Role of MEKK2-MEK5 in the regulation of TNF-alpha gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells.

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcepsilonRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcepsilonRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH(2)-terminal kinase (JNK) activation in response to cross-linking of FcepsilonRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcepsilonRI-induced activation of the tumor necrosis factor-alpha (TNF-alpha) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-alpha promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway.

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.

MEKK2 gene disruption causes loss of cytokine production in response to IgE and c-Kit ligand stimulation of ES cell-derived mast cells.

Ligation of the high-affinity IgE receptor (FcepsilonRI) or of c-Kit stimulates cytokine production in mast cells. We show that MEK kinase 2 (MEKK2), a MAPK kinase kinase (MAP3K) that regulates the JNK and ERK5 pathways, is required for cytokine production in embryonic stem (ES) cell-derived mast cells (ESMC). Targeted disruption of the MEKK2 or MEKK1 gene was used to abolish expression of the respective kinases in ESMC. Transcription of specific cytokines in response to IgE or c-Kit ligand was markedly reduced in MEKK2(-/-) ESMC relative to wild-type ESMC. Cytokine production in MEKK1(-/-) ESMC was similar to that of wild-type ESMC, demonstrating the specificity of MEKK2 in signaling cytokine gene regulation. MEKK2(-/-) ESMC also lost receptor-mediated stimulation of JNK. In contrast, JNK activation in response to UV irradiation was normal, showing that MEKK2 is required for receptor signaling but not for cellular stress responses. MEKK2 is the first MAP3K shown to be required for mast cell tyrosine kinase receptor signaling controlling cytokine gene expression.

Reovirus-induced apoptosis is mediated by TRAIL.

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton.

Cell shape change and the restructuring of the cytoskeleton are important regulatory responses that influence the growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun NH2-terminal kinase (JNK) pathway in response to exposure of cells to microtubule toxins, including taxol. MEKK1 expression is elevated 3-fold in mitosis and microtubule toxin-treated cells accumulated at G2/M of the cell cycle. Targeted disruption of MEKK1 expression in embryonic stem cells resulted in the loss of JNK activation and increased apoptosis in response to taxol. Targeted disruption of the MEK kinase 2 gene had no effect on activation of the JNK pathway in response to microtubule toxins demonstrating a specific role of MEKK1 in this response. Cytochalasin D-mediated disruption of actin fibers activates JNK and stimulates apoptosis similarly in MEKK1(-/-) and wild type cells. The results show that MEKK1 is required for JNK activation in response to microtubule but not actin fiber toxins in embryonic stem cells. MEKK1 activation can protect cells from apoptosis in response to change in the integrity of the microtubule cytoskeleton.

Organization and regulation of mitogen-activated protein kinase signaling pathways.

Mitogen-activated protein kinases (MAPKs) are components of a three kinase regulatory cascade. There are multiple members of each component family of kinases in the MAPK module. Specificity of regulation is achieved by organization of MAPK modules, in part, by use of scaffolding and anchoring proteins. Scaffold proteins bring together specific kinases for selective activation, sequestration and localization of signaling complexes. The recent elucidation of scaffolding mechanisms for MAPK pathways has begun to solve the puzzle of how specificity in signaling can be achieved for each MAPK pathway in different cell types and in response to different stimuli. As new MAPK members are defined, determining their organization in kinase modules will be critical in understanding their select role in cellular regulation.