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 down-stream 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 down-stream 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.