Activation of the Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion via integrin beta1.
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Activated Cdc42-associated kinase-2 (ACK-2) is a non-receptor tyrosine kinase that appears to be a highly specific target for the Rho-related GTP-binding protein Cdc42. In order to understand better how ACK-2 activity is regulated in cells, we have expressed epitope-tagged forms of this tyrosine kinase in COS-7 and NIH3T3 cells. We find that ACK-2 can be activated by cell adhesion in a Cdc42-dependent manner. However, unlike the focal adhesion kinase, which also is activated by cell adhesion, the activation of ACK-2 is F-actin-independent and does not require cell spreading. In addition, overexpression of ACK-2 in COS-7 cells did not result in the stimulation of extracellular signal-regulated kinase activity but rather activated the c-Jun kinase. Both anti-integrin beta1 antibody and RGD peptides inhibited the activation of ACK-2 by cell adhesion. In addition, ACK-2 was co-immunoprecipitated with integrin beta1. Overall, these findings suggest that ACK-2 interacts with integrin complexes and mediates cell adhesion signals in a Cdc42-dependent manner. (+info)
Bcl-xL blocks activation of related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2 and stress-activated protein kinase/c-Jun N-terminal protein kinase in the cellular response to methylmethane sulfonate.
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The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade. (+info)
The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin.
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In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin. (+info)
MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells.
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Smad proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor-beta (TGF-beta) superfamily of growth factors. Certain Smad proteins (e.g. Smad1, -2, and -3) can act as regulated transcriptional activators, a process that involves phosphorylation of these proteins by activated TGF-beta superfamily receptors. We demonstrate that the intracellular kinase mitogen-activated protein kinase kinase kinase-1 (MEKK-1), an upstream activator of the stress-activated protein kinase/c-Jun N-terminal kinase pathway, can participate in Smad2-dependent transcriptional events in cultured endothelial cells. A constitutively active form of MEKK-1 but not mitogen-activated protein kinase kinase-1 (MEK-1) or TGF-beta-activated kinase-1, two distinct intracellular kinases, can specifically activate a Gal4-Smad2 fusion protein, and this effect correlates with an increase in the phosphorylation state of the Smad2 protein. These effects do not require the presence of the C-terminal SSXS motif of Smad2 that is the site of TGF-beta type 1 receptor-mediated phosphorylation. Activation of Smad2 by active MEKK-1 results in enhanced Smad2-Smad4 interactions, nuclear localization of Smad2 and Smad4, and the stimulation of Smad protein-transcriptional coactivator interactions in endothelial cells. Overexpression of Smad7 can inhibit the MEKK-1-mediated stimulation of Smad2 transcriptional activity. A physiological level of fluid shear stress, a known activator of endogenous MEKK-1 activity in endothelial cells, can stimulate Smad2-mediated transcriptional activity. These data demonstrate a novel mechanism for activation of Smad protein-mediated signaling in endothelial cells and suggest that Smad2 may act as an integrator of diverse stimuli in these cells. (+info)
Dcdc42 acts in TGF-beta signaling during Drosophila morphogenesis: distinct roles for the Drac1/JNK and Dcdc42/TGF-beta cascades in cytoskeletal regulation.
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During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted transforming-growth-factor-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative transforming-growth-factor-beta-like receptors Thickveins and Punt activates a transforming-growth-factor-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the transforming-growth-factor-beta-like signaling pathway can disrupt dorsal closure. Our findings show that although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by the small Ras-like GTPase Drac1 and acts to assemble the cytoskeleton and specify the identity of the first row of cells of the epidermis prior to the onset of dorsal closure. Signaling in the transforming-growth-factor-beta-like pathway is mediated by Dcdc42, and acts during the closure process to control the mechanics of the migration process, most likely via its putative effector kinase DPAK. (+info)
Differential contribution of the ERK and JNK mitogen-activated protein kinase cascades to Ras transformation of HT1080 fibrosarcoma and DLD-1 colon carcinoma cells.
