Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in human platelets and other cardiovascular cells. To identify the VASP phosphorylation sites, purified VASP was phosphorylated by either protein kinase and subjected to trypsin, V8 and Lys-C proteolysis. The phosphorylated proteolytic fragments obtained were separated by reversed phase high performance liquid chromatography. Sequence analysis of the phosphorylated peptides and 32P measurement of the released 32P-labeled amino acids revealed three phosphorylation sites: a serine 1-containing site (LRKVSKQEEA), a serine 2-containing site (HIERRVSNAG), and a threonine-containing site (MNAVLARRRKATQVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated serine 2 rapidly and the threonine residue slowly, whereas cGK phosphorylated the serine 1 residue more rapidly than the cAK. These differences in the phosphorylation rates of VASP by the two protein kinases were also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitro cGK substrates and the serine 2-containing site as the site responsible for the phosphorylation-induced mobility shift of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with 32P-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selective activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in intact cells. However, important quantitative and qualitative differences between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinct functions of the two types of cyclic nucleotide-mediated VASP phosphorylation.
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PMID:cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. 818 57

DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000, has been shown to be phosphorylated on threonine-34, both in vitro with high efficiency by cAMP-dependent and cGMP-dependent protein kinases and in vivo by dopamine acting through cAMP-dependent protein kinase. In the present study, we investigated the nitric oxide (NO)/cGMP pathway for its ability to regulate the state of phosphorylation of DARPP-32 in slices of rat substantia nigra. DARPP-32 was phosphorylated on threonine-34 in these slices by sodium nitroprusside (SNP), an NO donor. The effect of SNP was abolished by preincubation of the slices with hemoglobin, indicating that the effect of SNP was due to released NO. The same concentration of SNP produced a 4-fold elevation of the cGMP level but did not alter the level of cAMP. The effect of SNP on DARPP-32 phosphorylation was mimicked by low concentrations of 8-bromo-cGMP and 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate, activators of cGMP-dependent protein kinase, but not by low concentrations of 8-bromo-cAMP, an activator of cAMP-dependent protein kinase. The data indicate a physiological role for the NO/cGMP pathway in the regulation of DARPP-32 phosphorylation in nerve terminals of striatonigral neurons. The results provide further evidence that the state of phosphorylation of DARPP-32 represents an important mechanisms for integration of information arriving at striatonigral neurons via a variety of neuronal pathways.
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PMID:Nitric oxide/cGMP pathway stimulates phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, in the substantia nigra. 838 74

New method for purification of phosphatase inhibitor 1 (PPI-1) was developed which avoid the phosphorylation of PPI-1 during the purification and provides a high yield of highly pure preparation. Using this preparation, it was shown that PPI-1 was stoichiometrically phosphorylated by cGMP-dependent protein kinase at Thr-35 and the phosphorylated PPI-1 potently inhibited protein phosphatase 1. Addition of the phosphorylated PPI-1 to beta-escin-skinned single smooth muscle cells resulted in force development of the cells at the submaximal pCa2+. The results suggest that the phosphorylation of PPI-1 can be the mechanism for modifying the Ca2+ sensitivity of smooth muscle contractile response.
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PMID:Enhancement of smooth muscle contraction with protein phosphatase inhibitor 1: activation of inhibitor 1 by cGMP-dependent protein kinase. 860 41

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells.
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PMID:Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia. 869 88

SCG10 is a neuron-specific, developmentally regulated protein which is highly enriched in growth cones. Sequence homology indicates that it is related to the phosphoprotein stathmin or Op18, an in vitro and in vivo substrate for several serine/threonine kinases which are involved in a variety of signaling pathways. As a first step to examine the biochemical properties of SCG10, the protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified protein was used in in vitro phosphorylation assays. SCG10 was phosphorylated by MAP kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, p34cdc2 kinase, DNA-dependent protein kinase, Ca2+/calmodulin kinase II, and casein kinase II. The protein was not a substrate for casein kinase I and protein kinase C. SCG10 was phosphorylated by src tyrosine kinase, which demonstrates that the protein can be phosphorylated in vitro on a tyrosine residue. Our data suggest that SCG10 is a phosphoprotein which might be involved in signal transduction in neurons.
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PMID:Purification, characterization, and in vitro phosphorylation of the neuron-specific membrane-associated protein SCG10. 912 8

