Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.
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PMID:Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. 132 24

The catalytic subunit (C) of cAMP-dependent protein kinase selectively phosphorylates vitronectin, a plasma protein that promotes cell adhesion and platelet aggregation, inhibits the inactivation of thrombin by antithrombin III, and participates in complement function. This specific phosphorylation is used here (a) to develop an enzymatic assay for vitronectin (with C and [gamma-32P]ATP) which can be used to identify the vitronectin-containing fractions at each stage of its purification; (b) to radioactively label vitronectin and differentiate between the intact and the nicked form of this protein in structure-function studies; and (c) to identify possible vitronectin-related proteins in the plasma of other animal species.
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PMID:An enzymatic assay for vitronectin based on its selective phosphorylation by protein kinase A. 169 53

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.
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PMID:Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C. 205 Jun 83

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.
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PMID:Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets. 216 Feb 34

Octimibate inhibited ADP- and collagen-induced platelet aggregation in human, rabbit and rat platelet-rich plasma. Washed human platelets treated with octimibate had elevated cyclic AMP (cAMP) levels and cAMP-dependent protein kinase activity. When whole platelets were incubated with radiolabeled phosphate, octimibate produced an increase in the phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. This pattern of protein phosphorylation is identical to that observed when the platelets were treated with forskolin, phosphodiesterase inhibitors or other compounds that elevate platelet cAMP levels. Octimibate also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets, which is consistent with octimibate's ability to elevate platelet cAMP levels. When isolated platelet plasma membranes were treated with octimibate, adenylate cyclase activity was stimulated, reaching maximal activation at 1 microM octimibate. (The maximal activation of adenylate cyclase observed with octimibate is 70-75% of that observed with 10 microM PGE1.) This stimulation of platelet adenylate cyclase activity was enhanced by GTP. Octimibate competed for radiolabeled prostaglandin E1 and lloprost binding to isolated platelet membranes at submicromolar concentrations, but did not compete with radiolabeled prostaglandin D2 binding. These studies suggest that octimibate inhibits platelet aggregation by activating platelet adenylate cyclase through stimulation of platelet prostacyclin receptors.
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PMID:Octimibate inhibition of platelet aggregation: stimulation of adenylate cyclase through prostacyclin receptor activation. 217 92

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.
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PMID:Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin. 246 67

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.
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PMID:Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets. 250 24

To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.
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PMID:In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. 254 93

Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.
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PMID:Effects of anagrelide on platelet cAMP levels, cAMP-dependent protein kinase and thrombin-induced Ca++ fluxes. 282 59

Rabbit serum is shown to contain a cAMP-dependent protein kinase (biochemically characterized as type II) that specifically phosphorylates a 135-kDa endogenous protein. This endogenous phosphorylation can be reproduced with platelet-rich plasma, after stimulation with thrombin, but not with plasma devoid of platelets. Stimulation of isolated platelets ("washed" by gel filtration) with either thrombin or ADP brings about a release of this kinase. The supernatant of these stimulated platelets, which contains the kinase, does not undergo a cAMP-dependent endogenous phosphorylation because it does not contain the 135-kDa protein substrate. On the other hand, plasma devoid of platelets does not contain cAMP-dependent protein kinase. By combining the supernatant of the physiologically stimulated platelets with the plasma devoid of platelets, it is possible to reconstitute the system and to reproduce the specific endogenous phosphorylation of the 135-kDa target substrate. On the basis of the above evidence it is proposed that upon physiological stimulation of platelets, they release into the blood a cAMP-dependent protein kinase in addition to the well-known release of MgATP. This kinase specifically phosphorylates the 135-kDa plasma protein.
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PMID:Platelet stimulation releases a cAMP-dependent protein kinase that specifically phosphorylates a plasma protein. 317 51


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