UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
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The cAMP-dependent protein kinases comprise two enzyme forms designated as type I and type II. The type II enzyme can catalyze an autophosphorylation reaction whereby phosphate is transferred from ATP to one seryl residue on each regulatory subunit monomer. Since this reaction can occur in the absence of cAMP-induced enzyme dissociation, it has been used as a probe to identify one site of interaction between the catalytic subunit (C) and the type II regulatory subunit (R11). The type I cAMP-dependent protein kinase does not catalyze an analogous reaction; however, if cGMP-dependent protein kinase is substituted for C, the type I regulatory subunit (R1) becomes phosphorylated. The effects of cyclic nucleotides on this reaction, coupled with the ability of R1 to serve as an inhibitor of cGMP-dependent protein kinase suggest that this phosphorylation also occurs within an important functional domain on R1. A comparison of the autophosphorylation site on R11 with the cGMP-dependent protein kinase catalyzed phosphorylation site on R1 indicates that each modification takes place within a similar proteolytically sensitive region. On each subunit, this sensitive "hinge" region lies distal to the functional domain responsible for regulatory subunit dimerization and proximal to that responsible for cAMP binding. Phosphorylation of the "hinge" region decreases the affinity of each regulatory subunit for C, although the magnitude of this change appears greater for R1 than for R11. Phosphorylation of R1 also reduces the stoichiometry of cAMP binding from two to one mole of cAMP bound per mole of R1 monomer. These results suggest that the "hinge" regions of both R1 and R11 form part of the interaction site between the regulatory subunit and C; and, in the case of R1, it also forms a portion of one of two cAMP-binding sites. The amino acid sequence surrounding the phosphorylated serine of each regulatory subunit has been determined: R11: D-R-R-V-S(P)-V R1: R-R-R-R-G-A-I-S(P)-A It is thought that the number and position of the basic amino acid residues proximal to the modified serine may be responsible, in part, for determining the susceptibility of each site to phosphorylation by cAMP or cGMP-dependent protein kinase. Both R1 and R11 exist as phosphoproteins in vivo. Dephosphorylation of purified "native" phospho-R1 is without effect on the ability of R1 to interact with either C or cAMP. The site phosphorylated in vivo is therefore distinct from that modified in vitro by cGMP-dependent protein kinase. In addition to the autophosphorylation site, R11 possesses a second, less enzymatically reactive, phosphorylation site that is modified in vivo. Dephosphorylation of this site is also without apparent effect on the functional properties of R11. The kinases responsible for catalyzing the phosphorylation of R1 and the cryptic site on R11 and the role that these modifications play in modulating kinase activity are currently unknown but are under active investigation.
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PMID:Phosphorylation of cAMP-dependent protein kinase subunits. 628 16

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11 beta-hydroxylation (11 beta)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11 beta was able to incorporate phosphate from ATP in the presence of C (Km = 7.5 microM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11 beta (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11 beta-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11 beta phosphorylation strikingly increases the P-450 11 beta-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11 beta-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.
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PMID:Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11 beta-hydroxylase) from bovine adrenal cortex. 628 91

Two isoforms (I and II) of soluble cAMP-dependent protein kinase with basal activity of 2.1 and 10.87 nmole of 32P/min/mg of protein, respectively, were detected in rabbit myometrium at functional rest. cAMP (5 microM) activates 1.5-fold both isoforms of the enzyme. The apparent Km values for ATP of isoforms I and II is 0.9 X 10(-5) M and 2.1 X 10(-5) M, respectively; Km for histone H1 are 0.15 and 0.29 mg/ml, respectively. The pH optimum for both isoforms lies at 7.3-7.6; the pI values are 5.0 and 5.5, respectively. Na-DS electrophoresis in polyacrylamide gel demonstrated that the molecular weight of the regulatory subunit (R) of isoform I is 47000, that of the catalytic subunit (C) is 31000. No difference in the electrophoretic mobility of C for forms I and II were found. The molecular weight of R II is 54000. Isoform II reveals the ability for autophosphorylation. The plot for the dependence of the reaction rate versus enzyme concentration is linear; up to 1.5 mole of 32P per mole of the holoenzyme is incorporated. The myometrium of pregnant rabbits contains one isoform of cAMP-dependent protein kinase which is identical to isoform II in terms of its elution profile on DEAE-cellulose, molecular weight of R, pI and the ability for autophosphorylation. The optimal conditions for the pregnant rabbit myometrium enzyme activity are as follows: pH 7.0-9.0, cAMP--10(-8) M, basal activity--3.68 nmole of 32P/min/mg of protein, cAMP activation--2.4-fold. The values of apparent Km for ATP and histone H1 are 5.6 X 10(-5) M and 0.42 mg/ml, respectively. During autophosphorylation 0.4 mole of 32P per mole of the holoenzyme is incorporated.
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PMID:[Soluble cAMP-dependent protein kinases from the rabbit myometrium]. 630 99

