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 steroid hormone receptor has been defined as an intracellular protein which associates with the corresponding ligand in a stereospecific manner. Its binding ability and affinity have been reported to be affected by many factors such as protein kinase, SH reducing agent and molybdate. Through ligand binding, hormone-receptor complexes undergo activation process resulting in its acquisition of high affinity toward DNA. Although the detailed biochemical mechanism of receptor activation remains to be elucidated, the reduction of their molecular size has been proposed to be an obligatory step for receptor activation. Recently, heat shock protein 90 K, has become known to bind with the oncogene product (pp 60 v-src). Furthermore, it has been demonstrated to be a common component of both nonactivated, and not activated steroid receptor. The recombinant gene technology has successfully determined the amino acid sequence of GR, ER and PgR. All steroid receptors consist of three domains, namely the immunogenic, DNA-binding and steroid-binding domains. Experiments using deletion mutant receptors revealed that the DNA binding domain has both abilities of DNA binding and transactivation, and that the steroid binding domain negatively regulates DNA binding activity. The purified GR has been found to bind with the specific region (GRE) of glucocorticoid-dependent genes. The consensus sequence of GRE has been observed to be TG-TTCT. Two other proteins which can be associated with some regions near GRE have been proposed to activate cooperatively the glucocorticoid responsive genes with GR. Therefore, the steroid hormone receptor systems are suitable for clarifying the molecular mechanism of gene activation.
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PMID:[Regulatory mechanism of steroid hormone receptor functions]. 282 24

The Raf-1 protein kinase participates in transduction of mitogenic signals, but its mechanisms of activation are incompletely understood. Treatment of human Raf-1 purified from insect Sf9 cells co-expressing c-H-Ras and Src(Y527F) (in which phenylalanine replaces tyrosine at residue 527) with either serine-threonine or tyrosine phosphatases resulted in enzymatic inactivation of Raf-1. Inactivation of purified Raf-1 was blocked by addition of either the 14-3-3 zeta protein or heat shock protein 90. Loading of plasma membranes from transformed cells with guanosine triphosphate (GTP) resulted in inactivation of endogenous or exogenous Raf-1; inactivation was blocked by inclusion of protein phosphatase inhibitors. These results suggest the existence of protein phosphatases in the cell membrane that are regulated by GTP and are responsible for Raf-1 inactivation.
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PMID:Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases. 760 63

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) when administered directly to a nuclear-free subcellular homogenate of guinea pig adipose tissue, caused a significant rise in protein kinase activities within 1-10 min. Such a rapid response was not expected, based on the classic transcriptional mechanism of action for TCDD, i.e. TCDD first binds with its cytosolic Ah-receptor, translocates into the nucleus, dimerizes with "arnt" (a nuclear transcription factor), and activates genes containing "xenobiotic-responsive element" (XRE). The above actions of TCDD on protein kinases were clearly blocked by two specific Ah-receptor blockers, even under cell- and nucleus-free conditions. TCDD-induced increases in protein phosphorylation occurred mainly in cytosolic preparations (i.e. 100,000 g supernatant) devoid of nucleus, microsomes and plasma membranes and were still observed in the presence of inhibitors of protein phosphatases. Furthermore, TCDD caused a rise in protein tyrosine kinase activity in a purified Ah-receptor preparation, as well as in an isolated heat shock protein 90 complex preparation containing the Ah-receptor. This activation took place in the presence of actinomycin D and cycloheximide, indicating a portion of TCDD's action that is unrelated to de novo protein synthesis in this process. We have also obtained evidence indicating that this action of TCDD triggers the protein kinase mediated growth factor signal transduction pathway, such as stimulation of mitogen activated protein kinase 2 and tyrosine kinase activity. These results clearly support the view that the basic action pathway for such a TCDD-induced activation of protein kinases is distinctly different from its conventional action pathway involving changes in gene transcription in the nucleus.
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PMID:Evidence for a second pathway in the action mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Significance of Ah-receptor mediated activation of protein kinase under cell-free conditions. 784 Aug 3

