Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.
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Exposure of human keratinocyte HaCaT cells to ultraviolet B-irradiation induced apoptotic morphologic changes. In this study, we found that the ultraviolet B irradiation (0.25 J per cm2) induced phosphorylation of p38 mitogen-activated protein kinase and c-jun N-terminal protein kinase, and also significant activation of caspase-3 (CPP32-like protease) and a small increase of caspase-1 (ICE-like protease) activity in the early stages of ultraviolet B-induced apoptosis. Pretreatments of the cells with a p38 mitogen-activated protein kinase inhibitor, SB203580, and a caspase-3 inhibitor, Ac-Asp-Met-Gln-Asp-1-aldehyde, suppressed the ultraviolet B irradiation-induced apoptosis by approximately 60% as estimated by nuclear staining and DNA laddering. Pretreatment with caspase-1 inhibitor, Ac-Tyr-Val-Lys-Asp-aldehyde was without effect. Ultraviolet B-induced caspase-3 activation resulted in cleavage of poly(ADP) ribose polymerase, which was abolished by the caspase-3 inhibitor. SB203580 pretreatment prevented activation of caspase-3 and caspase-1, and also suppressed the cleavage of poly(ADP) ribose polymerase. Neither ceramide generation nor sphingomyelinase activation (neutral and acid) was observed in the ultraviolet B-irradiated HaCaT cells. Also various antioxidants did not affect the caspase activation induced by ultraviolet B irradiation. These results indicated that activation of p38 mitogen-activated protein kinase upstream of caspases may play an important part in the apoptotic process of keratinocytes exposed to ultraviolet B irradiation. (+info)
Oncogenic Ras enhances NF-kappaB transcriptional activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways.
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Tumors frequently contain mutations in ras genes, resulting in constitutive activation of Ras-activated signaling pathways. The ultimate targets of these signal transduction cascades are transcription factors required for cellular proliferation. Understanding how constitutive activation of Ras contributes to tumorigenesis requires an understanding of both the signaling pathways that Ras activates and how these pathways in turn regulate gene expression. Gene expression from kappaB sites is enhanced in cells transformed with activated Ras and NF-kappaB activity is required for oncogenic Ras to transform NIH-3T3 and Rat-1 fibroblasts. Both dominant negative and constitutively active components of signaling pathways have been tested for their ability to regulate NF-kappaB. These experiments show that Ras utilizes Raf-dependent and Raf-independent pathways to activate NF-kappaB transcriptional activity, both of which require the stress-activated kinase p38 or a related kinase. In the case of Raf, activation of NF-kappaB by an autocrine factor stimulates kappaB-dependent transcriptional activity. (+info)
Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration.
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Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-gamma, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation. (+info)
Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis.
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The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g. anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis. (+info)
CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs.
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Tumor necrosis factor receptor-associated factors (TRAFs) associate with the CD40 cytoplasmic domain and initiate signaling after CD40 receptor multimerization by its ligand. We used saturating peptide-based mutational analyses of the TRAF1/TRAF2/TRAF3 and TRAF6 binding sequences in CD40 to finely map residues involved in CD40-TRAF interactions. The core binding site for TRAF1, TRAF2, and TRAF3 in CD40 could be minimally substituted. The TRAF6 binding site demonstrated more amino acid sequence flexibility and could be optimized. Point mutations that eliminated or enhanced binding of TRAFs to one or both sites were made in CD40 and tested in quantitative CD40-TRAF binding assays. Sequences flanking the core TRAF binding sites were found to modulate TRAF binding, and the two TRAF binding sites were not independent. Cloned stable transfectants of human embryonic kidney 293 cells that expressed wild type CD40 or individual CD40 mutations were used to demonstrate that both TRAF binding sites were required for optimal NF-kappaB and c-Jun N-terminal kinase activation. In contrast, p38 mitogen-activated protein kinase activation was primarily dependent upon TRAF6 binding. These studies suggest a role in CD40 signaling for competitive TRAF binding and imply that CD40 responses reflect an integration of signals from individual TRAFs. (+info)
MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo.
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Several growth factor- and calcium-regulated kinases such as pp90(rsk) or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF in vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(-/-)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2. (+info)
Effect of SB 203580 on the activity of c-Raf in vitro and in vivo.
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The inhibition of SAPK2a/p38 (a mitogen activated protein (MAP) kinase family member) by SB 203580 depends on the presence of threonine at residue 106. Nearly all other protein kinases are insensitive to this drug because a more bulky residue occupies this site (Eyers et al., 1998). Raf is one of the few protein kinases that possesses threonine at this position, and we show that SB 203580 inhibits c-Raf with an IC50 of 2 microM in vitro. However, SB 203580 does not suppress either growth factor or phorbol ester-induced activation of the classical MAP kinase cascade in mammalian cells. One of the reasons for this is that SB 203580 also triggers a remarkable activation of c-Raf in vivo (when measured in the absence of the drug). The SB 203580-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras, is not prevented by inhibitors of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase, and is not triggered by the binding of this drug to SAPK2a/p38. The paradoxical activation of c-Raf by SB 203580 (and by another structurally unrelated c-Raf inhibitor) suggests that inhibitors of the kinase activity of c-Raf may not be effective as anti-cancer drugs. (+info)
The N-terminal transactivation domain of ATF2 is a target for the co-operative activation of the c-jun promoter by p300 and 12S E1A.
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The adenovirus E1A proteins activate the c-jun promoter through two Jun/ATF-binding sites, jun1 and jun2. P300, a transcriptional coactivator of several AP1 and ATF transcription factors has been postulated to play a role in this activation. Here, we present evidence that p300 can control c-jun transcription by acting as a cofactor for ATF2: (1) Over-expression of p300 was found to stimulate c-jun transcription both in the presence and absence of E1A. (2) Like E1A, p300 activates the c-jun promoter through the junl and jun2 elements and preferentially activates the N-terminal domain of ATF2. (3) Co-immunoprecipitation assays of crude cell extracts indicate that endogenous p300/CBP(-like) proteins and ATF2 proteins are present in a multiprotein complex that can bind specifically to the jun2 element. We further demonstrate that the Stress-Activated-Protein-Kinase (SAPK) target sites of ATF2, Thr69 and Thr71 are not required for the formation of the p300/CBP-ATF2 multiprotein complex. These data indicate that E1A does not inhibit all transcription activation functions of p300, and, in fact, cooperates with p300 in the activation of the ATF2 N-terminus. (+info)