Phosphorylation site stoichiometries were determined for skeletal muscle glycogen synthase purified from control, alloxan-diabetic, and epinephrine-treated rabbits. One method of analysis was direct determination of the total in vivo phosphate content of each site after reverse phase high performance liquid chromatography separation of a complete tryptic digest of the purified synthase. The second method of analysis, in vitro phosphorylation, was based on the premise that in vitro 32P incorporation into each site would be inversely related to the in vivo phosphate content of that site. Glycogen synthase from control rabbits had the following distribution of in vivo phosphate (mole of phosphate/mol of site): site 1a, 0.29 +/- 0.08; site 5, 0.62 +/- 0.07; site 3, 0.46 +/- 0.06; site 1b, 0.23 +/- 0.03; and site 2, 0.43 +/- 0.07. Synthase from diabetic rabbits had 2-fold elevations of in vivo phosphate contents of sites 2 and 3. Epinephrine resulted in increased phosphorylation in vivo of site 1b (2.0-fold), site 2 (2.0-fold), and site 3 (1.5-fold). The in vitro phosphorylation analysis showed decreased 32P incorporation in vitro (indicative of increased in vivo phosphorylation) as follows: epinephrine, site 1a, site 3, site 1b, site 2; diabetic, site 3, site 2. The effect of diabetes on the in vitro phosphorylation of sites 2 and 3 was reversed by insulin treatment. We conclude that the major effect of epinephrine, phosphorylation of sites 1a, 1b, and 2, is mediated by the activation of the cAMP-dependent kinase. The mechanisms accounting for the phosphorylation of site 3 in response to epinephrine and phosphorylation of sites 2 and 3 in the diabetic state are under investigation.
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PMID:Effects of epinephrine, diabetes, and insulin on rabbit skeletal muscle glycogen synthase. Phosphorylation site occupancies. 632 4

A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was cAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles 32P per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its Km was 30 microM. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnCl2 and 4- to 9-fold by deoxymononucleotides. The nuclei of HeLa cells contained 64% of the total kinase of which 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a cAMP-independent protein kinase which is similar to glycogen synthase kinase.
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PMID:Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells. 643 81

Purified proteoglycan subunits from human articular, bovine articular and nasal cartilages, and a rat chondrosarcoma were phosphorylated in vitro by beef heart cAMP-dependent protein kinase in the presence of gamma 32P-ATP. In these experiments, a maximum of 1.7 moles of 32P were incorporated per mole of proteoglycan from human cartilage. Phosphorylation was dependent on the presence of cAMP. Analysis by autoradiography revealed that serine residues in the core protein of the proteoglycan were the sites of phosphorylation. Treatment of proteoglycan subunits with chondroitinase ABC and alkaline phosphatase prior to reaction with cAMP-dependent protein kinase increased the incorporation of 32P by 12-30% when compared with untreated proteoglycans. These data indicate that proteoglycans in cartilage can be phosphorylated by cAMP-dependent protein kinase.
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PMID:Phosphorylation of proteoglycans from human articular cartilage by a cAMP-dependent protein kinase. 647 53

