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)

We have examined the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioreductive metabolism of 17-allylamino-demethoxygeldanamycin (17-AAG). High-performance liquid chromatography (HPLC) analysis of the metabolism of 17-AAG by recombinant human NQO1 revealed the formation of a more polar metabolite 17-AAGH2. The formation of 17-AAGH2 was NQO1 dependent, and its formation could be inhibited by the addition of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based (suicide) inhibitor of NQO1. The reduction of 17-AAG to the corresponding hydroquinone 17-AAGH2 was confirmed by tandem liquid chromatography-mass spectrometry. 17-AAGH2 was relatively stable and only slowly underwent autooxidation back to 17-AAG over a period of hours. To examine the role of NQO1 in 17-AAG metabolism in cells, we used an isogenic pair of human breast cancer cell lines differing only in NQO1 levels. MDA468 cells lack NQO1 due to a genetic polymorphism, and MDA468/NQ16 cells are a stably transfected clone that express high levels of NQO1 protein. HPLC analysis of 17-AAG metabolism using cell sonicates and intact cells showed that 17-AAGH2 was formed by MDA468/NQ16 cells, and formation of 17-AAGH2 could be inhibited by ES936. No 17-AAGH2 was detected in sonicates or intact MDA468 cells. Following a 4-hour treatment with 17-AAG, the MDA468/NQ16 cells were 12-fold more sensitive to growth inhibition compared with MDA468 cells. More importantly, the increased sensitivity of MDA468/NQ16 cells to 17-AAG could be abolished if the cells were pretreated with ES936. Cellular markers of heat shock protein (Hsp) 90 inhibition, Hsp70 induction, and Raf-1 degradation were measured by immunoblot analysis. Marked Hsp70 induction and Raf-1 degradation was observed in MDA468/NQ16 cells but not in MDA468 cells. Similarly, downstream Raf-1 signaling molecules mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase and ERK also showed decreased levels of phosphorylation in MDA468/NQ16 cells but not in MDA468 cells. The ability of 17-AAG and 17-AAGH2 to inhibit purified yeast and human Hsp90 ATPase activity was examined. Maximal 17-AAG-induced ATPase inhibition was observed in the presence of NQO1 and could be abrogated by ES936, showing that 17-AAGH2 was a more potent Hsp90 inhibitor compared with 17-AAG. Molecular modeling studies also showed that due to increased hydrogen bonding between the hydroquinone and the Hsp90 protein, 17-AAGH2 was bound more tightly to the ATP-binding site in both yeast and human Hsp90 models. In conclusion, these studies have shown that reduction of 17-AAG by NQO1 generates 17-AAGH2, a relatively stable hydroquinone that exhibits superior Hsp90 inhibition.
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PMID:Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition. 1626 26

The phosphatidylinositol 3'-kinase (PI3K)/Akt pathway is often constitutively activated in malignant glioma cells, in many cases as a result of mutation of phosphatase and tensin homologue deleted on chromosome ten (PTEN), an endogenous inhibitor of Akt, which renders tumor cells resistant to cytotoxic insults, including those related to anticancer drugs. Pharmacological inhibition of this pathway may potentially restore or augment the effectiveness of conventional chemotherapy or other signaling-targeted agents. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the combination of the PI3K inhibitor LY294002 and the HSP90 inhibitor 17-allyl-aminogeldanamycin (17-AAG) would promote glioma cytotoxicity by decreasing both the activation status and levels of Akt, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of LY294002 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death, and irreversibly inhibited proliferative activity and colony forming ability of the glioma cell lines. Quantitative analysis revealed that enhancement by LY294002 of 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage together with the release of cytochrome c and apoptosis inducing factor (AIF). No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of epidermal growth factor receptor (EGFR), Raf-1, and mitogen activated protein kinase. Combination of 17-AAG and LY294002 did not modify phospho-JNK/SPK and phospho-p38. Cells exposed to 17-AAG and LY294002 displayed a significant reduction in cell-cycle regulatory proteins, such as retinoblastoma (Rb), cyclin dependent kinase (CDK)4, CDK6, cyclin D1, and cyclin D3. Taken together, these findings suggest that the PI3K/Akt pathway plays a critical role in regulating the apoptotic response to 17-AAG and that targeting this pathway could provide a potent strategy to treat patients with malignant gliomas.
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PMID:Synergistic interaction between 17-AAG and phosphatidylinositol 3-kinase inhibition in human malignant glioma cells. 1626 32

