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1.
Protein kinase activity associated with the purified rat hepatic glucocorticoid receptor. 总被引:1,自引:0,他引:1
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A Miller-Diener T J Schmidt G Litwack 《Proceedings of the National Academy of Sciences of the United States of America》1985,82(12):4003-4007
The Mr 94,000 steroid binding component of rat hepatic glucocorticoid receptor purified 5000-fold under-goes calcium-stimulated phosphorylation in vitro by [gamma-32P]ATP. Exogenous histones can be phosphorylated by this preparation without calcium. Calmodulin did not stimulate phosphorylation of the glucocorticoid receptor beyond that obtained with calcium alone. Although the specific calmodulin inhibitor calmidazolium had no effect, trifluoperazine and chlorpromazine, nonspecific calmodulin inhibitors, abolished the calcium-dependent phosphorylation of receptor. EGTA blocks the effect of calcium; magnesium cannot substitute for calcium. Cyclic nucleotides (cAMP or cGMP) do not stimulate phosphorylation of the receptor in the absence of calcium. Phosphorylation of the glucocorticoid receptor is steroid dependent. Triamcinolone acetonide elicited activation and phosphorylation of receptor in the presence of calcium, whereas the antagonists progesterone, cortexolone, and beta-lapachone did not. Sodium molybdate, which blocks the thermal activation step, inhibits phosphorylation of the receptor. The activated form of the glucocorticoid receptor is required for phosphorylation to occur. The ATP analogues 8-azido-ATP or fluorosulfonylbenzoyl adenosine, inhibit phosphorylation of the Mr 94,000 component, implying the presence of an ATP binding site inherent to the receptor. 相似文献
2.
Protein kinase activity associated with simian virus 40 T antigen. 总被引:22,自引:6,他引:22
J D Griffin G Spangler D M Livingston 《Proceedings of the National Academy of Sciences of the United States of America》1979,76(6):2610-2614
Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. 相似文献
3.
Protein kinase activity associated with the avian sarcoma virus src gene product. 总被引:194,自引:94,他引:194
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M S Collett R L Erikson 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(4):2021-2024
Incorporation of phosphorus from [gamma-32P]ATP into protein was catalyzed by specific immunoprecipitates from avian sarcoma virus (ASV)-transformed avian and mammalian cells. This incorporation was observed only when antiserum from tumor-bearing rabbits able to specifically precipitate the ASV sarcoma gene product, p60src, was used to immunoprecipitate antigens from transformed cell lysates. Immunoprecipitates of extracts from normal cells or cells infected with a transformation-defective ASV mutant showed no activity in this assay, nor did any immune complexes formed with normal rabbit serum and any of the cell extracts tested. The expression of the protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was growth temperature-dependent in cells infected with an ASV mutant temperature-sensitive for the transformation. These results on an enzymatic activity associated with the ASV transforming protein are discussed in terms of protein phosphorylation as a mechanism for viral transformation. 相似文献
4.
Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. 总被引:76,自引:16,他引:60
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S I Reed J A Hadwiger A T L?rincz 《Proceedings of the National Academy of Sciences of the United States of America》1985,82(12):4055-4059
Antibodies raised against the protein encoded by a lacZ-CDC28 in-frame fusion were shown to immunoprecipitate the CDC28 product from yeast cell lysates. The polypeptide p36CDC28 is a phosphoprotein of apparent Mr 36,000. Immune complexes prepared from yeast cell lysates by using anti-CDC28 antibody were found to possess a protein kinase activity, as determined by the transfer of label from [gamma-32P]ATP to a coprecipitated Mr 40,000 protein of unknown identity or function (p40). This activity was absent or thermolabile when extracts were prepared from several different cdc28 temperature-sensitive strains. The protein kinase activity was dependent on Zn2+ and transferred phosphate specifically to serine and threonine residues. 相似文献
5.
Tyrosine-specific protein kinase activity is associated with the purified insulin receptor. 总被引:27,自引:13,他引:27
M Kasuga Y Fujita-Yamaguchi D L Blithe C R Kahn 《Proceedings of the National Academy of Sciences of the United States of America》1983,80(8):2137-2141
Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [gamma-32P]ATP in the presence of MnCl2 (2 mM) and analyzed in NaDodSO4/acrylamide gel electrophoresis, only the Mr 95,000 band was labeled. Preincubation with several concentrations of insulin increased the 32P incorporation into this peptide in dose-dependent fashion, whereas insulin-like growth factors were approximately equal to 2% as potent and epidermal growth factor had little or no effect, consistent with their known affinities for the insulin receptor. Insulin stimulation of phosphorylation of the Mr 95,000 subunit of the receptor was observed also in immunoprecipitates of this highly purified insulin receptor by anti-insulin-receptor antibodies. Phosphoamino acid determination revealed only phosphotyrosine in both the basal and insulin-stimulated states. These data suggest that a tyrosine-specific protein kinase activity is closely associated with insulin receptor, and this may be important in the signal transmission required for insulin action. 相似文献
6.