Phospholamban (PLB), the regulator of the cardiac sarcoplasmic reticulum (SR) Ca2+ pump is specifically phosphorylated at Ser16 and Thr17 by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaMK), respectively. The regulation of this dual-site phosphorylation of amino acid residues in direct proximity is only poorly understood. In order to study the site-specific phosphorylation of PLB, we used a synthetic peptide (PLB-24) corresponding to the cytosolic part of the PLB monomer with the phosphorylation sites as a model substrate. PLB-24 possesses substrate properties as the native PLB as demonstrated by phosphorylation with exogenous, purified PKA, cGMP-dependent protein kinase (PKG) and a type II CaMK (CaMKII). In isolated vesicles of cardiac SR there was a rapid phosphorylation of the peptide by the endogenous PKA (SR-PKA) and CaMK (SR-CaMK), but not under conditions that activate PKG. Both SR-PKA and SR-CaMK incorporated the same amount of 32P into PLB-24, 0.60 +/- 0.01 nmol 32P/mg SR protein and 0.61 +/- 0.03 nmol 32P/mg SR protein, respectively. Phosphorylation by SR-PKA was abolished by the specific PKA inhibitor (IC50 = 0.2 microM), whereas SR-CaMK phosphorylation was inhibited by calmidazolium (IC50 = 1.6 microM) and a CaMKII-specific inhibitor peptide (IC50 = 2.5 microM). Phosphorylation by SR-PKA was exclusively at Ser, whereas SR-CaMK phosphorylated only Thr. After simultaneous activation of both SR-kinases 32P incorporation into PLB-24 was additive and occurred at Ser as well as at Thr. Sequential activation of SR-PKA and SR-CaMK also caused the additive phosphorylation of PLB-24 independently of which kinase was activated first. Thus, at the monomeric level of PLB the respective phosphorylation site appears to be accessible to its related SR protein kinase in vitro even when the adjacent site is phosphorylated.
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PMID:Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide- and Ca2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum. 920 42

Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCalpha may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.
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PMID:Inhibitory properties of the regulatory domains of human protein kinase Calpha and mouse protein kinase Cepsilon. 953 77

Recently, the CLN3 gene associated with Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL), a recessively inherited, progressive, neurodegenerative disorder of childhood, has been identified. The CLN3 gene encodes a novel protein (battenin) of a predicted 438 amino acids containing several potential posttranslational modifications. We have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein (GFP) to evaluate whether CLN3 protein is phosphorylated. By using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that the CLN3 protein is phosphorylated on both serine and threonine residues. We also demonstrate that CLN3 protein is not modified by mannose 6-phosphate. Furthermore, we show that phosphorylation of CLN3 protein is carried out by protein kinase A (cAMP-dependent protein kinase, PKA), protein kinase G (cGMP-dependent protein kinase, PKG), and casein kinase II and that it is enhanced by inhibition of protein phosphatase 1 (PP 1) or protein phosphatase 2A (PP 2A).
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PMID:Evidence for phosphorylation of CLN3 protein associated with Batten disease. 987 58

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exists almost exclusively in cerebellar Purkinje cells, where it is possibly involved in the induction of long-term depression. A G-substrate cDNA was identified by screening expressed sequence tag databases from a human brain library. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr-119) with amino acid sequences [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-substrate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chromosome 7p15 by radiation hybrid panel analysis. In vitro translation products of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE which was close to that of purified rabbit G-substrate (23 kDa). Bacterially expressed human G-substrate is a heat-stable and acid-soluble protein that cross-reacts with antibodies raised against rabbit G-substrate. Recombinant human G-substrate was phosphorylated efficiently by cGMP-dependent protein kinase exclusively at Thr residues, and it was recognized by antibodies specific for rabbit phospho-G-substrate. The amino acid sequences surrounding the sites of phosphorylation in G-substrate are related to those around Thr-34 and Thr-35 of the dopamine- and cAMP-regulated phosphoprotein DARPP-32 and inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein kinase inhibited protein phosphatase 2A more effectively than protein phosphatase 1, suggesting a distinct role as a protein phosphatase inhibitor.
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PMID:Molecular identification of human G-substrate, a possible downstream component of the cGMP-dependent protein kinase cascade in cerebellar Purkinje cells. 1005 66

PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMP-dependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.
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PMID:Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/parafusin by differential MALDI mass spectrometric peptide mapping. 1038 18


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