JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension.

MAP kinase (MAPK) cascades are composed of a MAPK, MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Despite the existence of numerous components and ample opportunities for crosstalk, most MAPKs are specifically and distinctly activated. We investigated the basis for specific activation of the JNK subgroup of MAPKs. The specificity of JNK activation is determined by the MAPKK JNKK1, which interacts with the MAPKKK MEKK1 and JNK through its amino-terminal extension. Inactive JNKK1 mutants can disrupt JNK activation by MEKK1 or tumor necrosis factor (TNF) in intact cells only if they contain an intact amino-terminal extension. Mutations in this region interfere with the ability of JNKK1 to respond to TNF but do not affect its activation by physical stressors. As JNK and MEKK1 compete for binding to JNKK1 and activation of JNKK1 prevents its binding to MEKK1, activation of this module is likely to occur through sequential MEKK1:JNKK1 and JNKK1:JNK interactions. These results underscore a role for the amino-terminal extension of MAPKKs in determination of response specificity.