R P Aaronson G Blobel 《Proceedings of the National Academy of Sciences of the United States of America》1975,72(3):1007-1011
Nuclear pore complexes have been isolated in association with a 150 A thick lamina by detergent and salt fractionation of nuclear envelopes from rat liver. The pore complexes exhibit characteristic morphology and appear to be attached in a highly specific orientation to the lamina, which extends over relatively large areas. The pore complex-lamina fraction is composed of three major and several minor polypeptides with little or no DNA, RNA, or phospholipid. It is suggested that the association of the pore complexes and the lamina reflects the structural arrangement of the nuclear periphery in vivo. 相似文献
7.
Ovarian cytosol from pseudopregnant rats was heated to 80-90 degrees C for 2 min and precipitated proteins removed by centrifugation. The supernatant of the heated ovarian cytosol contained no protein kinase C activity but when added to a control preparation containing protein kinase C, enzyme activity was increased to 200% of control. The stimulatory activity was stable to heating for 10 min, was retained on a centrifugal filtration device with a 100,000 M(r) cut-off, did not affect cAMP-dependent protein kinase, was not extractable in petroleum ether or chloroform/methanol (2:1), and enhanced the phosphorylation of protein kinase C-specific peptide substrates. The stimulatory factor was calcium-dependent and could substitute for phosphatidylserine and diacylglycerol in the protein kinase C assay. This stimulatory factor may provide a mechanism whereby the response of protein kinase C to hormonal activation could be regulated by the cell. 相似文献
8.
9.
Protein kinase C activity and hexamethylenebisacetamide-induced erythroleukemia cell differentiation. 总被引:2,自引:8,他引:2
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E Melloni S Pontremoli M Michetti O Sacco A G Cakiroglu J F Jackson R A Rifkind P A Marks 《Proceedings of the National Academy of Sciences of the United States of America》1987,84(15):5282-5286
Hexamethylenebisacetamide (HMBA) is a potent inducer of murine erythroleukemia (MEL) cell differentiation. The mechanism of action of HMBA is not known. In this study we provide evidence that protein kinase C has a role in inducer-mediated MEL cell differentiation: (i) HMBA induces the formation of a soluble, proteolytically activated form of protein kinase C that is catalytically active in the absence of Ca2+ and phospholipid; (ii) the protease inhibitor leupeptin blocks formation of this activated form of the kinase and inhibits HMBA-induced MEL cell hemoglobin accumulation; (iii) phorbol 12-myristate 13-acetate (PMA) inhibits HMBA-induced MEL differentiation and causes depletion of total protein kinase C activity; (iv) MEL cells depleted in protein kinase C activity by culture with PMA are resistant to induction by HMBA; (v) upon removal of PMA, restoration of MEL cell sensitivity to HMBA is correlated with reaccumulation of protein kinase C activity; and (vi) MEL cells grown to density arrest are both depleted of protein kinase C activity and resistant to HMBA. Together, these results suggest that HMBA-mediated MEL cell differentiation involves a protein kinase C-related mechanism and the proteolytically activated form of the kinase, which does not require Ca2+ or phospholipid for its catalytic activity. 相似文献
10.
Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene. 总被引:26,自引:12,他引:26
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C I Bargmann R A Weinberg 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(15):5394-5398
A single mutation altering the transmembrane domain of the receptor-like p185 protein encoded by the rat neu gene converts the normal neu gene into a potent oncogene. The biochemical consequences of this mutation were studied by examining phosphorylation of the normal and transforming p185 molecules in membrane preparations. Here we show that the transforming p185 is phosphorylated to a much higher extent in vitro than its normal counterpart. This preferential phosphorylation has the properties that would be expected of p185 autophosphorylation: it takes place on tyrosine and requires intact p185 kinase activity. The normal p185 protein does not demonstrate increased phosphorylation even when it coexists in a transformed cell with the transforming p185 protein. These data show that transforming p185 is specifically associated with an active tyrosine kinase activity and suggest that this activity is intrinsic to the transforming protein. Thus, the transmembrane domain of p185 appears to directly regulate its kinase activity. 相似文献
11.
Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte. 总被引:35,自引:31,他引:4
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G Kramer J M Cimadevilla B Hardesty 《Proceedings of the National Academy of Sciences of the United States of America》1976,73(9):3078-3082
Highly purified preparations of hemin-controlled repressor of rabbit reticulocyte contain a 3':5'-cyclic AMP-indenpendent protein kinase activity that phosphorylates the low-molecular-weight (about 38,000) polypeptide chain of the initiation factor that forms a ternary complex with GTP and Met-tRNAf. These preparations also phosphorylate several polypeptide components of reticulocyte 40S ribosomal subunits. However, no significant levels of phosphorylation are observed when casein, histones, Artemia salina 40S ribosomal subunits, or other initiation factor fractions are used as substrates although high levels of phosphorylation are obtained with cruder preparations of the repressor. An antibody to these highly purified preparations of repressor has been obtained from the serum of immunized goats. Preincubation with immune goat IgG results in the neutralization of the inhibitory activity of the repressor, while normal IgG has no effect. Preincubation with immune IgG also abolishes the protein kinase activity responsible for the phosphorylation of the initiation factor and reticulocyte 40S subunits. Histone phosphorylation by crude repressor preparations, on the other hand, is unaffected by preincubation with immune IgG. 相似文献
12.
Protein-tyrosine kinase activity tightly associated with human type II Fc gamma receptors. 总被引:2,自引:0,他引:2
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G Sármay I Pecht J Gergely 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(10):4140-4144
Stimulation of B cells by clustering their surface immunoglobulins (sIg) leads to enhanced phosphorylation of several cellular proteins on Ser and Tyr residues. The type II Fc gamma receptor (Fc gamma RII) is one of those proteins that undergo Ser phosphorylation. Upon affinity isolation of the Fc gamma RII, several molecular entities are coisolated from Triton X-100 lysates of BL41 Burkitt lymphoma line which undergo "in vitro" (cell free) phosphorylation in the immune complex-associated kinase assay. Furthermore, several molecules phosphorylated on Tyr upon sIgM cross-linking in the intact cells are coisolated with Fc gamma RII. The 59-kDa coprecipitated component is identified as the protein-tyrosine kinase (PTK) fyn. Clustering the sIgM molecules enhanced the in vitro phosphorylation of all molecules coprecipitated with Fc gamma RII as well as that of the exogenously added PTK substrate, enolase. Kinase renaturation assays suggest that at least two major renaturable protein kinases (59 kDa and 85-90 kDa) associate with Fc gamma RII. Whereas the 59-kDa component comigrates with the PTK fyn, the 85- to 90-kDa one is an unidentified Ser/Thr kinase. These data suggest that Fc gamma RII exists in the B-cell membrane as part of a multimolecular complex including protein kinases, activities of which are regulated by clustering of the antigen receptors. 相似文献
13.
A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase. 总被引:5,自引:2,他引:3
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M Sander J M Nolan T Hsieh 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(22):6938-6942
A protein kinase activity has been identified that is tightly associated with the purified Drosophila type II DNA topoisomerase. The kinase and topoisomerase activities are not separated when the enzyme is subjected to analytical chromatography (phosphocellulose, single-strand DNA agarose, and Sephacryl S-300) and analytical glycerol gradient sedimentation. These two activities are also inactivated to the same extent by either heat or N-ethylmaleimide treatment. The evidence, however, does not rule out the possibility that the kinase activity resides in a polypeptide other than the topoisomerase polypeptide. The topoisomerase-associated protein kinase activity is not stimulated by Ca2+ or cyclic nucleotides. It shows a broad substrate range, including the DNA topoisomerase itself, casein, phosvitin, and histones. Phosphoamino acid analysis identified phosphoserine and phosphothreonine in polypeptides modified by the topoisomerase-associated protein kinase. No similar activity has been identified previously in Drosophila melanogaster. 相似文献
14.
Y Sakanoue T Hatada T Horai Y Shoji M Kusunoki J Utsunomiya 《Scandinavian journal of gastroenterology》1992,27(4):275-280
Protein kinase C (PKC) activity was measured in the inflamed colonic mucosa of 24 patients with ulcerative colitis and in the normal colonic mucosa of 10 patients with other benign diseases. The particulate fraction activity in ulcerative colitis mucosa was significantly increased compared with that of normal mucosa (320 +/- 47 versus 200 +/- 30 pmol/min/mg protein; p less than 0.05). Inflamed ulcerative colitis mucosa also showed significantly increased total PKC activity in the particulate fractions compared with normal mucosa (147 +/- 26 versus 37 +/- 8 pmol/min/mg tissue; p less than 0.05). Mucosal samples from ulcerative colitis patients were divided into 12 with mild and 12 with severe inflammation by histologic examination. The particulate PKC activity of severely inflamed mucosa was significantly lower than that of mildly inflamed mucosa (p less than 0.05). These results indicate that colonic inflammation in ulcerative colitis may be associated with altered cellular PKC activity. 相似文献
15.
Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity. 总被引:12,自引:1,他引:12
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G E Bollag R A Roth J Beaudoin D Mochly-Rosen D E Koshland Jr 《Proceedings of the National Academy of Sciences of the United States of America》1986,83(16):5822-5824
The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor. 相似文献
16.
Biogenesis of the nuclear lamina: in vivo synthesis and processing of nuclear protein precursors. 总被引:17,自引:1,他引:16
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C F Lehner G Fürstenberger H M Eppenberger E A Nigg 《Proceedings of the National Academy of Sciences of the United States of America》1986,83(7):2096-2099
Utilizing antibodies against lamins A, B1, and B2, we have studied the biogenesis of the nuclear lamina in chicken embryo fibroblasts. (Lamins B1 and B2 have been identified recently as structurally distinct "lamin B" proteins.) We demonstrate that, unique among the nuclear proteins studied to date, lamin A is synthesized as a higher molecular mass precursor. A short-lived higher molecular mass variant (t 1/2 approximately equal to 3 min) accompanying the mature-size protein was also detected in the case of lamin B2 biosynthesis, but no precursor was found for lamin B1. By combining pulse-chase experiments with subcellular fractionation, we provide evidence that synthesis of lamin proteins occurs on free polysomes; subsequently, the newly synthesized proteins become rapidly associated with a crude nuclear fraction. The lamin A precursor is processed within the nucleus with a half-time of about 30 min. Concomitantly, lamin proteins acquire a characteristic resistance to detergent extraction, suggesting their insertion into a submembraneous protein network. The described biogenetic pathway involving precursor synthesis and processing is very unusual for nuclear proteins; it may have interesting implications for the mechanisms of transport and assembly of poorly soluble nuclear proteins. 相似文献
17.
C R Merril D Goldman M Ebert 《Proceedings of the National Academy of Sciences of the United States of America》1981,78(10):6471-6475
Patients having Lesch--Nyhan syndrome were studied by using enzymatic, immunologic, and two-dimensional electrophoretic techniques. Four hundred proteins were analyzed on each two-dimensional electrophoretogram for positional or quantitative variation. In autoradiograms of lymphocytes stimulated with phytohemagglutinin, there were 11 quantitative differences found in all patients that were significant at the 2P less than 0.01 level. A significant quantitative difference was also found in an analysis of silver-stained gels of unstimulated lymphocytes. Patients had trace amounts of erythrocyte hypoxanthine phosphoribosyl transferase (HPRT) activity and trace or no immunoprecipitable HPRT. However, HPRT was observed in silver-stained erythrocyte electrophoretograms and in autoradiograms from phytohemagglutinin-stimulated lymphocytes. Unstimulated lymphocytes contained 65% of the control HPRT concentration. Currently, the technology of two-dimensional electrophoresis detects a fraction of the total cellular proteins and defective proteins may not show electrophoretic alterations. However, specific secondary changes in other polypeptides may be observed and, when catalogued, will serve as an aid in the diagnosis and understanding of the pathophysiology of metabolic diseases. 相似文献
18.
19.
Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein. 总被引:26,自引:7,他引:26
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F Imani B L Jacobs 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(21):7887-7891
In this report we demonstrate that reovirus serotype 1-infected cells contain an inhibitor of the interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase. We provide evidence that suggests that the virus-encoded sigma 3 protein is likely responsible for this kinase inhibitory activity. We could not detect activation of the dsRNA-dependent protein kinase in extracts prepared from either interferon-treated or untreated reovirus serotype 1-infected mouse L cells under conditions that led to activation of the kinase in extracts prepared from either interferon-treated or untreated, uninfected cells. Extracts from reovirus-infected cells blocked activation of kinase in extracts from interferon-treated cells when the two were mixed prior to assay. The kinase inhibitory activity in extracts of reovirus-infected cells could be overcome by adding approximately 100-fold excess of dsRNA over the amount required to activate kinase in extracts of uninfected cells. Kinase inhibitory activity in extracts of interferon-treated, virus-infected cells could be overcome with somewhat less dsRNA (approximately 10-fold excess). Most of the inhibitory activity in the extracts could be removed by adsorption with immobilized anti-reovirus sigma 3 serum or immobilized dsRNA, suggesting that the dsRNA-binding sigma 3 protein is necessary for kinase inhibitory activity. Purified sigma 3 protein, when added to reaction mixtures containing partially purified kinase, inhibited enzyme activation. Control of activation of this kinase, which can modify eukaryotic protein synthesis initiation factor 2, may be relevant to the sensitivity of reovirus replication to treatment of cells with interferon and to the shutoff of host protein synthesis in reovirus-infected cells. 相似文献