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)

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.
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PMID:In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase. 200 28

Inhibiting cAMP-dependent protein kinase (A-kinase) in mammalian fibroblasts through microinjection of a modified specific inhibitor peptide, PKi(m) or the purified inhibitor protein, PKI, resulted in rapid and pronounced chromatin condensation at all phases of the cell cycle. Together with these changes in chromatin, a marked reorganization of microtubule network occurred, accompanied in G2 cells by extensive alterations in cell shape which have many similarities to the premitotic phenotype previously observed after activation of p34cdc2 kinase, including the lack of spindle formation and the persistence of a nuclear envelope. In order to examine whether A-kinase inhibition and p34cdc2 kinase form part of the same or different inductive pathways, PKI and p34cdc2 kinase were injected together. Co-injection of both components resulted in nuclear envelope disassembly, an event not observed with injection of either component alone. This result implies that p34cdc2 and A-kinase inhibition have complementary and additive effects on the process of nuclear envelope breakdown in living fibroblasts, a conclusion further supported by our observation of a pronounced dephosphorylation of lamins A and C in cells after injection of PKi(m). Taken together, these data suggest that down-regulation of A-kinase is a distinct and essential event in the induction of mammalian cell mitosis which co-operates with the p34cdc2 pathway.
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PMID:Inhibition of cAMP-dependent protein kinase plays a key role in the induction of mitosis and nuclear envelope breakdown in mammalian cells. 202 48

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.
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PMID:Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors. 204 6

Synthetic peptides corresponding to the autoinhibitory domains of calcium/calmodulin-dependent protein kinase II (CaMK-(281-309)), smooth muscle myosin light chain kinase (MLCK-(480-501)), and protein kinase C (PKC-(19-36)) as well as a peptide derived from the heat-stable inhibitor of cAMP-dependent protein kinase (PKI-tide) were tested for their inhibitory specificities. The inhibitory potencies of the four peptides were determined for each of the four protein kinases using both peptide substrates (at approximate Km concentrations) and protein substrates (at concentrations less than Km). In agreement with previous studies PKI-tide was a specific and potent inhibitor of only cAMP kinase, and none of the other inhibitory peptides gave significant inhibition of cAMP kinase at concentrations of less than 100 microM. With synthetic peptide substrates, PKC-(19-36) strongly inhibited native PKC (IC50 less than 1 microM) but also significantly inhibited autophosphorylated CaMK-II (IC50 = 30 microM) and proteolytically activated MLCK (IC50 = 35 microM). MLCK-(480-501) potently inhibited MLCK (IC50 = 0.25 microM) and also strongly inhibited both PKC and CaMK-II (IC50 = 1.4 and 1.7 microM, respectively). CaMK-(281-309) inhibited autophosphorylated CaMK-II, PKC, and proteolyzed MLCK almost equally (IC50 = 10, 38, and 48 microM, respectively). Qualitatively similar results were obtained with protein substrates. These studies validate the use of PKI-tide as a specific inhibitor of cAMP kinase in intact cell studies and suggest that PKC-(19-36) can also be used but only within a narrow concentration range. However, the autoinhibitory domain peptides from MLCK and CaMK-II are not sufficiently specific to be used in similar investigations.
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PMID:Specificities of autoinhibitory domain peptides for four protein kinases. Implications for intact cell studies of protein kinase function. 215 65