The multi-drug combination of oxaliplatin (OXA), 5-Fluorouracil (5-FU) and leucovorin (LF) is currently considered as the gold standard treatment for metastatic colorectal carcinoma. In previous studies, we have studied a chemotherapy regimen containing gemcitabine (GEM), OXA, LF, and 5-FU (named GOLF regimen) that has shown a good safety profile and highly significant anti-tumor activity. In the present study, we have investigated on the anti-tumour mechanisms of GOLF in human colon cancer HT-29 and WiDr cell lines. We have found that GOLF induced growth inhibition that was largely caused by apoptosis differently from other combinations. Moreover, the different drugs composing GOLF were highly synergistic in inducing growth inhibition. Apoptosis induced by GOLF combination was paralleled by PARP cleavage and caspase 9 and 3 activation that were not recorded in the other combinations. An about 85% decrease of the activity of Erk and Akt was found in GOLF-treated cells. These effects were likely due to decreased expression of the upstream activator Raf-1 and of Akt itself, respectively. The intracellular levels of these signalling components can be post-translationally regulated by ubiquitin-dependent degradation through proteasome. Therefore, we have evaluated the expression of some chaperone components and we have found that GOLF did not affect the expression of both heat shock protein (HSP) 90 and 27 but induced an about 90% increase of HSP70 levels suggesting the inactivation of the multi-chaperone complex. Moreover, an about 4-fold increase of the ubiquitination of Raf-1 was also found and the addition for 12 h of 10 microM proteasome inhibitor lactacystin caused an accumulation of the ubiquitinated isoforms of Raf-1. In conclusions, GOLF was a combination highly synergistic in inducing both growth inhibition and apoptosis of colon cancer cells. These effects likely occurred through the disruption of critical survival pathways and the inactivation of multi-chaperone complex.
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PMID:Chemotherapy regimen GOLF induces apoptosis in colon cancer cells through multi-chaperone complex inactivation and increased Raf-1 ubiquitin-dependent degradation. 1629 35

1. Related sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), have important effects on vascular smooth muscle. The aim of this study was to investigate the intracellular pathways regulated by S1P and SPC in rat cerebral artery. 2. In cerebral arteries, S1P increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation (5.2+/-1.4-fold increase) but did not activate p38 mitogen-activated protein kinase (p38MAPK) as assessed by immunoblotting. In contrast, SPC increased p38MAPK phosphorylation (3.0+/-0.3-fold increase) but did not stimulate ERK1/2. This differential activation was confirmed by measuring activation of heat shock protein (HSP) 27, a known downstream target of p38MAPK. Only SPC, but not S1P, activated HSP27. 3. In enzymatically dispersed cerebral artery myocytes, SPC increased [Ca2+]i in a concentration-dependent manner (peak response at 10 microM: 0.4+/-0.02 ratio units) as determined using the Ca2+ indicator, Fura 2. In contrast to S1P, the SPC-induced [Ca2+]i increase did not involve intracellular release but was due to Ca2+ influx via L-type Ca2+ channels. 4. Despite differences in signalling, both S1P and SPC phosphorylated the transcription factor cAMP response element-binding protein (CREB). S1P-induced CREB activation was dependent on ERK1/2 and Ca2+-calmodulin-dependent protein kinase (CaMK) activation. CREB activation by SPC required both p38MAPK and CaMK activation, but not ERK1/2. 5. In conclusion, S1P and SPC activate distinct MAP kinase isoforms and increase [Ca2+]i via different mechanisms in rat cerebral artery. This does not affect the ability of S1P or SPC to activate CREB, although this occurs via different pathways.
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PMID:Sphingolipids differentially regulate mitogen-activated protein kinases and intracellular Ca2+ in vascular smooth muscle: effects on CREB activation. 1640 45

