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)

The cytosolic free Ca2+ concentration [( Ca2+]i) was measured in cultured human umbilical vein smooth muscle cells using fura-2 as a Ca2+ indicator and microscopic digital image analysis system. Activation of cells with histamine and vasopressin resulted in a prompt though transient rise in [Ca2+]i 10- to 12-fold higher than the resting [Ca2+]i. The [Ca2+]i then declined rapidly during the first 30-40 seconds after hormonal stimulation and then gradually decreased to near resting levels in 2-3 minutes in the continued presence of hormones. The magnitude of the increase in peak [Ca2+]i was similar in buffered salt solution containing 1.8 mM Ca2+, zero Ca2+, and zero Ca2+ buffered salt solution containing 10 mM La3+, suggesting that receptor-mediated increase in [Ca2+]i is primarily due to the release of Ca2+ from the intracellular stores. Addition of La3+ produced oscillations in [Ca2+]i in approximately half the cells in response to both hormones. Addition of 10 microM forskolin did not significantly affect the resting [Ca2+]i, the hormone-stimulated peak [Ca2+]i, or the time course of hormone-stimulated [Ca2+]i transients. These data suggest that mechanisms involved in A-kinase-mediated smooth muscle relaxation may be subsequent to the changes in [Ca2+]i. Activation of C-kinase by 1 microM 12 deoxyphorbol 13-isobutyrate-20 acetate (DPBA) did not affect the resting [Ca2+]i, though it attenuated the histamine and vasopressin-mediated peak elevation in [Ca2+]i. Since DPBA inhibited the peak [Ca2+]i response to both the hormones to the same extent, it would appear that C-kinase activation may uncouple the receptor-mediated activation of phospholipase C.
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PMID:Regulation of cytosolic free Ca2+ concentration in vascular smooth muscle cells by A- and C-kinases. 273 23

Evidence is presented for a testicular protein kinase activity capable of stimulating the activity in vitro of a partially purified preparation of the testicular galactolipid sulphotransferase. This enzyme is responsible for the synthesis of the major mammalian testicular glycolipid, sulphogalactosylglycerol, and is an early marker of differentiation during spermatogenesis. This stimulatory activity has been separated by affinity chromatography, using 3',5'-ADP-agarose, from both the detergent-solubilized microsomes (microsomal fractions) and the soluble fraction of the testicular homogenate. The stimulator was eluted from the affinity matrix by either a salt, or, more selectively, a cyclic AMP gradient. Thus this matrix can function as an analogue of 3',5'-cyclic AMP. The activity of the sulphotransferase stimulator was ATP-dependent and coincident with protein kinase activity. Sulphotransferase activity was also stimulated in the presence of commercial preparations of cyclic AMP-dependent protein kinase and stimulation was prevented in the presence of kinase inhibitors. Our results suggest that sulphogalactolipid biosynthesis is regulated by a phosphorylation process during spermatogenesis. In addition, our results suggest that affinity chromatography on 3',5'-ADP-agarose may provide a general method for the purification of cyclic AMP-dependent kinases.
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PMID:Modulation of testicular galactolipid sulphotransferase activity by phosphorylation. Stimulation of enzyme activity in vitro by an endogenous kinase. 277 26

This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with synaptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase. In the present study, we examined the high-affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 microM) increased uptake 1 by approximately 36%. This increase could be mimicked by adenosine-5'-O-(3-thiotriphosphate) tetralithium salt (ATP gamma S), an analogue of ATP which can be utilized by protein kinases, and not by 5'-adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP, GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATP gamma S, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATP gamma S blocked the activation produced by ATP gamma S alone. Kinetic analysis showed that ATP gamma S pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase.
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PMID:Extracellular ATP stimulates norepinephrine uptake in PC12 cells. 279 17

