MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.
ABSTRACT: The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway.
Project description:The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin.
Project description:Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38? is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3-/- cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3-/- and MKK6-/- mice by micro-CT analysis. Bone loss was partially inhibited in MKK3-/- as well as MKK6-/- mice, despite normal osteoclastogenesis in MKK6-/- cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation.
Project description:The p38 mitogen-activated protein kinase (MAPK) is activated in vitro by three different protein kinases: MKK3, MKK4, and MKK6. To examine the relative roles of these protein kinases in the mechanism of p38 MAP kinase activation in vivo, we examined the effect of disruption of the murine Mkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway. We show that MKK3 and MKK6are essential for tumor necrosis factor-stimulated p38 MAPK activation. In contrast, ultraviolet radiation-stimulated p38 MAPK activation was mediated by MKK3, MKK4, and MKK6. Loss of p38 MAPK activation in the mutant cells was associated with defects in growth arrest and increased tumorigenesis. These data indicate that p38 MAPK is regulated by the coordinated and selective actions of three different protein kinases in response to cytokines and exposure to environmental stress.
Project description:TP53 is universally recognized as a pivotal protein in cell-cycle fate and apoptotic induction and, unsurprisingly, it is one of the most commonly hijacked control mechanisms in cancer. Recently, the kinase MKK3 emerged as a potential therapeutic target in different types of solid tumor being linked to mutant p53 gain-of-function. In this review, we summarize the delicate relationship among p53 mutational status, MKK3/MKK6 and the downstream activated master kinase p38MAPK, dissecting a finely-tuned crosstalk, in a potentially cell-context dependent scenario that urges towards a deeper characterization of the different molecular players involved in this signaling cascade and their interactions.
Project description:The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced beta-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2'-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38alpha-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCdelta (protein kinase Cdelta), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1beta and interferon-gamma, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.
Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) are prevalent causes of late-onset Parkinson's disease. Here, we show that LRRK2 binds to MAPK kinases (MKK) 3, 6, and 7, and that LRRK2 is able to phosphorylate MKK3, 6 and 7. Over-expression of LRRK2 and MKK6 increased the steady state levels of each protein beyond that observed with over-expression of either protein alone. Co-expression increased levels of MKK6 in the membrane more than in the cytoplasm. The increased expression of LRRK2 and MKK6 requires MKK6 activity. The disease-linked LRRK2 mutations, G2019S, R1441C and I2020T, enhance binding of LRRK2 to MKK6. This interaction was further supported by in vivo studies in C. elegans. RNAi knockdown in C. elegans of the endogenous orthologs for MKK6 or p38, sek-1 and pmk-1, abolishes LRRK2-mediated protection against mitochondrial stress. These results were confirmed by deletion of sek-1 in C. elegans. These data demonstrate that MKKs and LRRK2 function in similar biological pathways, and support a role for LRRK2 in modulating the cellular stress response.
Project description:<h4>Background</h4>Conventional p38? inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.<h4>Methods</h4>BMDM from wild type (WT), MKK3-/-, and MKK6-/- mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D?±?SB203580.<h4>Results</h4>Pre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3-/- BMDM compared with either WT or MKK6-/- cells. IL-10 mRNA expression was modestly reduced in MKK3-/- BMDM but was preserved in MKK6-/- cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3-/- and p38 inhibitor pre-treated cells, but not MKK6-/- cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6-/- cells.<h4>Conclusion</h4>Unlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA.
Project description:The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.
Project description:A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway.
Project description:Notch1 genes encode receptors for a signaling pathway that regulates various aspects of cell growth and differentiation; however, the role of Notch1 signaling in p38 mitogen-activated protein kinase (MAPK) signaling pathway is still not well defined. In this study, we found that Notch1 intracellular domain (Notch1-IC) prevents oxidative stress-induced cell death through the suppression of the Apoptosis signal-regulating kinase (ASK) 1 signaling pathway. Notch1-IC inhibited H2O2-induced activation of ASK1 and the activation of downstream kinases in the p38 MAPK signaling cascade. The results of both in vivo binding and kinase studies have revealed that ASK1 is the direct target of Notch1-IC, whereas it produced no effect on either MAP kinase kinase (MKK) 3 or p38 MAPK. Notch1-IC blocked both the homooligomerization of ASK1 and inhibited ASK1 activity. Furthermore, Notch1-IC facilitated the translocation of activated ASK1 toward the nucleus. Notch1 knockdown was determined to be highly susceptible to oxidative stress-induced activation of ASK1-MKK3/MKK6-p38 MAPK signaling cascade and cell death. Taken together, our findings suggest that Notch1-IC may act as a negative regulator in ASK1 signaling cascades.