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)

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.
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PMID:Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. 1061 55

The rate limiting step in steroidogenesis is cholesterol transport through the outer to the inner mitochondrial membrane and the cytochrome P450 side chain cleavage (P450scc) complex. The protein factor responsible for this transport, and as such necessary for regulating the acute production of steroids, has been identified and named the steroidogenic acute regulatory protein (StAR). We investigated the expression of StAR in functional and non-functional adrenal neoplasms and compared the expression with that of P450scc. Poly A RNA was extracted from normal adrenal glands (NAG, n=5), aldosterone producing adenomas (APA, n=4), cortisol producing adenomas (CPA, n=5), adrenocortical carcinomas (ACC, n=6) and non-functional adenomas (NFA, n=3), electrophoresed through a 1% agarose gel, blotted and hybridised with a PCR-generated cDNA labelled with [(32)P]CTP. The blots were stripped and re-hybridised with a P450scc cDNA and a mouse beta-actin probe. Compared with P450scc, StAR mRNA expression showed little variability in the magnitude of expression and did not correlate with the endocrine profiles (NAG: StAR 100+/-16%, P450scc 100+/-14%; APA: StAR 80+/-3%, P450scc 94+/-13%; CPA: StAR 71+/-10%, P450scc 109+/-15%; NFA: StAR 64+/-9.5%, P450scc 18+/-5%; means+/-s.e.m.). ACC expressed low levels of both genes probably as a result of dedifferentiation (StAR 29+/-9%, P450scc 46+/-18%). Incubation of the NCI-h295 tumour cell line with 10nmol ACTH and 10micromol forskolin induced an increase in the abundance of StAR and P450scc mRNA, demonstrating gene regulation by the cAMP protein kinase A pathway. Furthermore, we incubated the NCI-h295 tumour cell line with the adrenostatic compounds, aminoglutethimide and metyrapone. We could not detect an effect on the expression of StAR mRNA, whereas the expression of P450scc mRNA was significantly reduced. We conclude that, in contrast to P450scc, StAR seems to be evenly expressed in adrenocortical adenomas. Therefore, the endocrine activity of a given tumour cannot be explained by the abundance of StAR expression. In ACC, both StAR and P450scc expression is low, explaining the relatively inefficient steroid production of these tumours.
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PMID:Steroidogenic acute regulatory protein mRNA expression in adrenal tumours. 1070 Jul 25

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.
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PMID:Biochemical evidence for ATPase activity in CFTR-enriched apical membrane vesicles from tracheal epithelium. 1093 May 4

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have previously shown that the P protein of VSV, expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present study we present evidence that the P protein, in addition to being phosphorylated, binds covalently to GTP only when it is phosphorylated. Competition experiments show that ATP, ADP, GTP, and GDP can compete for the binding site(s) of GTP but not AMP, GMP, CTP, or UTP. Interestingly, once GTP is bound to P protein it cannot be displaced by unlabeled GTP. The GTP binding site has been mapped within the domain where the phosphorylation of P protein by CKII occurs. Finally, we show that phosphorylation negative P mutants P3A (P60A, P62A, P64A), P3E (P60E, P62E, P64E), and P3R (P60R, P62R, P64R) failed to bind to GTP, indicating that phosphorylation of P is indeed essential for binding to GTP. Although the precise role of binding of GTP to P is unclear, it appears that phosphorylation of P may initiate a structural change within the P protein allowing GTP to bind, thus manifesting biological function to the transcription factor.
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PMID:Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitis virus. 1217 45

When capsaicin, the pungent compound in hot pepper, is applied to epithelia it produces pain, allodynia, and hyperalgesia. We investigated, using whole cell path clamp, whether some of these responses induced by capsaicin could be a consequence of capsaicin blocking I(A) currents, a reduction in which, such as occurs in injury, increases neuronal excitability. In capsaicin-sensitive (CS) rat trigeminal ganglion (TG) neurons, capsaicin inhibited I(A) currents in a dose-dependent manner. I(A) currents were reduced 49% by 1 microM capsaicin. In capsaicin-insensitive (CIS) rat TG neurons, or small-diameter mouse VR1-/- neurons, 1 microM capsaicin inhibited I(A) currents 9 and 3%, respectively. These data suggest that in CS neurons the vast majority of the capsaicin-induced inhibition of I(A) currents occurs as a consequence of the activation of vanilloid receptors. Capsaicin (1 microM) did not alter the I(A) conductance-voltage relationship but shifted the inactivation-voltage curve about 15 mV to hyperpolarizing voltages, thereby increasing the number of inactivated I(A) channels at the resting potential. I(A) currents were relatively unaffected by 1 mM CTP-cAMP or 500 nM phorbol-12, 13-dibuterate (a protein kinase C agonist) but were inhibited by 20-30% with either 1 mM CTP-cGMP or 25 microM N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide HCl (a calcium-calmodulin kinase inhibitor). In the presence of 0.5 microM KT5823, an inhibitor of protein kinase G (PKG) pathways, 1 microM capsaicin inhibited I(A) by only 26%. In summary, in CS neurons, capsaicin decreases I(A) currents through the activation of vanilloid receptors. That activation, partially through the activation of cGMP-PKG and calmodulin-dependent pathways should result in increased excitability of capsaicin-sensitive nociceptors.
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PMID:Modulation of IA currents by capsaicin in rat trigeminal ganglion neurons. 1262 18

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Delta ura8Delta mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Delta ura8Delta mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from (32)P(i)-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.
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PMID:Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A. 1617 39

TRPM7 is a Ca(2+)- and Mg(2+)-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg(2+), the functional roles of intracellular levels of Mg.ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg.ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg(2+). Mg.ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg(2+) and Ca(2+) overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP > or = GTP > or = UTP > ITP approximately free Mg(2+) alone. These nucleotides also enhanced TRPM7 inhibition by free Mg(2+), suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg(2+)-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg.NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg(2+) binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg(2+) binding site extrinsic to that domain.
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PMID:TRPM7 channel is regulated by magnesium nucleotides via its kinase domain. 1653 98

The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5'-triphosphate, were used to affinity label purified Avena phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys(75),Ala(25)) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome.
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PMID:Affinity labeling of Avena phytochrome with ATP analogs. 1659 43

CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.
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PMID:Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation. 1718 48

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.
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PMID:Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3. 1768 42


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