Exposure of beta-adrenergic receptors (beta ARs) to agonists causes rapid desensitization of the receptor-stimulated adenylyl cyclase response. Three main mechanisms have been implicated in this process: phosphorylation of the receptors by the cAMP-dependent protein kinase (PKA), phosphorylation by the specific agonist-dependent beta AR kinase, and sequestration of the receptors away from the cell surface. By applying inhibitors of these processes to digitonin-permeabilized A431 cells we investigated their contributions to beta AR desensitization. Each process could be selectively inhibited: PKA-dependent phosphorylation by an inhibitor peptide (amino acids 1-24 of the heat-stable inhibitor of PKA (PKI], beta AR kinase-dependent phosphorylation by heparin, and sequestration by concanavalin A. In permeabilized cells, heparin plus PKI completely blocked agonist-induced phosphorylation of the beta ARs. Desensitization was assessed by quantitating the signal transduction efficacy of the system. At high agonist concentrations (approximately 1 microM) up to 70% desensitization occurred. Complete blockade of this desensitization required the concurrent inhibition of all three pathways. When individual pathways were blocked it could be demonstrated that either the PKA or beta AR kinase mechanisms alone resulted in 40-50% desensitization whereas sequestration alone caused 20-30% desensitization. At low agonist concentrations (approximately 10 nM), the PKA pathway was selectively activated. These data indicate that while desensitization mediated via the three different mechanisms can occur independently, the quantitative contributions are not additive. Such findings suggest distinct but overlapping physiological roles for each mechanism in controlling receptor function.
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PMID:Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. 215 73

Cyclic nucleotides (both cAMP and cGMP) stimulate the phosphorylation of several proteins of 65-70, 50-52, 21, 13, and 12 kD in rod outer segments (ROS) of the frog retina. Subcellular fractionation showed that phosphopeptides of 67, 21, 13, and 12 kD were soluble and phosphopeptides of 69, 67, 50-52, and 12 kD were membrane associated at physiological ionic strength. Components I and II, 13 and 12 kD, respectively, are the major cyclic nucleotide-dependent phosphoproteins of ROS and have been reported to be phosphorylated in the dark and dephosphorylated in the light. Under unstimulated conditions, phosphorylated Components I and II were found in the soluble fraction. Cyclic nucleotide stimulation of phosphorylation resulted in increased phospho-Components I and II in the soluble fraction, and phospho-Component II on the membrane. Light had no effect on the phosphorylation level of soluble Components I and II, but it caused a depletion within 1 s of the membrane-bound phospho-Component II. A half-maximal decrease in membrane-bound Component II was seen at 5 x 10(5) rhodopsins bleached per outer segment. The cyclic nucleotide-dependent protein kinase(s) were found primarily in the peripheral membrane fraction of ROS proteins. 8-bromo cyclic AMP was two orders of magnitude more effective than 8-bromo cyclic GMP at stimulating Component I and II phosphorylation. An active peptide of the Walsh inhibitor of cAMP-dependent protein kinase [PKI(5-22)amide] blocked the phosphorylation with an IC50 of 10 nM. Photoaffinity labeling studies with 8-N3-cAMP and 8-N3-cGMP revealed the presence of a 52-kD band specifically labeled with 8-N3-cAMP, but no specific 8-N3-cGMP labeling. These data suggest that cyclic nucleotide-dependent protein phosphorylation in ROS occurs via the activation of a cAMP-dependent protein kinase.
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PMID:Regulation by light of cyclic nucleotide-dependent protein kinases and their substrates in frog rod outer segments. 215 94

A new eukaryotic initiation factor 2 kinase has been purified for the first time from calf brain cytosol. The purification of a nonabundant novel protein kinase activity, designated as PKI, that phosphorylates the alpha subunit of eukaryotic initiation factor 2 is described. The protein kinase activity was assayed using purified initiation factor 2 as a substrate and was purified by ammonium sulphate precipitation, conventional chromatography in heparin-Sepharose and phosphocellulose and by high performance size exclusion and anion exchange chromatographies. The protein kinase activity elutes in the region of 140,000 in the size exclusion chromatography and is associated with two different polypeptides a and b, with relative molecular masses of 38,000 and 20,000 and an approximate ratio of 2.5-3.0:1. The protein kinase does not phosphorylate casein or histones and it is independent of cyclic nucleotides. It can be classified as a serine kinase since the phosphorylation of the alpha subunit of eIF-2 is produced in serine residues. Under these conditions none of the kinase subunits are phosphorylated.
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PMID:Purification of a novel eIF-2 alpha protein kinase from calf brain. 230 40

We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2'-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.
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PMID:Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. 242 Jun 61

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.
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PMID:Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase. 246 99

Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells. 247 35


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