To assess the interaction of human casein kinase II (CKII) with the heat shock protein 90 (HSP90) class of chaperone proteins, human CKII alpha and beta subunits and beta S2A mutant were expressed and purified separately or from a tandem coexpression construct in Escherichia coli. Recombinant human HSP90 beta and recombinant yeast HSP90 as His6 constructs were also expressed in and purified from E. coli. The rhCKII S2A mutant removed the regulatory beta subunit autophosphorylation site but had no effect on catalytic efficiency with peptide or protein substrates. As a CKII substrate, recombinant hHSP90 beta displayed a Km of 9.8 microM and a kcat of 4.1 min-1 and was phosphorylated to 1.5 mol/mol, whereas ryHSP90, lacking the known serine CKII sites of hHSP90, was phosphorylated at a 19-fold lower kcat/Km ratio to levels of 0.8 mol/mol. The endoplasmic reticulum HSP90 family member Grp94 was phosphorylated to 1.4 mol/mol but, in contrast, HSC70 and FKBP25 chaperones were phosphorylated to < 0.01 mol/mol. Neither phospho nor dephospho forms of hHSP90 showed significant activation of CKII toward the peptide substrate RRREEETEEE in contrast to a previous report that activation was observed at high molar ratios of chaperone to kinase.
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PMID:Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions. 814 88

Protein kinase CK2 is a ubiquitous protein kinase responsible for the phosphorylation of Ser and Thr residues specified by acidic side chains in many proteins, including several key enzymes, growth factor receptors, transcription factors and cytoskeletal proteins. The holoenzyme is composed of two catalytic and two regulatory subunits, the latter having antagonistic roles. CK2 is constitutively active and its targeting seems to be modulated through association with a variety of cellular proteins (e.g. heat shock protein 90 and p53). CK2 is abnormally elevated in proliferating and neoplastic tissues and recent studies suggest that mice overexpressing CK2 develop leukemia. Specific inhibitors of CK2, currently being developed, may have therapeutic potential.
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PMID:Protein kinase CK2. 936 31

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was found to activate protein kinases under cell- and nucleus-free conditions in isolated C57 mouse liver cytosol (100,000 x g supernatant). This action of TCDD was found to be aryl hydrocarbon receptor (AHR) dependent, concentration dependent, and inhibited by genistein, a tyrosine kinase inhibitor. The lowest concentration of TCDD to produce a statistically significant increase in protein phosphorylation was 10 pM. We also investigated the possibility that a protein kinase is physically associated with the cytosolic AHR complex. Kinase renaturation tests designed to detect reactivated protein kinases after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of a 60-kDa kinase in the washed immunoprecipitate obtained from liver cytosol using anti-AHR antibody (IgG) and protein A/G/agarose beads but not when a nonspecific IgG was used instead of anti-AHR antibody. The same 60-kDa band was present in an immunoprecipitate prepared in a similar manner from the same cytosol but with anti-heat shock protein 90 antibody (IgM). This 60-kDa kinase was found to be activated by TCDD treatment of whole cytosol from untreated mice. Moreover, pp60(src) immunoprecipitated from cytosol that had been previously treated with TCDD under cell-free conditions exhibited 2-fold more kinase activity than the equivalent preparation treated with a solvent control. Again, such an effect of TCDD could not be detected when a nonspecific IgG was used in place of an anti-pp60(src) antibody. Increased protein phosphorylation was observed after direct TCDD treatment of immunoprecipitates obtained using antibodies to AHR and pp60(src), respectively, but not when a nonspecific IgG was used for immunoprecipitation in either case. This observation is consistent with the idea that in cytosol, the AHR and pp60(src) coexist as part of a multimeric protein complex that can be specifically coimmunoprecipitated. These results provide evidence that (i) TCDD activates protein kinases in murine hepatic cytosol, (ii) a 60-kDa protein kinase is associated with the cytosolic form of the AHR complex, (iii) ligand binding directly activates this kinase because TCDD treatment of immunoprecipitated AHR complex results in increased protein kinase activity, and (iv) the AHR-associated protein kinase seems to be pp60(src) kinase. The current findings provide a clue to a potentially important mechanism by which TCDD can exert rapid, pleiotropic effects through the AHR-associated kinase to alter functions of many proteins through a cascade of protein phosphorylations.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced activation of a protein tyrosine kinase, pp60src, in murine hepatic cytosol using a cell-free system. 938 30

