 27


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