Balance between MKK6 and MKK3 mediates p38 MAPK associated resistance to cisplatin in NSCLC

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.     

MEK kinase 3 directly activates MKK6 and MKK7, specific activators of the p38 and c-Jun NH2-terminal kinases.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no substrates for MEKK3 have been identified. We have examined the regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK kinases specific for JNK and p38, respectively. Coexpression of MKK7 with MEKK3 in COS-7 cells enhanced MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6 autophosphorylation and in vitro activation of p38alpha were also observed following coexpression of MKK6 with MEKK3. MEKK2, a closely related homologue of MEKK3, also activated MKK7 and MKK6 in COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2 directly activated recombinant MKK7 and MKK6 in vitro. These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and MKK6 in the JNK and p38 pathways. We have also examined whether MEKK3 or MEKK2 activates p38 in intact cells using MAPK-activated protein kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin, sorbitol, or the expression of MEKK3 in HEK293 cells enhanced MAPKAPK2 phosphorylation, whereas MEKK2 was less effective. Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular stress was abolished by the p38 inhibitor SB-203580, suggesting that MEKK3 is coupled to p38 activation in intact cells.     

The interaction of p38 with its upstream kinase MKK6

Mitogen‐activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full‐length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.     

TGF-β1 induces endothelial cell apoptosis by shifting VEGF activation of p38(MAPK) from the prosurvival p38β to proapoptotic p38α

TGF-β1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-β1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-β1 induces endothelial cell expression of VEGF, which mediates TGF-β1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-β1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, β, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-β1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, β, and γ. VEGF activates p38β, whereas TGF-β1 activates p38α. TGF-β1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38β is activated, and the surviving cells become refractory to TGF-β1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-β1 induction of apoptosis, whereas downregulation of p38β or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38β and p38γ transduce survival signaling. TGF-β1 activation of p38α is mediated by VEGF, which in the absence of TGF-β1 activates p38β. Therefore, these results show that TGF-β1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38β to the proapoptotic p38α.     

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

Physiological roles of MKK4 and MKK7: insights from animal models

c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of numerous physiological processes during development and in response to stress. Its activity is increased upon phosphorylation by the MAPK kinases, MKK4 and MKK7. Similar to the early embryonic death of mice caused by the targeted deletion of the jnk genes, mice lacking mkk4 or mkk7 die before birth. The inability of MKK4 and MKK7 to compensate for each other's functions in vivo is consistent with their synergistic effect in mediating JNK activation. However, the phenotypic analysis of the mutant mouse embryos indicates that MKK4 and MKK7 have specific roles that may be due to their selective regulation by extracellular stimuli and their distinct tissue distribution. MKK4 and MKK7 also have different biochemical properties. For example, whereas MKK4 can activate p38 MAPK, MKK7 functions as a specific activator of JNK. Here we summarize the studies that have shed light on the mechanism of activation of MKK4 and MKK7 and on their physiological functions.     

Differential involvement of p38 mitogen-activated protein kinase kinases MKK3 and MKK6 in T-cell apoptosis

The p38 mitogen-activated protein kinase (p38MAPK) is activated in response to various stimuli, including cellular stress, inflammatory cytokines and cell surface receptors. The activation of p38MAPK is predominantly mediated by the two upstream MAPK kinases MKK3 and MKK6. To study the role of the p38MAPK pathway in vivo, we generated Mkk6^–/– mice. We examined whether T-cell apoptosis is affected in these mice and in our previously reported Mkk3^–/– mice. Strikingly, in vivo deletion of double positive thymocytes in Mkk6^–/– mice was impaired, whereas Mkk3^–/– mice showed no apparent abnormality. Conversely, CD4⁺T cells from Mkk3^–/– but not from Mkk6^–/– mice were resistant to activation-induced cell death and cytokine-withdrawal-induced apoptosis. In peripheral CD4⁺T cells, MKK3 is induced upon stimulation, whereas MKK6 is downregulated. These results suggest a novel mechanism regulating T-cell apoptosis differentially through the p38MAPK pathway by MKK3 and MKK6.     

