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)

Kinetics of the catalytic activities of creatine kinase (CK;EC 2.7.3.2) for three CK-3 and two CK-2 isoforms in serum were studied in 20 patients with myocardial infarction randomly assigned to receive either intracoronary urokinase (group A) or conventional therapy (group B). The temporal characteristics of isoform changes described were (a) time at which the isoform activities are significantly greater than initial values, (b) maximal rate (Ka) at which isoforms are released into blood, (c) time lag from onset of pain until maximum activity value, (d) peak value of each serum isoform, and (e) rate (Kd) at which each isoform is cleared from serum. Thrombolytic treatment induced earlier peak times in group A: for CK-3(3), 7.4 vs 20.0 h; for CK-3(2), 11.6 vs 24.8; for CK-3(1), 18.6 vs 34.3; for CK-2(2), 9.1 vs 17.8; and for CK-2(1), 11.8 vs 26.8 (numbers given are medians; for all isoforms, P less than 0.05). Ka values were at least twofold greater and the first increase was significantly earlier in the urokinase group. Consequently, the ratio for CK-3(3) to CK-3(1) activities peaked significantly earlier in group A. Isoform peak activities and Kd were not significantly different between the two groups.
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PMID:Isoforms of creatine kinase isoenzymes in serum in acute myocardial infarction after intracoronary thrombolysis. 367 76

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

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms alpha and beta were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC alpha and PKC beta were present. In the particulate fraction two peaks of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC alpha immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC alpha whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC alpha in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC alpha and PKC beta, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG.
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PMID:Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes. 904 17

Thromboxane A2 (TxA2) is a potent vasoconstrictor and platelet agonist. Its biological function is tightly regulated. G protein-coupled membrane receptors transduce the effects of TxA2. However, although a single thromboxane receptor (TP) gene has been identified, two splice variants have been cloned from human placenta and megakaryocytic lines (TPalpha) and from human endothelial cells (TPbeta). These differ in the length of their carboxyl-terminal extensions (15 versus 79 residues), which contain multiple potential sites for receptor phosphorylation. Given that TP agonists activate protein kinase C (PKC), it would seem possible that PKC-dependent phosphorylation of TPs might play a central role in homologous desensitization of these receptors. To determine if the TP isoforms were differentially phosphorylated in response to agonist in vivo, human embryonic kidney (HEK) 293 cells were stably transfected with TPalpha and TPbeta. Isoform-specific anti-peptide antibodies were developed and used to immunoprecipitate the phosphorylated receptors. U46619, a PGH2/TxA2 mimetic, induced specific phosphorylation of both isoforms. Phosphorylation of the two isoforms was similar in dose and time dependence, reaching a plateau at around 100 nM U46619. Inhibition of PKC with either GF 109203X (5 microM) or RO 31-8220 (5 microM) or of protein kinase A with H-89 (50 microM) marginally influenced agonist-dependent phosphorylation of either isoform and failed to modulate homologous desensitization of agonist-induced stimulation of inositol phosphate formation. Similar results were obtained when PKC was down-regulated by long term incubation with the phorbol ester, phorbol myristate acetate. Although short term stimulation with phorbol myristate acetate caused PKC-dependent phosphorylation of TPs in vivo, thrombin stimulation of the TP-transfected HEK cells in vivo failed to phosphorylate either of the TP isoforms. Thus, despite the capacity of PKC to phosphorylate TPs in HEK 293 cells and the likely activation of PKC by TP stimulation, this enzyme, like protein kinase A, contributes marginally to rapid, agonist-induced phosphorylation of either TP isoform.
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PMID:Rapid, agonist-dependent phosphorylation in vivo of human thromboxane receptor isoforms. Minimal involvement of protein kinase C. 905 15

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.
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PMID:L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells. 913 5

Conflicting evidence exists as to whether "conventional" protein kinase C isoforms (cPKCs) function as monomers or oligomers. In this report, we demonstrate that purified cPKC isoforms can be rapidly cross-linked by the sulfhydryl-selective cross-linker bis(maleimido)hexane, but only in the presence of both Ca(2+) and phosphatidylserine; cross-linking was minimal in the presence of either of these activators alone. In addition, cross-linking of these cPKCs did not require Mg(2+) or ATP. Among the various phospholipids tested, phosphatidylserine was found to be the most effective in the promotion of cPKC self-association and for the stimulation of protein kinase activity toward the exogenous substrate histone. Phosphatidic acid and phosphatidylinositol were less effective in this regard, whereas phosphatidylcholine exhibited little ability to induce cPKC self-association or to stimulate kinase activity. An examination of the mechanism by which the cPKC isoforms self-associate in the presence of phospholipid/Ca(2+) revealed that this process occurred independently of phospholipid aggregation. Moreover, self-association was not inhibited by saturating the enzyme active site with a peptide substrate, suggesting that self-association is distinct from an enzyme-substrate interaction. Isoform-specific antibodies revealed that all cPKC isoforms (alpha, beta, and gamma) self-associate and that, in a mixture of cPKC isoforms, PKC-alpha forms primarily alpha-alpha homodimers. Besides cPKC interactions detected with purified enzyme, PKC-alpha also appeared capable of self-association in murine B82L fibroblasts that were treated with calcium ionophore, phorbol ester, or epidermal growth factor but not in untreated cells. Collectively, these data indicate that self-association occurs in parallel with cPKC activation, that self-association is not mediated by the substrate binding site, and, at least in the case of PKC-alpha, that the formation of isoform homodimers predominates.
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PMID:Calcium and phosphatidylserine stimulate the self-association of conventional protein kinase C isoforms. 1050 5