FKBP65 is a member of the FK506-binding protein class of immunophilins and is the only member reported to contain four peptidylprolyl cis-trans isomerase domains and an unrelated COOH-terminal domain. In this report, we show that the heat shock protein hsp90 and the serine/threonine protein kinase c-Raf-1 are components of FKBP65 immune complexes. The NH2-terminal regulatory domain of c-Raf-1 appears to be required for its interaction with FKBP65. Using GST-FKBP65 fusion protein and purified Raf proteins, we show that full-length FKBP65 can interact with c-Raf-1 but not B-Raf. The activation kinetics of c-Raf-1 after v-H-RasV12 injection of Xenopus oocytes appear to correlate with FKBP65/c-Raf-1 interaction, suggesting that FKBP65 may preferentially associate with forms of c-Raf-1 that are more posttranslationally modified. The interaction of FKBP65 with the c-Raf-heat shock protein 90 heterocomplex implicates this immunophilin in signal-transduction processes.
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PMID:The immunophilin FKBP65 forms an association with the serine/threonine kinase c-Raf-1. 943 87

17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a compound that is proposed for clinical development, shares the ability of geldanamycin to bind to heat shock protein 90 and GRP94, thereby depleting cells of p185erbB2, mutant p53, and Raf-1. Urine and plasma from mice treated i.v. with 17AAG contained six materials with absorption spectra similar to that of 17AAG. Therefore, in vitro metabolism of 17AAG by mouse and human hepatic preparations was studied to characterize: (a) the enzymes responsible for 17AAG metabolism; and (b) the structures of the metabolites produced. These materials had retention times on high-performance liquid chromatography of approximately 2, 4, 5, 6, 7, and 9 min. When incubated in an aerobic environment with 17AAG, murine hepatic supernatant (9000 x g) produced each of these compounds; the 4-min metabolite was the major product. This metabolism required an electron donor, and NADPH was favored over NADH. Metabolic activity resided predominantly in the microsomal fraction. Metabolism was decreased by approximately 80% in anaerobic conditions and was essentially ablated by CO. Microsomes prepared from human livers produced essentially the same metabolites as produced by murine hepatic microsomes, but the 2-min metabolite was the major product, and the 4-min metabolite was next largest. There was no metabolism of 17AAG by human liver cytosol. Metabolism of 17AAG by human liver microsomes also required an electron donor, with NADPH being preferred over NADH, was inhibited by approximately 80% under anaerobic conditions, and was essentially ablated by CO. Liquid chromatography/mass spectrometry analysis of human and mouse in vitro reaction mixtures indicated the presence of materials with molecular weights of 545, 601, and 619, compatible with 17-(amino)-17-demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The metabolite with retention time of 4 min was identified as 17AG by cochromatography and mass spectral concordance with authentic standard. Human microsomal metabolism of 17AAG was inhibited by ketoconazole, implying 3A4 as the responsible cytochrome P450 isoform. Incubation of 17AAG with cloned CYP3A4 produced metabolites 4 and 6. Incubation of 17AAG with cloned CYP3A4 and cloned microsomal epoxide hydrolase produced metabolites 2 and 4, with greatly decreased amounts of metabolite 6. Incubation of 17AAG with human hepatic microsomes and cyclohexene oxide, a known inhibitor of microsomal epoxide hydrolase, did not affect the production of metabolite 4 but decreased the production of metabolite 2 while increasing the production of metabolite 6. These data imply that metabolite 2 is a diol and metabolite 6 is an epoxide. Mass spectral fragmentation patterns and the fact that 17AG is not metabolized argue for the epoxide and diol being formed on the 17-allylamino portion of 17AAG and not on its ansamycin ring. These data have implications with regard to preclinical toxicology and activity testing of 17AAG as well as its proposed clinical development because: (a) production of 17AG requires concomitant production of acrolein from the cleaved allyl moiety; and (b) 17AG, which was not metabolized by microsomes, has been described as being as active as 17AAG in decreasing cellular p185erbB2.
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PMID:Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations. 962 79

Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamycin depleted cellular Raf-1 of rat splenic cells without affecting the steady state levels of hsp90 and downstream mitogen activated protein (MAP) kinases, ERK1 and ERK2. In parallel, it inhibited mitogen-induced nuclear factor-kappa B (NF-kappa B) activation in these cells. Geldanamycin also had a potent suppressive effect on recall antigen-induced T cell proliferation, with an IC50 value of 1 nM. In vivo, geldanamycin suppressed the progression of adjuvant-induced arthritis dose-dependently. These results suggest that geldanamycin exerts an immunosuppressive effect partly through destabilizing Raf-1, and raise a new strategy for the prevention of inflammatory diseases.
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PMID:Immunosuppressive effects of the heat shock protein 90-binding antibiotic geldanamycin. 1031 10

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.
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PMID:KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules. 1038 57


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