Cyclic-nucleotide-elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium-derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade. Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441-448) established that in human platelets cyclic-nucleotide-elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP- and cGMP-dependent protein kinases. Of particular interest was a membrane-bound 50-kDa protein whose phosphorylation was increased both by cAMP- and cGMP-elevating vasodilators in intact platelets and by endogenous cAMP- and cGMP-dependent protein kinase in platelet membranes. Since the molecular mechanism of action of cyclic-nucleotide-elevating vasodilators is unknown, this 50-kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye-ligand chromatography. The purified protein migrated as a 46-kDa protein in SDS/PAGE, was an excellent substrate for both cAMP- and cGMP-dependent protein kinases and migrated in SDS/PAGE as a 50-kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50-kDa protein phosphorylated by both cAMP- and cGMP-dependent protein kinases in platelet membranes and in response to cAMP- and cGMP-elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP- or cGMP-dependent protein kinase. The availability of this vasodilator-regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic-nucleotide-elevating vasodilators.
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PMID:Purification of a vasodilator-regulated phosphoprotein from human platelets. 280 62

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.
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PMID:Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes. 284 6

During cellular remodeling that accompanies cornification of epidermal cells, the highly phosphorylated protein, profilaggrin, is dephosphorylated and proteolytically cleaved to filaggrin, the keratin matrix protein. Using rat filaggrin phosphorylated by bovine casein kinase II (CK II) as a substrate, we have partially purified a phosphatase from rat epidermis which dephosphorylates rat profilaggrin in vitro. Anion exchange, hydroxylapatite, and gel filtration chromatography yielded a 100-fold purification of phosphatase from a low-salt extract. Further purification led to loss of activity; therefore, only the partially purified phosphatase was characterized. Two forms of the phosphatase, with molecular weights of approximately 170 and 40 kDa, were resolved during gel filtration. The 170-kDa form could be converted to the 40-kDa form in the presence of dithiothreitol. Both forms had pH optima of 6.6, and were strongly inhibited by NaCl (50% inhibition at 35-40 mM). Neither form hydrolyzed para-nitrophenylphosphate or dephosphorylated casein or the synthetic peptide arg3-glu3-thr-glu3, which were phosphorylated by casein kinase II. The two forms were similarly inhibited by known inorganic phosphatase inhibitors, with 22%-36% inhibition by 0.1 mM Na+/K+ tartrate, 55%-60% inhibition by 0.1 mM NaF, and 75% inhibition by 0.1 mM Na pyrophosphate. Para-chloromercuribenzoate also inhibited the activity, suggesting that reduced thiols may be important in catalysis. One mM calcium chloride altered the activity in a complex manner depending on the pH, suggesting a possible role for calcium in regulating enzyme activity.
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PMID:Characterization of an epidermal phosphatase specific for filaggrin phosphorylated by casein kinase II. 284 73

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.
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PMID:Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation. 288 24

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.
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PMID:Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein. 296 1

By c.d. studies it is shown that liver-pyruvate-kinase-related peptide substrates of cyclic AMP-dependent protein kinase have a high tendency towards non-random structures in non-aqueous media. When phosphorylated, the conformation tendencies decrease. This structural change is explained in terms of the formation of strong intrapeptide phosphate-guanidinium salt links. It is proposed that similar events occur at the catalytic site of protein kinase and that such an interaction could facilitate the removal of the phosphorylated products.
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PMID:The role of the phosphate group for the structure of phosphopeptide products of adenosine 3',5'-cyclic monophosphate-dependent protein kinase. 299 37

To define the roles of subnuclear structure in SV40 infection, the relative distribution of T-antigen (T-ag) in various subnuclear fractions obtained from both lytically infected and transformed African green monkey kidney cells was determined. Depending on the differential sensitivity of nuclear T-ag to extraction by salt and detergent, nuclear T-ag could be separated into nucleoplasmic T-ag, salt-sensitive T-ag and matrix-bound T-ag subclasses. At least fivefold less matrix-bound T-ag was found in transformed cells than in lytically infected cells. While a cAMP-independent protein kinase was detected in the nuclear matrix, the matrix-bound T-ag (94K) could not be phosphorylated in vitro. The removal of cellular chromosomes by DNase caused changes in the interaction of T-ag with nuclear components. The results suggest that the compartmentalization of nuclear T-ag may be determined by its interaction with host chromosomes.
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PMID:Association of phosphorylated simian virus 40 T-antigen with subnuclear fractions of infected and transformed cells. 299 93


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