p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function.
(1/1291)
Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular. (+info)
Salmonella typhimurium and lipopolysaccharide stimulate extracellularly regulated kinase activation in macrophages by a mechanism involving phosphatidylinositol 3-kinase and phospholipase D as novel intermediates.
(2/1291)
Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuated Salmonella strains. Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen. Furthermore, the pathways used by Salmonella and LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS. Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase. Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation. (+info)
Involvement of tyrosine phosphorylation in HMG-CoA reductase inhibitor-induced cell death in L6 myoblasts.
(3/1291)
Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, causes myopathy in rabbits and kills L6 myoblasts. The present study was designed to elucidate the molecular mechanism of HCRI-induced cell death. We have demonstrated that simvastatin induces the tyrosine phosphorylation of several cellular proteins within 10 min. These phosphorylations were followed by apoptosis, as evidenced by the occurrence of internucleosomal DNA fragmentation and by morphological changes detected with Nomarski optics. Simvastatin-induced cell death was prevented by tyrosine kinase inhibitors. The MTT assay revealed that the addition of mevalonic acid into the culture medium partially inhibited simvastatin-induced cell death. Thus, these results suggested that protein tyrosine phosphorylation might play an important role in the intracellular signal transduction pathway mediating the HCRI-induced death of myoblasts. (+info)
Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors.
(4/1291)
Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis. (+info)
Tyrosine kinase-dependent modulation by interferon-alpha of the ATP-sensitive K+ current in rabbit ventricular myocytes.
(5/1291)
We examined the effects of interferon-alpha on the ATP-sensitive K+ current (IK,ATP) in rabbit ventricular cells using the patch-clamp technique. IK,ATP was induced by NaCN. Whole-cell experiments indicated that interferon-alpha (5 x 10(2) - 2.4 x 10(4) U/ml) inhibited IK,ATP in a concentration-dependent manner (60.7+/-7.5% with 2.4 x 10(4) U/ml). In cell-attached configuration, interferon-alpha (2.4 x 10(4) U/ml) applied to the external solution also inhibited the activity of the single ATP-sensitive K+ (KATP) channel by 56.0+/-5.8% without affecting the single channel conductance. The inhibitory effect of IK,ATP by interferon-alpha was blocked by genistein and herbimycin A, tyrosine kinase inhibitors, but was not affected by N-(2-metylpiperazyl)-5-isoquinolinesulfoamide (H-7), an inhibitor of protein kinase C and cAMP-dependent protein kinase. These findings suggest that interferon-alpha inhibits the cardiac KATP channel through the activation of tyrosine kinase. The tyrosine kinase-mediated inhibition of IK,ATP by cytokines may aggravate cell damage during myocardial ischemia. (+info)
Cellular and molecular characterization of the scurfy mouse mutant.
(6/1291)
Mice hemizygous (Xsf/Y) for the X-linked mutation scurfy (sf) develop a severe and rapidly fatal lymphoproliferative disease mediated by CD4+CD8- T lymphocytes. We have undertaken phenotypic and functional studies to more accurately identify the immunologic pathway(s) affected by this important mutation. Flow cytometric analyses of lymphoid cell populations reveal that scurfy syndrome is characterized by changes in several phenotypic parameters, including an increase in Mac-1+ cells and a decrease in B220+ cells, changes that may result from the production of extremely high levels of the cytokine granulocyte-macrophage CSF by scurfy T cells. Scurfy T cells also exhibit strong up-regulation of cell surface Ags indicative of in vivo activation, including CD69, CD25, CD80, and CD86. Both scurfy and normal T cells are responsive to two distinct signals provided by the TCR and by ligation of CD28; scurfy cells, however, are hyperresponsive to TCR ligation and exhibit a decreased requirement for costimulation through CD28 relative to normal controls. This hypersensitivity may result, in part, from increased costimulation through B7-1 and B7-2, whose expression is up-regulated on scurfy T cells. Although the specific defect leading to this hyperactivation has not been identified, we also demonstrate that scurfy T cells are less sensitive than normal controls to inhibitors of tyrosine kinases such as genistein and herbimycin A, and the immunosuppressant cyclosporin A. One interpretation of our data would suggest that the scurfy mutation results in a defect, which interferes with the normal down-regulation of T cell activation. (+info)
Stretch-induced overproduction of fibronectin in mesangial cells is mediated by the activation of mitogen-activated protein kinase.
