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 inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. 149 92

Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.
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PMID:Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. 151 24

During chemically induced differentiation of murine erythroleukemia (MEL) cells, cAMP-dependent protein kinase activity increases, and the enzyme's isozyme pattern changes. To examine the enzyme's role during MEL cell differentiation, we stably transfected MEL cells with recombinant plasmids in which the mouse metallothionein I promoter controlled expression of either a mutant form of the type I regulatory subunit of cAMP-dependent protein kinase (RI) or the enzyme's specific peptide inhibitor (PKI); expressing either sequence rendered cells cAMP-dependent protein kinase-deficient. Chemically induced differentiation of MEL cells as assessed by beta-globin mRNA and hemoglobin accumulation was inhibited in RI mutant and PKI transfectants; adding zinc further inhibited differentiation in the transfectants but had no effect on parental MEL cells. The inhibition of differentiation correlated with the amount of RI mutant mRNA and protein in the RI mutant transfectants and with the cells' degree of cAMP-dependent protein kinase deficiency in both the RI mutant and PKI transfectants. Overexpression of wild type RI did not interfere with differentiation or enzyme activity. We conclude that cAMP-dependent protein kinase activity is important for chemically induced differentiation of MEL cells and that the down-regulation of RI protein which occurs during MEL cell differentiation is not essential for differentiation to proceed.
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PMID:Chemically induced murine erythroleukemia cell differentiation is severely impaired when cAMP-dependent protein kinase activity is repressed by transfected genes. 164 3

The theme of this study is an evaluation of the involvement of cAMP and cAMP-dependent protein kinase (PKA) in the regulation of the human heat shock protein (hsp) 70 gene promoter. Expression of a highly specific protein inhibitor of PKA (pRSVPKI) inhibited the basal as well as heat- and cadmium-induced expression of the cotransfected pHBCAT, a human hsp 70 promoter-driven reporter gene; this inhibition was dependent on the amount of pRSVPKI used. The effect of an expression vector of the RI regulatory subunit of PKA, pMTREV, was similar to that of pRSVPKI; pMTREV inhibited both the basal as well as the heat-induced expression of pHBCAT. The specificity of effects of these expression vectors was demonstrated by the lack of effect of a mutant PKI gene and by the unaffected expression of a reference gene (pRSV beta gal) under these conditions. Analysis of the effects of dibutyryl cAMP (1 mM), forskolin (10 microM), and 8-Br-cAMP (1 mM) on the transient expression of pHBCAT showed that these cAMP-elevating agents stimulated the hsp 70 promoter activity, whereas cAMP (1 mM) was without effect. Chloramphenicol acetyltransferase gene constructs with truncated or mutated hsp 70 promoter were used to define the cis-acting DNA element(s) that confer this cAMP stimulation; the heat induced (42 degrees C) expression was used as a control. Mutation of the adenovirus transcription factor element (pLSN-40/-26) greatly reduced the basal level of expression; forskolin had little or no effect on this adenovirus transcription factor-minus promoter, although the promoter activity was very heat inducible. The absence of a functional heat shock consensus element (HSE) in the construct pLSPNWT rendered the promoter heat insensitive; this construct was forskolin responsive although the magnitude of this stimulation was reduced when compared with that of a control construct with HSE. These results were corroborated by studies using consensus sequence of ATF (ATFE) and HSE as competitors to titrate our cellular factors that may interact with these elements. We showed that cotransfection with ATFE and HSE depressed the basal (37 degrees C) expression of pHBCAT by 25 and 60%, respectively. The heat-induced expression of pHBCAT was not significantly affected by the cotransfection of ATFE and was reduced by 60% when HSE was cotransfected. ATFE and HSE reduced the forskolin-induced pHBCAT expression by 70 and 40%, respectively. The implications of these findings as they relate to the action of cAMP and cAMP-dependent protein kinase in the control of heat shock gene expression are discussed.
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PMID:cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity. 164 17

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.
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PMID:Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. 164 31

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII. PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 microM-free Ca2+ and 1 microgram/ml of the modulator. The stimulatory effect of the Ca(2+)-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80. PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca(2+-)-calmodulin dependent protein kinase, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.
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PMID:Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa. 165 80

