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 human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.
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PMID:Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase. 815 97

Rat adipocyte cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) appears to be dually regulated in intact cells by serine phosphorylations induced by isoprenaline and insulin, respectively (Degerman, E., Smith, C. J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 533-537; Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C. (1991) J. Biol. Chem. 266, 13385-13390). Since cAMP-dependent protein kinase (cAMP-PK) catalyzes the beta-adrenergic effects, the site in the isolated cGI-PDE phosphorylated by this kinase was explored. A peptide, LRRSSGASGLLTSEHHSR (P18), corresponding to the amino acid sequence Leu423-Arg440 in the putative regulatory domain of the rat adipocyte cGI-PDE was synthesized. It contains a consensus substrate sequence -RRXS- for cAMP-PK within two tryptic cleavage sites and was readily phosphorylated by cAMP-PK. Two phosphopeptides, identified as RS-[32P]SGASGLLTSEHHSR and S-[32P]SGASGLLTSEHHSR, were obtained after stoichiometric phosphorylation and trypsinization of the peptide. These two peptides and the two main tryptic phosphopeptides obtained from immunoisolated [32P]cGI-PDE phosphorylated with cAMP-PK in a solubilized crude adipocyte membrane fraction were immuno-precipitated by an affinity-purified polyclonal antibody raised against P18 and exhibited the same chromatographic and electrophoretic profiles in three different separation systems. Similar radiosequencing profiles indicated that the second most N-terminal serine, corresponding to Ser-427 in the intact cGI-PDE, was phosphorylated by cAMP-PK in both P18 and authentic cGI-PDE. It is concluded that serine 427 is the target for cAMP-PK phosphorylation of the rat adipocyte cGI-PDE in vitro.
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PMID:Identification of the phosphorylation site in vitro for cAMP-dependent protein kinase on the rat adipocyte cGMP-inhibited cAMP phosphodiesterase. 816 98

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.
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PMID:The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone. 822 96

To investigate the role of cAMP-dependent protein kinase (PKA) and cAMP levels in ATP-dependent mitogenesis, Swiss 3T3 cells were transfected with an expression vector coding for (i) a mutated regulatory subunit of PKA (PKA mutant) or (ii) a yeast low Km cAMP phosphodiesterase gene (PDE mutant). The PKA mutant showed 70% reduced PKA activity. Phosphodiesterase activity increased 2.5-fold in the PDE mutant, leading to a great reduction of cAMP levels stimulated by ATP and other cAMP-increasing agents. The mitogenic responses of PKA and PDE mutants to insulin, epidermal growth factor, or 12-O-tetradecanoylphorbol-13-acetate were not significantly changed. However, the further stimulation by ATP, ADP, and adenosine 5'-(beta,gamma-imido)triphosphate in the presence of these growth factors was reduced by > 80%. Mitogenic effect of prostaglandin E2, forskolin, cholera toxin, or adenosine was inhibited in both mutants. The mitogenic stimulation by dibutyryl cAMP, which is resistant to phosphodiesterase, was inhibited in the PKA mutant, but not in the PDE mutant. A partial reduction of platelet-derived growth factor- or bombesin-stimulated mitogenesis, which involves protein kinase C as well as the cAMP signal, was observed in the mutants. These genetic results confirm pharmacological data on the role of PKA and cAMP levels in mitogenesis due to ATP and other growth factors.
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PMID:Role of adenosine 3':5'-monophosphate-dependent protein kinase and cAMP levels in ATP-dependent mitogenesis in Swiss 3T3 cells. 827 49

The distribution and phosphoprotein band patterns of low Km, cGMP-inhibited cAMP phosphodiesterase (cGI PDE) activity were examined in cytosolic and microsomal fractions of human, canine, rabbit and guinea pig left ventricular myocardium following phosphorylation by cAMP-dependent protein kinase, immunoprecipitation with anti-cGI PDE antibodies and SDS-PAGE. The recovery of cGI PDE activity in cytosolic and microsomal fractions was comparable in all four species. Microsomal cGI PDE was comprised chiefly of a approximately 135 kDa phosphoprotein. Cytosolic cGI PDE was comprised solely of approximately 116 kDa and lower molecular weight phosphoproteins. The approximately 135 kDa phosphoprotein probably corresponds to the holoenzyme encoded by the recently cloned cDNA for human myocardial cGI PDE, whose predicted molecular weight is 126 kDa. The approximately 116 kDa phosphoprotein may result from deletion or removal of putative membrane-association domains from the N-terminal region of the holoenzyme. These results suggest that the cytosolic and sarcoplasmic reticulum-associated forms of mammalian myocardial cGI PDE are separate molecular species.
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PMID:Cytosolic and sarcoplasmic reticulum-associated low Km, cGMP-inhibited cAMP phosphodiesterase in mammalian myocardium. 838 Dec 78

Two distinct but related cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) cDNAs were cloned from rat adipose tissue cDNA libraries. The open reading frame (3324 base pairs) of RcGIP1 encodes 1108 amino acids, including a hydrophobic membrane-associated domain in the NH2-terminal portion and, in the COOH-terminal portion, a putative catalytic domain conserved among all mammalian PDEs which is preceded by a putative regulatory domain that contains three consensus cAMP-dependent protein kinase phosphorylation sites and followed by a hydrophilic COOH-terminal domain. The carboxyl-terminal portion including the conserved domain was expressed as a glutathione S-transferase fusion protein and exhibited cAMP PDE activity which was inhibited by cilostamide, a specific cGI PDE inhibitor. RcGIP1 cDNA hybridizes strongly with RNA from isolated adipocytes, and its mRNA increases dramatically during differentiation of 3T3-L1 adipocytes. The deduced sequence of the second partial cDNA clone (RcGIP2 clone 53B) is highly homologous to the corresponding region of human cardiac cGI PDE cDNA. RcGIP2 cDNA hybridized strongly with rat cardiac tissue RNA and weakly if at all with RNA from rat adipocytes or 3T3-L1 fibroblasts or adipocytes. We suggest that RcGIP1 represents the hormone-sensitive, membrane-associated rat adipocyte cGI PDE and RcGIP2, a cGI PDE from vascular elements in rat adipose tissue.
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PMID:Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase. 839 9

The distinct phosphodiesterase isoenzyme activities in guinea-pig lung were identified and characterised. We demonstrate that protein kinase A catalyses the activation of lung Type V cyclic GMP phosphodiesterase. This occurs via a marked change in the Vmax for cyclic GMP hydrolysis. The sensitivity of the activated PDE to inhibition by zaprinast is also markedly reduced (zaprinast inhibits in PDE activity via a mixed mechanism). We suggest that activation of the PDE by protein kinase A involves a mechanism that leads to alteration in the regulatory action of a non-catalytic cyclic GMP binding site.
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PMID:Lung phosphodiesterase isoenzymes. 839 18

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.
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PMID:Photoaffinity labelling of cyclic GMP-binding proteins in human platelets. 839 9

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).
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PMID:Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. 862 20

A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.
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PMID:Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation. 866 27


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