Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

Oxaliplatin down-regulates survivin by p38 MAP kinase and proteasome in human colon cancer cells

Oxaliplatin, a platinum derivative cancer drug, has been used for treating human colorectal cancers. Survivin has been proposed as a cancer target, which highly expressed in most cancer cells but not normal adult cells. In this study, we investigated the regulation of survivin expression by exposure to oxaliplatin in human colon cancer cells. Oxaliplatin (3–9 μM for 24 h) markedly induced cytotoxicity, proliferation inhibition and apoptosis in the human RKO colon cancer cells. The survivin protein expression of RKO cells is dramatically reduced by oxaliplatin; however, the survivin gene expression is slightly altered. The survivin blockage of oxaliplatin elevated caspase-3 activation and apoptosis in RKO cells. Over-expression of survivin proteins by transfection with a survivin-expressed vector resisted the oxaliplatin-induced cancer cell death. Meantime, oxaliplatin elicited the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB202190, a specific p38 MAP kinase inhibitor, restored the survivin protein level and attenuated oxaliplatin-induced cancer cell death. In addition, oxaliplatin increased the levels of phospho-p53 (Ser-15) and total p53 proteins. Inhibition of p53 expression by a specific p53 inhibitor pifithrin-α reduced the phosphorylated p38 MAP kinase and active caspase-3 proteins in the oxaliplatin-exposed RKO cells. In contrast, SB202190 did not alter the oxaliplatin-induced p53 protein level. Furthermore, treatment with a specific proteasome inhibitor MG132 restored survivin protein level in the oxaliplatin-treated colon cancer cells. Taken together, our results demonstrate for the first time that survivin is down-regulated by p38 MAP kinase and proteasome degradation pathway after treatment with oxaliplatin in the human colon cancer cells.     

MAP kinase cascades in elicitor signal transduction

 Protein kinases play important roles in elicitor signal transduction. In this article, I describe the current view of the role of mitogen-activated protein kinase (MAPK) cascades in elicitor signal transduction of plant cells based on our own research and recent developments in this field. In the past several years, it has become apparent that MAPK cascades play important roles in elicitor signal transduction in plants. Our early studies demonstrated the identification of p47 MAPK in tobacco as an elicitor-responsive protein kinase and possible involvement of p47 MAPK in elicitor signal transduction to induce defense responses, including defense gene expression and hypersensitive cell death. However, the molecular identity of p47 MAPK is still unclear. Recent important studies suggest that tobacco MAPK cascades that include SIPK, and/or WIPK, and NtMEK2, an upstream kinase for both SIPK and WIPK, have a crucial function in induction of defense responses and hypersensitive cell death. The orthologs of these protein kinases in Arabidopsis and alfalfa are also suggested to have similar functions. Furthermore, the identification of loss-of-function mutation in Arabidopsis reveals a negative regulatory role for putative MAPK cascades in plant defense mechanisms. Received: February 7, 2002 / Accepted: February 25, 2002     

Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase

Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Here we show that inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signalling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signalling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction.     

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.     

p38 MAPK信号传导通路

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COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells

Interaction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 microg/cm(2)) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. We also show that specific inhibition of p38 MAPK by 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole (SB203580) or by overexpression of a dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells.     

Inhibitors of unactivated p38 MAP kinase

Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.     

Oxalate selectively activates p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signal transduction pathways in renal epithelial cells

Oxalate, a metabolic end product, is an important factor in the pathogenesis of renal stone disease. Oxalate exposure to renal epithelial cells results in re-initiation of the DNA synthesis, altered gene expression, and apoptosis, but the signaling pathways involved in these diverse effects have not been evaluated. The effects of oxalate on mitogen- and stress-activated protein kinase signaling pathways were studied in LLC-PK1 cells. Exposure to oxalate (1 mM) rapidly stimulated robust phosphorylation and activation of p38 MAPK. Oxalate exposure also induced modest activation of JNK, as monitored by phosphorylation of c-Jun. In contrast, oxalate exposure had no effect on phosphorylation and enzyme activity of p42/44 MAPK. We also show that specific inhibition of p38 MAPK by 4(4-(fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazole (SB203580) or by overexpression of a kinase-dead dominant negative mutant of p38 MAPK abolishes oxalate induced re-initiation of DNA synthesis in LLC-PK1 cells. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as MAPK-activated protein kinase-2 activity in cell extracts. Thus, this study not only provides the first demonstration of selective activation of p38 MAPK and JNK signaling pathways by oxalate but also suggests that p38 MAPK activity is essential for the effects of oxalate on re-initiation of DNA synthesis.     

