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

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus. 253 84

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 role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.
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PMID:Kinetics of protein phosphorylation in microvessels isolated from rat brain: modulation by second messengers. 283 36

It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP-dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393-394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287-290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin-extracted membranes showed ATP-dependent Ca2+ uptake as well as cGMP-dependent protein kinase (G-kinase) activity. The molecular masses of the major protein substrates of the G-kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+-ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I-calmodulin overlay technique and an antibody against human erythrocyte Ca2+-ATPase. Ca2+-uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP-dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP-dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady-state phosphorylation levels at 37 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees C. However, the cGMP-dependent thiophosphorylation failed to influence the Ca2+-uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP-dependent protein kinase and is not regulated by the cGMP-dependent phosphorylation of membrane proteins.
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PMID:The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities. 296 69

The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggests a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP [ADAP peptide(645-661)] on Ser-655. Ca2+/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. This peptide was virtually ineffective as a substrate for cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, or insulin receptor protein-tyrosine kinase. When a homogenate of rat cerebral cortex was used as the source of protein kinase, phosphorylation of ADAP peptide(645-661) was stimulated by calcium/phosphatidylserine/diolein to a level 4.6-fold above the basal level of phosphorylation, consistent with a prominent stimulation by protein kinase C. Using rat cerebral cortex synaptosomes prelabeled with 32Pi, a 32P-labeled phosphoprotein of approximately equal to 135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.
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PMID:Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca2+/calmodulin-dependent protein kinase II. 313 67

Several endogenous substrate proteins of cilia from axenically grown Paramecium tetraurelia were phosphorylated in vitro by inherent protein kinases (PKs). Labeling was stimulated by cAMP and to a lesser extent by cGMP. ATP breakdown was most rapid in cilia and subciliary fractions. Using multiple substrate additions during incubations it was shown that phosphorylation was almost completed within 30 s. Very little dephosphorylation by phosphoprotein phosphatases occurred during 5 min of incubation. Proteins of molecular weight of 103 000 and 46 000 were shown to be particularly associated with axonemal structures of the cilia. No distinct differences in phosphorylation patterns were apparent in ciliary membrane vesicles of low and high buoyant density, which exhibit differential enzyme patterns. cAMP receptor proteins were identified by use of the photoaffinity label 8-azido-[32P]cAMP. Receptor proteins with apparent molecular weights of 43 000, 39 000, 37 000, 31 000 and 30 000 were probably related to the regulatory subunits of cAMP-dependent protein kinases as evidenced by inhibition of incorporation of the photoaffinity label by low concentrations of cAMP. Tagging of a protein of 85 000 molecular weight was specifically inhibited by cGMP, thus in all likelihood it corresponded to a cGMP-dependent protein kinase. Corresponding autophosphorylated protein bands were observed with gamma-[32P]ATP. A functional role for protein phosphorylation in cilia of Paramecium remains to be established.
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PMID:Phosphorylation of endogenous proteins of cilia from Paramecium tetraurelia in vitro. 631 38

In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium. 779 56

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

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

Natural cytotoxicity (NC) against cancer involves receptor-ligand interactions between lymphohemopoietic cells that mediate NC against tumor cells. The only candidate for a receptor on cells mediating NC is NC-1.1, identified using mAb 1C4. In this study we showed that mAb 1C4 blocked NC-1.1+ cell conjugation to WEHI-164 tumor cells, indicating that NC-1.1 is a surface protein required for cell-cell interaction. Affinity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good in vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-dependent protein kinase (PKA). Phosphopeptide mapping revealed one phosphopeptide phosphorylated by PKG and PKA, and two additional peptides phosphorylated by PKC. Phosphorylation by PKG or PKA abolished phosphorylation at the PKC sites, while coincubation of NC-1.1 with both PKG and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosphoprotein after immunoprecipitation from intact spleen cells and its phosphorylation was increased after cell stimulation with PKC or PKG activators (phorbol esters or 8-bromo-cGMP). The possible consequences of intracellular signaling were tested in functional assays for NC. Phorbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both phorbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolished NC without affecting cell conjugation. These results suggest that NC-1.1 is a receptor for a ligand on certain tumor cells and reveal that key intracellular signaling pathways involving PKC, PKG, and PKA interact to effect a coordinated control of NC.
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PMID:Phosphorylation of the NC-1.1 receptor and regulation of natural cytotoxicity by protein kinase C and cyclic GMP-dependent protein kinase. 903 46


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