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)

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.
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PMID:Characterization of synthetic peptide substrates for p34cdc2 protein kinase. 204 31

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.
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PMID:Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases. 308 Sep 87

Injection of whole adenovirus DNA into Xenopus oocytes results in the synthesis of large amounts of the early region 2A DNA-binding protein (E2A-DBP) and smaller amounts of polypeptide IX. The lack of synthesis of any functional messenger RNAs transcribed from the major late promotor at 16.3 map units is remarkable. Cleavage of the adenovirus DNA outside the E2A gene proper by restriction enzymes decreases synthesis of the DBP to about 10% of the amount produced after injection of intact DNA. On the other hand, presence of the terminal (Bellett) protein on the injected template enhances DBP synthesis considerably. Experiments with injected DNA restriction fragments, as well as reconstructed genes cloned into pBR322, indicate that efficient synthesis of DBP in oocytes requires the presence of either or both of the two main promoters from which the E2A gene is transcribed plus an intact 3' end of the gene. In the absence of any known promotor, 100-fold lower amounts of otherwise normal DBP are produced. Unlike in a regular infection, synthesis of DBP in oocytes does not require the product of the E1A gene. The same series of experiments also demonstrates that the DBP, a phosphoprotein, is the substrate of a cellular rather than a virus-encoded protein kinase. Two minor E2A proteins, although colinear with the major DBP, are synthesized independently. Synthesis of a 44,000 Mr protein, probably corresponding to the carboxy-terminal 360 amino acid residues of the DBP, is not decreased after injection of "promotorless" E2A genes. Unlike the 44,000 Mr protein, production of a 67,000 Mr protein (carboxy-terminal 483 amino acid residues) by one DNA-construct is probably directed by a T-A-T-A-A-A-T-A sequence in the vector DNA.
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PMID:Analysis of expression of adenovirus DNA (fragments) by microinjection in Xenopus oocytes. Independent synthesis of minor early region 2 proteins. 630 67

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.
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PMID:Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis. 711 96

Exposure of mammalian cells to ionizing radiation causes a delay in progression through the cycle at several checkpoints. Cells from patients with ataxia-telangiectasia (A-T) ignore these checkpoint controls postirradiation. The tumour suppressor gene product p53 plays a key role at the G1/S checkpoint preventing the progression of cells into S phase. The induction of p53 by radiation is reduced and/or delayed in A-T cells, which appears to account for the failure of delay at the G1/S checkpoint. We have investigated further this defect in radiation signal transduction in A-T. While the p53 response was defective after radiation, agents that interfered with cell cycle progression such as mimosine, aphidicolin and deprivation of serum led to a normal p53 response in A-T cells. None of these agents caused breaks in DNA, as determined by pulse-field gel electrophoresis, in order to elicit the response. Since this pathway is mediated by protein kinases, we investigated the activity of several of these enzymes in control and A-T cells. Ca+2-dependent and -independent protein kinase C activities were increased by radiation to the same extent in the two cell types, a variety of serine/threonine protein kinase activities were approximately the same and anti-tyrosine antibodies failed to reveal any differences in protein phosphorylation between A-T and control cells. It is not evident what is the nature of the defect in signal transduction in A-T cells. However, it is clear that the p53 response is normal in these cells after exposure to some agents and it is mediated through protein kinase C or another serine/threonine kinase.
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PMID:Defect in radiation signal transduction in ataxia-telangiectasia. 753 Jul 54

