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 MKK6 are 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. Several groups of mitogen-activated protein kinase (MAPK) signal transduction pathways have been identified in mammals, including extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38 MAPK. Each of these groups of MAPK is activated by dual phosphorylation on Thr and Tyr within a tripeptide motif (Thr-Xaa-Tyr) located within the activation loop of the MAPK. This phosphorylation is mediated by seven MAPK kinases (MAPKKs) that have specificity for individual MAPK isoforms. Thus, ERK1 and ERK2 are activated by MEK1 and MEK2, ERK5 is activated by MEK5, JNK is activated by MKK4 and MKK7, and p38 MAPK is activated by MKK3 and MKK6 (Schaeffer and Weber 1999; Kyriakis and Avruch 2001). These MAPKKs and MAPKs can create independent signaling modules that may function in parallel.The mechanism that accounts for the specificity of MAPKKs to activate individual MAPK isoforms is mediated, in part, by an interaction between an N-terminal region located on the MAPKK and a docking site located on the MAPK (Bardwell and Thorner 1996; Enslen and Davis 2001). Recently, structural insight into the mechanism of interaction between a MAPKK and a MAPK has been achieved by X-ray crystallography (Chang et al. 2002). This analysis demonstrated that there is a direct interaction of the N-terminal region of the MAPKK with a docking groove present on the surface of the MAPK distant from the catalytic active site (Weston et al. 2002). A second determinant of MAPKK specificity is the structure of the MAPK activation loop that contains the ThrXaa-Tyr dual phosphorylation motif (Enslen et al. 2000). The specificity of these interactions mediate, in part, the ability of an individual MAPKK to activate a particular MAPK selectively.It is interesting that mammalian MAPK signaling modules include more than one MAPKK because in yeast only a single MAPKK appears to activate each MAPK. The role of this pathway complexity in mammals is unclear. However, it may be significant that individual yeast MAPK isoforms are activated by only a limited group of extracellular stimuli, but mammalian MAPK isoforms are activated by a wide array of extracellular stimuli. It is there...

The p38 mitogen-activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38alpha, p38beta2, p38gamma and p38delta). These p38 MAPK isoforms appear to mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and also to selective activation by MAPK kinases (MAPKKs). Here we report the identification of two factors that contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N-terminus of the MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T-loop) of p38 MAPK isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK.

Mixed-lineage protein kinase 3 (MLK3) is a member of the mitogen-activated protein (MAP) kinase kinase kinase group that has been implicated in multiple signaling cascades, including the NF-B pathway and the extracellular signal-regulated kinase, c-Jun NH 2 -terminal kinase (JNK), and p38 MAP kinase pathways. Here, we examined the effect of targeted disruption of the murine Mlk3 gene. Mlk3 ؊/؊ mice were found to be viable and healthy. Primary embryonic fibroblasts prepared from these mice exhibited no major signaling defects. However, we did find that MLK3 deficiency caused a selective reduction in tumor necrosis factor (TNF)-stimulated JNK activation. Together, these data demonstrate that MLK3 contributes to the TNF signaling pathway that activates JNK.The mechanism of c-Jun NH 2 -terminal kinase (JNK) activation caused by tumor necrosis factor (TNF) is incompletely understood. It is established that JNK is activated by dual phosphorylation on the T-loop within the motif Thr-Pro-Tyr (7). This phosphorylation is mediated by the actions of two different mitogen-activated protein (MAP) kinase kinases: MKK4 and MKK7 (35). These MAP kinase kinases can be activated by MAP kinase kinase kinases (MAP3K), but the identity of the relevant TNF-stimulated MAP3K is unclear.Three MAP3K have been implicated in the activation of JNK caused by TNF. First, apoptosis signal-regulating kinase 1 is thought to be involved in the late phase of JNK activation in response to TNF, most likely as a result of the generation of reactive oxygen species (34). The immediate activation of JNK caused by TNF may be mediated by TAK1 and/or by one or more members of the mixed-lineage protein kinase (MLK) family. MLKs may be selectively involved in TNF-stimulated JNK activation (25), while TAK1 is implicated as a common TNF-stimulated activator of JNK, p38 MAPK,29). The relative roles of TAK1 and MLKs in TNF signaling are unclear. In this study, we have examined the possible contribution of a MLK to TNF signaling.There are three subgroups of MLKs (10). The MLK group (MLK1, MLK2, MLK3, and MLK4) shares similar structural domains, including an SH3 domain and a Crib motif that binds Cdc42 and Rac1. The DLK group (DLK and LZK) is structurally distinct and lacks the SH3 and Crib sequences. The third group of protein kinases consists of a single member (ZAK) that is distinctive because of the presence of a SAM domain. Many of these protein kinases are expressed in only a limited number of tissues; for example, MLK1 is expressed in epithelial cells and DLK is expressed in neurons (10). However, one member of this gene family is ubiquitously expressed, consistent with a possible role as a mediator of TNF signaling in many tissues-MLK3. This possibility is consistent with previous studies showing that TNF activates MLK3 (25), that MLK-family protein kinases (like TNF) (35) can selectively activate MKK7 (14, 18), and that a small-molecule MLK inhibitor can block TNF-stimulated JNK activation (25). However, recent RNA interference (RNAi)-based studies have su...