Specific regulation of cytokine-dependent p38 MAP kinase activation by p62/SQSTM1

We have previously shown that p62/SQSTM1 binds to p38. In this study, we identified two association domains of p62 to p38 by conducting co-immunoprecipitation experiments. One domain comprises the amino acids 173-182, named N-terminal p38 interaction (NPI) domain, and the other domain comprises the amino acids 335-344, named C-terminal p38 interaction (CPI) domain. An aspartic acid tripeptide located at 335-337 was required for their association. However, the direct interaction was only observed between the recombinant p38 and the peptide of the NPI domain, but not that of the CPI domain in the surface plasmon resonance analyses. These results suggest that the CPI domain may serve to form a certain conformation suitable for the association with p38. Furthermore, we showed that knockdown of p62 expression by siRNA led to impaired p38 phosphorylation only when HeLa cells were stimulated by cytokine. The critical role of p62 in cytokine-dependent p38 signalling pathway was further confirmed by measuring IL-8 mRNA. Cytokine mRNA is often stabilized via p38 pathway. In the absence of p62, IL-8 mRNA induced by IL-1beta became more fragile. These data show that p62 specifically regulates cytokine-dependent p38 signalling pathway.     

PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases

We examined how pigment epithelium derived factor (PEDF), an effective endogenous antiangiogenic protein, decreases survival of primary cultures of human umbilical vein endothelial cells (HUVECs) in a low serum environment supplemented with the endothelial cell growth factor (VEGF). We provide evidence that induction of apoptosis by PEDF is associated with activation of p38 followed by cleavage of caspases 3, 8, and 9 by treatment with PEDF, and PEDF's actions are caspase dependent. A key mediator in the executioner effects of PEDF is p38 since the inhibition of p38 activity blocked apoptosis and prevented cleavage of caspases 3, 8, and 9. Although PEDF-induced phosphorylation of JNK1, the inhibition of JNK1 had no effect on apoptosis, even though it prevented phosphorylation of JNK1 by PEDF. Based on these findings, we propose that the antiangiogenic action of PEDF is dependent on activation of p38 MAPkinase which regulates cleavage of multiple caspases cascades.     

p38 MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells

We have previously reported that interleukin (IL)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.     

'Reverse' alpha-ketoamide-based p38 MAP kinase inhibitors

We have identified a second series of potent p38 inhibitors. As with our first generation series, these compounds are based on an alpha-ketoamide scaffold. The reversal of the ketoamide order, however, introduces more chemical flexibility and in addition results in improve potencies against p38.     

Regulation of p21-activated kinase-independent Rac1 signal transduction by nischarin

Nischarin regulates Rac1-dependent cell motility by interaction with and inhibition of the p21-activated kinase (PAK1). In addition to regulating the activation of PAK1, Rac1 controls multiple downstream pathways to regulate cell growth and differentiation, as well as cell motility. Signaling by a constitutively activated Rac1 mutant deficient in PAK binding (Rac1Q61L-40C) was examined to determine whether Nischarin impinges on these other Rac1 effector pathways. Nischarin formed immunoprecipitatable complexes with Rac1Q61L and Rac1Q61L-40C when the proteins were co-expressed. In NIH3T3 cells, Rac1Q61L and Rac1Q61L-40C stimulation of a minimal NF-kappaB response element or the cyclin D1 promoter, a downstream target of NF-kappaB, was inhibited by co-expression of Nischarin. Additionally, suppression of endogenous Nischarin protein with small interfering RNA in PC12 cells enhanced Rac1Q61L and Rac1Q61L-40C activation of NF-kappaB. In further support of Nischarin suppressing PAK independent Rac signaling, foci formation in monolayers of NIH3T3 cells by Rac1Q61L-40C in cooperation with c-Raf/CAAX was inhibited by the presence of Nischarin. Nischarin alters the cellular localization of Rac1Q61L and Rac1Q61L-40C to vesicles and this positively correlates with the repression of the Rac1 signal. Thus, Nischarin, in addition to regulating the PAK strand of Rac1 signaling, can also regulate other links in the web of Rac1 signaling pathways.