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 multienzyme polypeptide CAD is phosphorylated at two sites by cyclic AMP (cAMP)-dependent protein kinase. Site 2 has two interesting features: it is located in a 'linking region' between two discretely folded enzyme domains, and a histidine, instead of the more usual arginine, is found three positions N-terminal to the phosphorylated serine. A synthetic peptide corresponding to the sequence around site 2 has an extended or random structure in solution, and the proton n.m.r. chemical shift of the histidine residues can be titrated against pH in the range 6.0-8.0. The peptide is phosphorylated more rapidly by cAMP-dependent protein kinase at lower pH values, indicating that the protonated histidine side chain corresponds to the arginine in the consensus recognition sequence for the kinase. Kemptide, a specific synthetic substrate for the kinase, was phosphorylated with a higher affinity and at a similar rate at all pH values. CAD was a better substrate than the synthetic peptide, and labelling was not affected by the pH of the incubation conditions. The results indicate that the phosphorylation site in the interdomain linker is sufficiently exposed to the solvent to ensure accessibility to the kinase, but that secondary or tertiary structure in the intact protein allows the histidine residue to remain protonated at physiological pH and enhances recognition of the phosphorylatable serine residue.
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PMID:A protonated histidine residue in a phosphorylation site for cyclic AMP-dependent protein kinase. Comparison of a synthetic peptide with the exposed linking region in the multienzyme polypeptide CAD. 135 77

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.
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PMID:Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. 334 46

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.
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PMID:Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis. 409 95

Collateral blood vessels supplement normal coronary blood flow and coronary blood flow compromised by coronary artery disease, thereby protecting the myocardium from ischemia. Collateral vessel formation is the result of angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a secreted mitogen specific for endothelial cells and an extremely potent angiogenic factor. In the present study, VPF/VEGF mRNA and protein were demonstrated to be markedly stimulated in primary rat cardiac myocytes in vitro in response to reduction of the oxygen tension to 1% or inhibition of the electron transport chain. Four isoforms of VPF/VEGF were coordinately regulated by hypoxia, including a novel isoform not previously described. Phorbol ester and the depolarizing agent veratridine, stimulators of protein kinase C and calcium influx, respectively, were found to markedly increase VPF/VEGF mRNA expression in cardiac myocytes. Forskolin, a potent stimulator of adenylate cyclase, produced a small but significant increase in VPF/VEGF mRNA expression in the cardiac myocytes. However, only H7, an inhibitor of protein kinase C, inhibited the hypoxic induction of VPF/VEGF mRNA; inhibitors of calcium influx and the calcium-calmodulin-dependent protein kinase II as well as inhibition of protein kinase A did not block the hypoxic induction of VPF/VEGF mRNA. This suggests that more than one signal transduction pathway is involved in regulating VPF/VEGF expression. The sensor that regulates the expression of hypoxia-responsive genes has been proposed to be a heme protein. Consistent with this model, transition metals initiate a genetic program similar to hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of vascular endothelial growth factor in cardiac myocytes. 772 92

The proto-oncogene Raf-1 is a cytoplasmic serine/threonine kinase implicated in the signaling process in cell proliferation. To determine if Raf-1 is sufficient and necessary to transmit mitogenic signals to growth-responsive genes, we examined the effect of constitutively activated (v-raf) or inhibitory (Raf-C4) Raf-1 proteins on reporter gene activation in transient expression assays of NIH 3T3 cells. In serum-starved cells, v-raf strongly activated transcription from the promoters of the immediate-early genes c-fos and egr-2, as well as the proximal or B promoter of the late growth response gene rep-3 (rep-3b). Two other late response gene promoters, cad and dhfr, were only modestly activated by v-raf, however. An individual serum response element from the c-fos or egr-2 promoter conferred both serum-inducibility and v-raf-responsiveness to a heterologous promoter. Consistent with the degree to which antisense c-raf-1 RNA and dominant-negative Raf-1 proteins interfere with NIH 3T3 cell proliferation, Raf-C4 reduced serum-induced transcription from the egr-2 and rep-3b promoters in a dose-dependent manner by 50%. In contrast, Raf-C4 did not significantly reduce transcription from the c-fos or cad promoters or the serum response element-driven heterologous promoters. We conclude that Raf-1 is both sufficient and necessary to activate a subset of early and late growth response genes.
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PMID:An inhibitory Raf-1 mutant suppresses expression of a subset of v-raf-activated genes. 834 Mar 92