Soluble complexes of poly (U) and adenylic nucleotides in NaCl solutions were studied by scanning microcalorimetry. The melting enthalpies, delta Hm, of poly (U) complexes with adenosine, 2',3' -cAMP, 2'(3')-AMP, 5-AMP, ADP, ATP in 1 M NaCl are 50.5; 45.0; 42.9; 28.6; 26.1 and 25.6 kJ/mole triplets, respectively. Delta Hm is independent of the complex melting temperature, Tm. The calorimetric enthalpies are considerably lower than the apparent delta Hv.H. obtained from Tm dependence on free monomer concentration. The enthalpy of complex formation in 1 M NaCl depends neither ob the number nor on the degree of ionization of the phosphate groups but is essentially determined by their 5' - or 2'(3')-position. In contrast to 2'(3')- AMP. 2 poly (U), delta Hm of 5'AMP. 2 poly (U) increases considerably at lowering Na+ concentration. The enthalpy of poly (U) double helix melting in 1 M NaCl is 8.8 kJ/mole pairs which is 2.5 times lower than that in MgCl2 solutions.
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PMID:Calorimetric study of the complexes between polyuridylic acid and adenylic nucleotides. 730 78

Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.
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PMID:Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance. 758 61

Protein kinase C has been previously shown both to phosphorylate and to desensitize the ability of the human 5-HT1A receptor to inhibit adenylyl cyclase [Raymond, J. R. (1991) J. Biol. Chem. 266, 14747-14753]. In this study, we examined the effects of short-term treatment with protein kinase A activators on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the human serotonin 5-HT1A receptor in CHO cells that stably express 1200 fmol of receptor/mg of protein. Forskolin induced a concentration- and time-dependent phosphorylation of the receptor that was detectable at 5 min and maximal at 15-30 min with a half-maximal concentration of 10-20 microM. Phosphorylation was also induced by Sp-cAMPS or dibutyryl-cAMP, and blocked by Rp-cAMPS and a pseudosubstrate inhibitor of PKA, but not by heparin (inhibitor of receptor kinase) or sphingosine (inhibitor of PKC). The stoichiometry of phosphorylation induced by forskolin was 1 mol of phosphate per mole of receptor. PKA activators did not induce a measurable desensitization of 5-HT1A receptor-inhibited adenylyl cyclase activity. However, forskolin augmented the desensitization caused by a submaximal concentration of phorbol 12-myristate 13-acetate (300 nM PMA) as evidenced by a rightward shift of the concentration-response curve for 5-HT, and approximately doubled the amount of phosphate incorporated into the receptor by PMA. Forskolin did not augment desensitization or increase the degree of phosphorylation induced by a maximal concentration of PMA (5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein kinase A induces phosphorylation of the human 5-HT1A receptor and augments its desensitization by protein kinase C in CHO-K1 cells. 772 77

The gene encoding human protease inhibitor 4 (kallistatin; gene symbol PI4), a novel serine proteinase inhibitor (serpin), has been isolated and completely sequenced. The kallistatin gene is 9618 bp in length and contains five exons and four introns. The structure and organization of the kallistatin gene are similar to those of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, and alpha 1-antitrypsin. The kallistatin gene is also similar to the genes encoding rat and mouse kallikrein-binding proteins. The first exon of the kallistatin gene is a noncoding 89-bp fragment, as determined by primer extension. The fifth exon, which contains 308 bp of noncoding sequence, encodes the reactive center of kallistatin. In the 5'-flanking region of the kallistatin gene, 1125 bp have been sequenced and a consensus promoter segment with potential transcription regulatory sites, including CAAT and TATA boxes, an AP-2 binding site, a GC-rich region, a cAMP response element, and an AP-1 binding site, has been identified within this region. The kallistatin gene was localized by in situ hybridization to human chromosome 14q31-q32.1, close to the serpin genes encoding alpha 1-antichymotrypsin, protein C inhibitor, alpha 1-antitrypsin, and corticosteroid-binding globulin. In a genomic DNA Southern blot, kallistatin-related genes were identified in monkey, mouse, rat, bovine, dog, cat, and a ground mole. The patterns of hybridization revealed clues of human serpin evolution.
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PMID:Molecular cloning, sequence analysis, and chromosomal localization of the human protease inhibitor 4 (kallistatin) gene (PI4). 783 86

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