ZD1839 ("Iressa") is an orally active, selective epidermal growth factor (EGF) receptor-tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with HSP90 antagonist, 17-AAG in malignant human glioma cell lines. ZD1839 independently produced a dose-dependent inhibition of cellular proliferation in glioma cells grown in culture with time- and dose-dependent accumulation of cells in G(1) phase of the cell cycle on flow cytometric analysis, although the concentrations required for optimal efficacy were at or above the limits of clinically achievable levels. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the HSP90 inhibitor 17-AAG would potentiate ZD 1839-mediated glioma cytotoxicity by decreasing the activation status of EGF receptor, as well as down regulating the levels of other relevant signaling effectors. We, therefore, examined the effects of ZD1839 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death and quantitative analysis revealed that interaction between ZD1839 and 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and PARP cleavage. No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of EGFR receptor, Raf-1 and mitogen activated protein kinase (MAPK). Cells exposed to 17-AAG and ZD1839 displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that ZD1839, an EGF receptor tyrosine kinase inhibitor, plays a critical role in regulating the apoptotic response to 17-AAG and that multi-site targeting of growth signaling and cell survival pathways could provide a potent strategy to treat patients with malignant gliomas.
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PMID:Cooperative inhibitory effect of ZD1839 (Iressa) in combination with 17-AAG on glioma cell growth. 1655 Jun 10

Histone deacetylases (HDACs) are enzymes that regulate the functions of histones as well as nonhistones by catalyzing the removal of acetyl groups from lysine residues. HDACs regulate many biological processes, including the cell division cycle and tumorigenesis. Although recent studies have implicated HDAC8 in tumor cell proliferation, the molecular mechanisms linking HDAC8 to cell growth remain unknown. Here, we report that the human ortholog of the yeast ever-shorter telomeres 1B (EST1B) binds HDAC8. This interaction is regulated by protein kinase A-mediated HDAC8 phosphorylation and protects human EST1B (hEST1B) from ubiquitin-mediated degradation. Phosphorylated HDAC8 preferentially recruits Hsp70 to a complex that inhibits the CHIP (C-terminal heat shock protein interacting protein) E3 ligase-mediated degradation of hEST1B. Importantly, HDAC8 regulation of hEST1B protein stability modulates total telomerase enzymatic activity. Our findings reveal a novel mechanism by which HDAC8 contributes to tumorigenesis by regulating telomerase activity.
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PMID:Histone deacetylase 8 safeguards the human ever-shorter telomeres 1B (hEST1B) protein from ubiquitin-mediated degradation. 1680 64

Treatment of PBMCs with TNF-alpha decreased the levels of heat shock protein (HSP) 27, but had little effect on the level of HSP70. Parallel to the decrease of HSP27, TNF-alpha increased the level of HSP27 in the incubation medium of the cells. The decrease of HSP27 induced by TNF-alpha was suppressed by the pretreatment of PBMCs with the specific protein kinase C (PKC) inhibitor, GF109203X. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, decreased the levels of HSP27. To investigate the effect of TNF-alpha on the oligomerization state of HSP27 in PBMCs, we performed sucrose density gradient centrifugation with subsequent fractionation and immunoassay. Extract of vehicle-treated PBMCs contained mainly dissociated forms of HSP27. The amounts of dissociated forms of HSP27 in PBMCs was decreased by TNF-alpha, while the amounts of aggregated form of HSP27 was little changed. In intact PBMCs, HSP27 is constitutively phosphorylated at Ser78, but not at Ser15 or at Ser82. The amount of phosphorylated HSP27 at Ser78 was decreased by TNF-alpha. These results indicate that TNF-alpha reduces HSP27 in PBMCs through PKC activation. This decrease may be due to efflux of dissociated form of HSP27, phosphorylated HSP27 at Ser78, from the cells.
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PMID:TNF-alpha decreases hsp 27 in human blood mononuclear cells: involvement of protein kinase c. 1706 61