The report is a discussion of previously published and newly analyzed results concerning the association between heart diseases and alterations in the force-frequency relation (FFR). The optimum stimulation frequency of the FFR is measured and compared in isolated left ventricular myocardium from non-failing hearts with atrial septal defect, coronary artery disease (without and with insulin dependent diabetes mellitus) and from failing hearts with mitral regurgitation, or idiopathic dilated cardiomyopathy. Specifically, we examine the role of altered control of the excitation-contraction coupling system in blunting the force-frequency relation. We use the percent slope of the FFR as a measure of changes in the frequency sensitivity of this control. Our finding of a linear, direct relation between optimum stimulation frequency and % slope across all disease types suggests both parameters are coupled to the same underlying mechanism. To investigate the possible role of altered control of the calcium pump in this mechanism, we analyzed the detailed relation between isometric twitch relaxation kinetics and stimulation frequency in mitral regurgitation myocardium (MR). In the presence of 0.5 microM forskolin the depressed slope and optimum frequency of the FFR and the prolonged half-time of twitch relaxation were all restored to values found in non-failing myocardium. We use the kinetics of isometric twitch relaxation as an index of changes in pumping rate that occur in response to changes in stimulation frequency or in intracellular cyclic adenosine monophosphate concentration. A mathematical model based on the Hill relations for calcium pump uptake rate and for isometric tension as a function of intracellular pCa is developed to simulate isometric twitch relaxation in MR and non-failing myocardium. The success of this model in simulating non-failing and failing twitch relaxation supports a proposed mechanism for the prolonged relaxation time and depressed FFR in MR involving depressed protein kinase-A activity (due to lowered cAMP or to a defect in the Ser16 site of phospholamban) as a mechanism of altered control of the calcium pump in MR heart disease.
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PMID:Role of cAMP in modulating relaxation kinetics and the force-frequency relation in mitral regurgitation heart failure. 920 49

Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation.
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PMID:Site-directed substitution of Ser1406 of hamster CAD with glutamic acid alters allosteric regulation of carbamyl phosphate synthetase II. 921

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.
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PMID:Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability. 935 52

The endothelial cells of the vascular system are responsible for many biological activities that maintain vascular homeostasis. Responding to a variety of chemical and physical stimuli, the endothelium elaborates a host of vasoactive agents. One of these agents, endothelium-derived relaxing factor, now accepted as nitric oxide, influences both cellular constituents of the blood and vascular smooth muscle. A principal intracellular target for nitric oxide is guanylate cyclase, which, when activated, increases the intracellular concentration of cyclic guanosine monophosphate, which in turn activates protein kinase G. Acting by this pathway, nitric oxide induces relaxation of vascular smooth muscle and inhibits platelet activation and aggregation. Derangements in endothelial production of nitric oxide are implicated as both cause and consequence of vascular diseases, including hypertension, atherosclerosis, and coronary artery disease.
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PMID:Nitric oxide and regulation of vascular tone: pharmacological and physiological considerations. 950 27

Adenomatous polyposis coli (APC) mutations are present in >70% of colon cancers. The APC protein binds to beta-catenin (beta-cat), a protein first identified because of its role in E-cadherin (E-cad) cell adhesion. In some colon cancers lacking APC defects, mutations in presumptive glycogen synthase kinase 3beta phosphorylation sites near the beta-cat NH2 terminus appear to render beta-cat resistant to regulation by APC and glycogen synthase kinase 3beta. In cells with APC or beta-cat defects, beta-cat is stabilized and, in turn, binds to and activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef) transcription factors. To further explore the role of APC, beta-cat, Tcf, and E-cad defects in gastrointestinal cancers, we assessed gastric and pancreatic cancers for constitutive Tcf transcriptional activity (CTTA). Two of four gastric and two of eight pancreatic cancer lines showed CTTA. One gastric and one pancreatic cancer had mutations in the NH2-terminal phosphorylation sites of beta-cat. The other gastric cancer with CTTA had a missense mutation at serine 28 of gamma-cat, a potential phosphorylation site in this beta-cat-related protein. Although E-cad is an important binding partner for beta-cat and gamma-cat, E-cad inactivation did not result in CTTA. The beta-cat and gamma-cat mutant proteins identified in our studies strongly activated Tcf transcription in vitro, whereas beta-cat mutant proteins with large NH2-terminal deletions had only modest effects on Tcf. Our results suggest a role for Tcf deregulation in gastric and pancreatic cancer, resulting from beta-cat and gamma-cat mutations in some cases and, in others, from yet to be defined defects. Furthermore, these data imply that the consequences of APC and beta-cat mutations are distinct from the effects of E-cad inactivation.
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PMID:Beta- and gamma-catenin mutations, but not E-cadherin inactivation, underlie T-cell factor/lymphoid enhancer factor transcriptional deregulation in gastric and pancreatic cancer. 1039 98


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