We have previously demonstrated that cells from patients with ataxia-telangiectasia (A-T) fail to show initial delay at several cell cycle checkpoints post-irradiation. In addition a defect in the induction of p53 by ionizing radiation was evident. We demonstrate here that the radiation signal transduction pathway operating through p53, its target gene WAF1, cyclin-dependent kinases and the retinoblastoma (Rb) protein is defective in A-T cells. The defective p53 induction after ionizing radiation, observed previously in A-T cells, was also reflected at the functional level using p53-DNA binding activity, transactivation and transfection with wild type p53. Correction of the defect at the G1/S checkpoint was observed when wild type p53 was constitutively expressed in A-T cells. Exposure of control cells to radiation gave rise to p53 induction and as a consequence increased expression of WAF1 mRNA and protein, but A-T cells were defective in this response. As expected the WAF1 response in irradiated control cells resulted in an inhibition of cyclin-dependent kinase activity including cyclin E-cdk2, which plays an important role in the transition from G1 to S phase. No inhibition of cyclin-dependent kinase activity was observed in A-T cells correlating with the delayed WAF1 response. On the contrary an enhancement of cyclin-dependent kinase activity was seen in A-T cells post-irradiation. An accumulation of the hypophosphorylated form of Rb protein occurred in irradiated control cells compatible with the G1/S phase delay observed in these cells after exposure to radiation. In unirradiated A-T cells the amount of Rb protein was much higher compared to controls and it was mainly in the hyperphosphorylated (functionally inactive) form. In addition, accumulation of the hypophosphorylated form of Rb in A-T cells post-irradiation was defective, consistent with the lack of cell cycle arrest. Thus the failure of the G1/S checkpoint in A-T cells after exposure to ionizing radiation is consistent with a defective radiation signal transduction pathway operating through p53.
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PMID:Nature of G1/S cell cycle checkpoint defect in ataxia-telangiectasia. 765 23

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-C-->C-G or T-A-->G-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine had G-C-->A-T mutations at non-CpG sites; in addition, EMS induced several A-T-->G-C, A-T-->T-A, and G-C-->T-A substitutions. A single ICR191-induced mutant analyzed had a unique A-T-->G-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.
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PMID:Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens. 797 5

The recent description of a novel gene (ATM) mutated in ataxia-telangiectasia (A-T), with homologies to genes encoding proteins involved in both G1/S and G2/M checkpoint control, points to a common defect in cell cycle control in A-T operating through the cyclin-dependent kinases. In this report we demonstrate that cyclin-dependent kinases are resistant to inhibition by ionizing radiation exposure in A-T cells, and this appears to be due to insufficient induction of WAF1. Exposure of control lymphoblastoid cells to radiation during S phase and in G2 phase causes a rapid inhibition of cyclin A-Cdk2 and cyclin B-Cdc2 activities, respectively. Irradiation led to a 5-20-fold increase in Cdk-associated WAF1 in these cells, which accounts at least in part for the decrease in cyclin-dependent kinase activity. In contrast, radiation did not inhibit any of the cyclin-dependent kinase activities in S phase or G2 phase in A-T cells at short times after irradiation nor was there any significant change in the level of Cdk-associated WAF1 compared to unirradiated cells. These results are similar to those reported previously for the G1 checkpoint and provide additional evidence for the involvement of ATM at multiple points in cell cycle regulation.
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PMID:Defect in multiple cell cycle checkpoints in ataxia-telangiectasia postirradiation. 870 89

The gene mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently (Savitsky et al., 1995a) and the complete coding sequence of this gene, ATM, has been reported (Savitsky et al., 1995b). The derived amino acid sequence demonstrates significant homologies to several proteins containing a phosphatidylinositol 3-kinase (PI3-kinase) domain, including the yeast TOR proteins and the human protein FRAP. Since the TOR and FRAP proteins are targets for the immunosuppressive drug rapamycin, we have investigated the effects of this compound on A-T cells. We report here that 3 A-T cell lines are more resistant than control cells to rapamycin's growth inhibiting effects but were more sensitive to the PI3-kinase inhibitor wortmannin. As expected rapamycin (1 nM) inhibited the rate of exit of control cells from G1 phase but failed to perturb the progression of A-T cells. This difference in cell cycle progress after rapamycin treatment is reflected in ribosomal S6 protein kinase (p70S6k) by both a downward mobility shift on SDS-PAGE and inhibition of activity. Furthermore, the G1 phase cyclin-dependent kinase, cyclin E-cdk2, was rapidly inhibited in control cells post-treatment, whereas in A-T cells it took considerably longer to observe inhibition. There was no evidence that a GST-FKBP12 fusion protein specifically precipitated the ATM protein in the presence of rapamycin in either cell type. These results demonstrate that the ATM protein is not a direct target for rapamycin but its functional loss renders cells more resistant to this compound.
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PMID:Rapamycin resistance in ataxia-telangiectasia. 880 86

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.
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PMID:The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes. 884 91


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