1. The characteristics have been examined of the high threshold calcium channel current in cultured rat dorsal root ganglion (DRG) neurones recorded in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM in the patch pipette). This current, termed IBa, GTP gamma S, was slowly activating and showed little inactivation over 100 ms. 2. External application of forskolin (10 microM) to elevate internal cyclic AMP levels increased the amplitude of IBa, GTP gamma S whereas it had no effect on the control IBa. This cyclic AMP-dependent protein kinase (PKI; 25 microM). 3. The cyclic AMP-dependent phosphorylation induced enhancement of IBa, GTP gamma S was voltage dependent and either did not occur or was observed only transiently at a holding potential (VH) of -30 mV. The forskolin-stimulated enhancement seen at VH -80 mV was lost with a t1/2 of about 1 min when VH was depolarized to -30 mV. Cholera toxin pre-treatment also increased the amplitude of IBa, GTP gamma S at VH -80 mV but not at VH -30 mV. 4. The calcium channel antagonist (-)-202-791 (5 microM) increased the amplitude of IBa, GTP gamma S when applied at VH -80 mV, but either not, or only transiently, at VH -30 mV, as previously observed. This 'agonist' effect of (-)-202-791 was prevented by PKI and was occluded by prior enhancement of IBa, GTP gamma S with forskolin. (-)-202-791 did not increase cyclic AMP levels in DRG neurones. 5. The 'agonist' response of IBa, GTP gamma S to D600 (10 microM) was also occluded by application of forskolin (10 microM) in the patch pipette. Forskolin alone, applied in this manner, increased IBa, GTP gamma S to a similar extent to D600 applied alone. 6. The agonist effect of (+)-202-791 (5 microM) on IBa, GTP gamma S was not prevented by prior enhancement with forskolin, nor was it prevented by PKI. 7. In conclusion, internal GTP gamma S activates G proteins which may interact directly with calcium channels to influence the kinetics of activation and to reduce steady-state inactivation of the channels. There is also an indirect effect on the generation of second messengers such as cyclic AMP. It is likely that forskolin enhances IBa, GTP gamma S by increasing activated Gs coupling to adenylyl cyclase and increasing cyclic AMP generation. The mechanism of action of (-)-202-791 to enhance IBa, GTP gamma S also involves cyclic AMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ca2+ channel currents in rat sensory neurones: interaction between guanine nucleotides, cyclic AMP and Ca2+ channel ligands. 165 19

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.
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PMID:Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine. 165 25

Primary uterine cell cultures were used to study multifactor regulation of progesterone receptor (PR) and the signal transduction pathways which may serve to mediate that regulation. Increases in intracellular cAMP, brought about by treatment with cholera toxin plus isobutyl methyl xanthine or by addition of 8-bromo-cAMP, result in 6- to 7-fold increases in the intracellular content of PR as monitored by [3H]R5020 binding and by Western immunoblot using anti-PR antibodies. In these primary cultures of uterine cells isolated from 19-day-old immature rats, 8-bromo-cAMP evokes significant increases in PR by 8 h with maximal increases by 24 h. This time course and magnitude of PR stimulation are similar to those evoked by maximally effective concentrations of estradiol (3 x 10(-9) M) or IGF-I (20 ng/ml). Dose-response studies reveal that 10(-6) to 10(-4) M concentrations of 8-bromo-cAMP (8-Br-cAMP) elicit a maximal response. In contrast, 8-bromo-cGMP over a wide concentration range was unable to elevate cellular PR levels. Under these culture conditions, cell proliferation was not altered by treatment with any of these agents. Although estrogen, cAMP, and insulin-like growth factor I (IGF-I) may act via different pathways to increase PR, the effects evoked by maximally effective concentrations of these agents are not additive implying involvement of a common component. The increases in PR evoked by estradiol, cAMP, or IGF-I are markedly suppressed by treatment with antiestrogen (ICI 164,384) or the cyclic nucleotide-dependent protein kinase inhibitor H8 or the protein kinase A inhibitor PKI, indicating the involvement of the estrogen receptor and phosphorylation pathways in PR regulation by these three agents. The present studies identify cAMP, as well as estrogen and IGF-I, as important regulators of the level of PR in uterine cells and suggest that multiple factors, including those affecting intracellular cAMP levels, might influence responsiveness to progestins via regulation of the intracellular PR content.
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PMID:Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. 170 63

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.
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PMID:Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts. 191 18


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