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (delta) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous beta-estradiol. The vast majority of deltaRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of deltaRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from deltaA-Raf:ER than either deltaRaf-1:ER or deltaB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the deltaB-Raf:ER and deltaRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in deltaRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the deltaRaf:ER proteins as removal of beta-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.
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PMID:Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism. 1076 50

Previous study results have demonstrated that cigarette smoke or acetaldehyde rapidly stimulates protein kinase C (PKC)-mediated release of interleukin-8 (IL-8) in bovine bronchial epithelial cells (BECs). Low concentrations of acetaldehyde combine synergistically with malondialdehyde to increase significantly maximal BEC PKC activity at 48 to 96 h stimulation. Because more than 95% of alcoholics are cigarette smokers, we hypothesized that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, both a product of ethanol metabolism and a component of cigarette smoke, might stimulate PKC-mediated IL-8 release in BECs by malondialdehyde-acetaldehyde (MAA) adduct formation, rather than as free aldehydes. Protein kinase C activity is maximally elevated in BECs treated with 50 microg/ml of BSA-MAA from approximately 1 to 3 h. This activity subsequently begins to decrease by 4 to 6 h, with a return to baseline unstimulated kinase activity levels by 24 h. No activation of cyclic AMP-dependent protein kinase (PKA) or cyclic GMP-dependent protein kinase (PKG) was observed in BSA-MAA-treated BECs. The MAA adduct activation of PKC was followed by a fourfold to tenfold greater release of IL-8 over that observed for both BECs exposed to media only and BSA control-treated BECs. Protein kinase C activation and IL-8 release were blocked by pretreating BECs with 1 microM calphostin C or 100 nM of the PKC alpha-specific inhibitor, Go 6976. Isoform-specific inhibitors to PKC beta, PKC delta, and PKC zeta failed to inhibit completely MAA adduct-stimulated PKC or IL-8 release. Results of these studies indicate that metabolites derived from ethanol and cigarette smoke, such as acetaldehyde and malondialdehyde, form adducts that stimulate airway epithelial cell PKC alpha-mediated release of promigratory cytokines.
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PMID:Malondialdehyde-acetaldehyde-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells. 1183 59

The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons. Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5prie; exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles. There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N-terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N. This review will summarize the current information on isoform characteristics of both dUTPase and uracil-DNA glycosylase. It will also focus on evidence for phosphorylation and speculate as to the purpose of these post-translational events.
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PMID:The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity. 1236 30

In Caenorhabditis elegans, the gene unc-89 is required for A-band organization of striated muscle. In mammals, a likely homolog of UNC-89, called obscurin, has been described and found to be localized at both the M-lines and Z-discs of striated muscle. Here, we show that the coding sequence for unc-89 is larger than originally thought, and that the gene encodes at least four major isoforms: UNC-89-A (original isoform, 732 kDa), UNC-89-B (potentially 900 kDa), and UNC-89-C and UNC-89-D (each 156 kDa). UNC-89-C and -D, except for unique N-terminal tails of eight and 11 residues, respectively, are co-linear with the C terminus of UNC-89-B. The unc-89 complex transcription unit contains at least three promoters: one directing UNC-89-A and -B primarily in body-wall and pharyngeal muscle, one internal promoter directing expression of UNC-89-C primarily in body-wall muscle, and one internal promoter directing expression of UNC-89-D primarily in a few muscle cells of the tail. Isoform-specific RNA interference resulted in a muscle structural phenotype similar to a typical unc-89 mutant, but with varying degrees of severity. Antibodies generated to the interkinase region shared by the UNC-89-B, -C and -D isoforms localize to the middle of A-bands, like previously-described UNC-89 antibodies, and detect proteins on immunoblots consistent with the proposed gene organization and additional isoforms. The three new UNC-89 isoforms contain two protein kinase domains, of the myosin light chain kinase (MLCK) family. UNC-89-B contains two complete protein kinase domains, designated PK1 and PK2. UNC-89-C and -D begin with partial kinase domains, PK1-C and PK1-D. Homology modeling suggests that PK2 is catalytically active, PK1 is inactive, and that PK1-C and PK1-D have similar structures at their N termini that may create sites for interaction with other proteins.
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PMID:Three new isoforms of Caenorhabditis elegans UNC-89 containing MLCK-like protein kinase domains. 1531 9


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