(7/1291)
An excessive production of extracellular matrix (ECM) proteins in glomerular mesangial cells is considered to be responsible for the development of mesangial expansion seen in diabetic nephropathy. Mechanical stretch due to glomerular hypertension has been proposed as one of the factors leading to an increase in the production of ECM proteins in mesangial cells, but the precise mechanism of stretch-induced overproduction of ECM proteins has not been elucidated. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) may play a key role in the overproduction of fibronectin (FN) in mesangial cells exposed to mechanical stretch. MAPK, also termed extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), was activated by mechanical stretch in time- and intensity-dependent manners. Stretch-induced activation of ERK was inhibited by herbimycin A, a tyrosine kinase inhibitor, but not by GF109203X or calphostin C, the inhibitors of protein kinase C. Mechanical stretch also enhanced DNA-binding activity of AP-1, and this enhancement was inhibited by PD98059, an inhibitor of MAPK or ERK kinase (MEK). Furthermore, mechanical stretch stimulated the expression of FN mRNA followed by a significant increase in its protein accumulation. PD98059 could prevent stretch-induced increase in the expression of FN mRNA and protein. These results indicate that the activation of ERK may mediate the overproduction of ECM proteins in mesangial cells exposed to mechanical stretch, an in vitro model for glomerular hypertension seen in diabetes. (+info)
Decreases in cAMP phosphodiesterase activity in hepatocytes cultured with herbimycin A due to cellular microtubule polymerization related to inhibition of tyrosine phosphorylation of alpha-tubulin.
(8/1291)
The increase in cellular cAMP concentration during 10-min incubation of rat hepatocytes with glucagon or forskolin was enhanced markedly when the hepatocytes had been cultured for several hours with herbimycin A. This effect of herbimycin was accompanied by inhibition of tyrosine-phosphorylation of cellular proteins including alpha-tubulin, antagonized by coaddition of Na3VO4 plus H2O2, which also antagonized the herbimycin-induced tyrosine phosphorylation, and overcome by the addition to the 10-min incubation medium of a certain inhibitor of cAMP phosphodiesterase (PDE), which caused a huge accumulation of cAMP. The effective PDE inhibitors were 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and 4-(3-butyloxy-4-methoxyphenyl)-2-imidazolidinone (Ro-20-1724, a PDE4 inhibitor), in addition to 3-isobutyl-1-methylxanthine (a nonselective inhibitor). Rapid breakdown of the once-accumulated cAMP in cultured hepatocytes during the subsequent incubation without PDE inhibitors was progressively prevented when the concentration of herbimycin was increased from 0.3 to 10 microM during prior culture. This effect of herbimycin to inhibit PDE activity in intact cells was abolished by coaddition of a microtubule-disrupting agent, either colchicine or vinblastine, into the culture, but remained unchanged if the vinblastine-containing medium was further supplemented with taxol, a microtubule-stabilizing agent, which by itself mimicked the effect of herbimycin. None of these agents, which thus affected PDE activity in intact cells, inhibited the PDE activity assayable in the cell lysates. The taxol-like and vinblastine-suppressible action of herbimycin to stimulate microtubular assembly was antagonized by Na3VO4/H2O2, as confirmed by confocal microscopic images of the cells stained with fluorescein-bound anti-(alpha-tubulin). Thus, 4-h culture of hepatocytes with herbimycin inhibits phosphorylation of the C-terminal tyrosine residue of alpha-tubulin, thereby stimulating formation of a microtubular network which is responsible for the inhibition of PDE4 in the intact cells by an unknown mechanism. (+info)