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Although an important contribution of ERK and JNK mitogen-activated protein kinase (MAPK) activation in Ras transformation of rodent fibroblasts has been determined, their role in mediating oncogenic Ras transformation of human tumor cells remains to be established. We have utilized the human HT1080 fibrosarcoma and DLD-1 colon carcinoma cell lines, which contain endogenous mutated and oncogenic N- and K-ras alleles, respectively, to address this role. Study of these cells is advantageous over Ras-transformed rodent model cell systems for two key reasons. First, the ras mutations occurred naturally in the progression of the tumors from which the cell lines were derived, rather than due to overexpression of an exogenously introduced gene. Second, although these tumor cells possess defects in multiple genetic loci, it has been established that mutated Ras contributes significantly to the transformed phenotype of these cells. Clonal variant lines of HT1080 and DLD-1 have been isolated which have lost the oncogenic ras allele and exhibit a corresponding impairment in growth transformation in vitro and in vivo. We found that upregulation of Raf/MEK/ERK and JNK correlated with expression of oncogenic Ras in HT1080, but not DLD-1 cells. Furthermore, inhibition of ERK activation in parental HT1080 cells caused the same changes in cell morphology and actin stress fiber organization seen with loss of expression of activated N-Ras(61K). Thus, we suggest that constitutive activation of the Raf/MEK/ERK and JNK pathways is necessary for Ras-induced transformation of HT1080 but not DLD-1 cells. These results emphasize that cell type differences exist in the signaling pathways by which oncogenic Ras causes transformation. (+info)
Limited role of ceramide in lipopolysaccharide-mediated mitogen-activated protein kinase activation, transcription factor induction, and cytokine release.
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The involvement of ceramide in lipopolysaccharide-mediated activation of mouse macrophages was studied. Lipopolysaccharide, cell-permeable ceramide analogs, and bacterial sphingomyelinase led to phosphorylation of the extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 kinase and induced AP-1 DNA binding in C3H/OuJ (Lpsn) but not in C3H/HeJ (Lpsd) macrophages. Lipopolysaccharide and ceramide mimetics showed distinct kinetics of mitogen-activated protein kinase phosphorylation and AP-1 induction and activated AP-1 complexes with different subunit compositions. Lipopolysaccharide-activated AP-1 consisted of c-Fos, Jun-B, Jun-D, and c-Jun, while C2-ceramide induced Jun-D and c-Jun only. Lipopolysaccharide and, less potently, C2-ceramide or sphingomyelinase, stimulated AP-1-dependent reporter gene transcription in RAW 264.7 cells. Unlike lipopolysaccharide, C2-ceramide failed to activate NF-kappaB and did not induce production of tumor necrosis factor or interleukin-6. The lipopolysaccharide antagonist, Rhodobacter sphae-roides diphosphoryl lipid A, inhibited lipopolysaccharide activation of NF-kappaB and AP-1 but did not block C2-ceramide-induced AP-1. Pretreatment of C3H/OuJ macrophages with C2-ceramide greatly diminished AP-1 induction following subsequent C2-ceramide stimulation. However, lipopolysaccharide-induced transcription factor activation and cytokine release were not influenced. In contrast, lipopolysaccharide pretreatment inhibited both lipopolysaccharide- and C2-ceramide-mediated responses. Thus, ceramide partially mimics lipopolysaccharide in activating the mitogen-activated protein kinases and AP-1 but not in mediating NF-kappaB induction or cytokine production, suggesting a limited role in lipopolysaccharide signaling. (+info)
The Jun kinase cascade is responsible for activating the CD28 response element of the IL-2 promoter: proof of cross-talk with the I kappa B kinase cascade.
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Costimulation of TCR/CD3 and CD28 receptors leads to activation of the Jun kinase (JNK) cascade, which plays a key role in T cell activation, including activation of the IL-2 promoter. We demonstrate that the JNK cascade plays a central role in the activation of the CD28 response element (CD28RE) in the IL-2 promoter. This response element is linked to an activating protein-1 (AP-1) site, which functions synergistically with the CD28RE. The role of the JNK cascade in the activation of this composite element is twofold: 1) activation of the AP-1 site through transcriptional activation of c-Jun, and 2) activation of the CD28RE through selective cross-talk with I kappa B kinase-beta (IKK beta). Dominant-negative versions of JNK kinase, c-Jun, and IKK beta interfered In CD3- plus CD28-induced CD28RE/AP-1 luciferase activity in Jurkat cells. In contrast, the dominant-active JNK kinase kinase, MEKK1, induced CD28RE/AP-1 luciferase activity, in parallel with induction of c-Jun and c-Rel binding to this combined promoter site. Dominant-active MEKK1 also induced transfected IKK beta, but not IKK alpha, activity. In contrast to the JNK cascade, the extracellular signal-regulated kinase (ERK) cascade did not exert an affect on the CD28RE/AP-1 site, but did contribute to activation of the distal NF-AT/AP-1 site. (+info)