抑制p38MAPK信号通路对Bpiv3复制的影响

由牛副流感病毒3型(Bovine parainfluenza virus type 3,Bpiv3)感染引起的牛副流感病已成为各国牛场最重要的传染病之一,每年都会给世界养牛业造成巨大的经济损失,但关于该病致病的分子机制研究较少。本研究通过观察Bpiv3感染对MDBK细胞中丝裂原活化蛋白激酶(MKK3)及其下游分子p38丝裂酶原活化的蛋白激酶(p38MAPK)的表达的影响,探讨相关的信号转导机制,对p38 MAPK通路在Bpiv3感染过程中的作用进行了初步研究。Bpiv3感染细胞后,采用Western Blot检测MKK3,p38 MAPK在蛋白水平的表达变化,并采用ELISA法检测细胞上清中IL-6,IL-8,IL-13和TNF-α的水平变化,采用SPSS 12软件进行统计学分析。结果表明,Bpiv3在感染后能够诱导MKK3的激活以及p38的磷酸化,激活了p38 MAPK信号通路。而且p38 MAPK信号通路参与了Bpiv3的复制过程。ELISA检测Bpiv3感染后以及使用抑制剂SB202190处理后的细胞上清中IL-6、IL-8、IL-13和TNF-α的水平发现,p38 MAPK信号通路参与了Bpiv3诱导的炎症反应。研究证实Bpiv3感染能够激活p38 MAPK通路,显著上调MKK3的表达并诱导p38发生磷酸化,进一步激活下游分子发挥生物学活性,促进Bpiv3的复制及诱导促炎细胞因子的产生。p38 MAPK信号通路的激活可能是Bpiv3感染诱发炎症反应的机制之一。     

Effect of pressure overload-induced hypertrophy on the expression and localization of p38 MAP kinase isoforms in the mouse heart

p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: α, β, γ, and δ. To date, the prominent isoform in heart has been thought to be p38α. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38γ and p38δ were expressed at protein levels comparable to p38α and 38β, respectively. In the early phase of pressure-overload hypertrophy (1–7 days after constriction of the transverse aorta), the abundance of p38β, p38γ and p38δ mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38α and p38γ in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7–28 days after constriction of the transverse aorta), p38γ immunoreactivity accumulated in the nucleus whereas the distribution of p38α remained unaffected. Hence, both p38α and p38γ are prominent p38 isoforms in heart and p38γ may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.     

Role of C/EBP-β, p38 MAPK, and MKK6 in IL-1β-mediated C3 gene regulation in astrocytes

Complement component C3, the central player in the complement cascade and the pro-inflammatory cytokine IL-1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL-1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38-α MAPK, and C/EBP-β isoforms (LAP1, LAP2, or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL-1β, led to the induction of C3 mRNA and protein expression as determined by real-time RT-PCR and Western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190-HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL-1β also induced C3 promoter activity in U373 cells in a MAP kinase- and C/EBP-β-dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, that is, MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP-β, LAP(T235A) showed reduction in IL-1β-mediated C3 promoter activation. These results suggest that the p38α, MAPK, and MKK6 play prominent roles in IL-1β and C/EBP-β-mediated C3 gene expression in astrocytes.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAK1-MKK3-p38 MAPK pathway

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C(2)C(12) cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C(2)C(12) myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation.     

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.     

The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal Kinase Kinases

The c-Jun NH₂-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH₂ termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α isoforms lack an NH₂-terminal extension that is present in the other MKK7 isoforms. This NH₂-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.     

Impaired synergistic activation of stress-activated protein kinase SAPK/JNK in mouse embryonic stem cells lacking SEK1/MKK4: different contribution of SEK2/MKK7 isoforms to the synergistic activation.

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) family, plays an important role in a stress-induced signaling cascade. SAPK/JNK activation requires the phosphorylation of Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 (MKK4) and MKK7 (SEK2) have been identified as the upstream MAPK kinases. Here we examined the activation and phosphorylation sites of SAPK/JNK and differentiated the contribution of SEK1 and MKK7alpha1, -gamma1, and -gamma2 isoforms to the MAPK activation. In SEK1-deficient mouse embryonic stem cells, stress-induced SAPK/JNK activation was markedly impaired, and this defect was accompanied with a decreased level of the Tyr phosphorylation. Analysis in HeLa cells co-transfected with the two MAPK kinases revealed that the Thr and Tyr of SAPK/JNK were independently phosphorylated in response to heat shock by MKK7gamma1 and SEK1, respectively. However, MKK7alpha1 failed to phosphorylate the Thr of SAPK/JNK unless its Tyr residue was phosphorylated by SEK1. In contrast, MKK7gamma2 had the ability to phosphorylate both Thr and Tyr residues. In all cases, the dual phosphorylation of the Thr and Tyr residues was essentially required for the full activation of SAPK/JNK. These data provide the first evidence that synergistic activation of SAPK/JNK requires both phosphorylation at the Thr and Tyr residues in living cells and that the preference for the Thr and Tyr phosphorylation was different among the members of MAPK kinases.     

The role of RIP2 in p38 MAPK activation in the stressed heart

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.