We applied the novel ProteinChip technology (SELDI-MS) to investigate and identify differentially regulated proteins upon myocardial remodelling in different heart regions. Tissue samples were isolated from the atria, the interventricular septum, and the right and left ventricles three months after surgically-induced myocardial infarction (MI) in rats. Corresponding protein extracts from control versus MI hearts were analysed on two different ProteinChip surfaces. In each of the functionally distinct cardiac regions, we obtained specific protein profile alterations upon myocardial remodelling. Most alterations were observed in the non-infarcted right ventricle, where down-regulation occurred more frequently than up-regulation of protein expression. Three of the differentially regulated proteins were identified: the metabolic enzyme triosephosphate isomerase (TIM), the cell signalling protein Raf-1 kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine binding protein (PEBP), and the small heat shock protein alphaB-crystallin. These proteins showed a pronounced tissue-dependent regulation. TIM was down-regulated only in the atrium and in the left ventricle, RKIP/PEBP was down-regulated only in the right ventricle and in the interventricular septum, and alphaB-crystallin was up-regulated only in the right and in the left ventricle. A simple correlation of peak intensity changes using two of the identified peaks demonstrated the diagnostic potential of SELDI-MS. We conclude that this novel proteomic method is a powerful high-throughput tool for the fast detection of region-specific cardiac protein profiles in small biopsy samples, and that SELDI-MS may become a useful complementary technique for the diagnosis and prognosis of cardiac diseases.
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PMID:Region-specific alterations of global protein expression in the remodelled rat myocardium. 1708 28

Interaction of human 14-3-3gamma with the small heat shock protein Hsp20 was analyzed by means of size-exclusion chromatography and chemical crosslinking. Unphosphorylated Hsp20 and its mutant S16D mimicking phosphorylation by cAMP-dependent protein kinase did not interact with 14-3-3. Phosphorylated Hsp20 formed a tight complex with 14-3-3 in which dimer of 14-3-3 was bound to dimer of Hsp20. 14-3-3 did not affect the chaperone activity of unphosphorylated Hsp20 but increased the chaperone activity of phosphorylated Hsp20 if insulin was used as a model substrate. Estimation of the effect of 14-3-3 on the chaperone activity of Hsp20 with other model substrates was complicated by the fact that under in vitro conditions isolated 14-3-3 possessed its own high chaperone activity. Taken into account high content of Hsp20 in different muscles it is supposed that upon phosphorylation Hsp20 might effectively compete with multiple protein targets of 14-3-3 and by this means indirectly affect many intracellular processes.
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PMID:Small heat shock protein Hsp20 (HspB6) as a partner of 14-3-3gamma. 1710 79

Diabetes mellitus (DM) and high glucose (HG) are known to reduce the bioavailability of nitric oxide (NO) by modulating endothelial nitric-oxide synthase (eNOS) activity. eNOS is regulated by several mechanisms including its interaction with heat shock protein (Hsp) 90. We previously discovered that DM in vivo and HG in vitro induced the translocation of Hsp90alpha to the outside of aortic endothelial cells. In this report we tested the hypothesis that translocation of Hsp90alpha is responsible for the decline in NO production observed in HG-treated cells. We found that HG increased phosphorylation of Hsp90alpha in a cAMP-dependent protein kinase A-dependent manner, and that this event was required for translocation of Hsp90alpha in porcine aortic endothelial cells. Furthermore, preventing translocation of Hsp90alpha protected from the HG-induced decline in eNOS.Hsp90alpha complex and NO production. Notably, DM increased phosphorylation of Hsp90alpha and reduced its association with eNOS in the aortic endothelium of diabetic rats. These studies suggest that translocation of Hsp90alpha is a novel mechanism by which HG and DM impair eNOS activity and thereby reduce NO production.
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PMID:Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes. 1720 41


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