The N-terminal ERR-binding site of MEK1 is required for efficient feedbackphosphorylation by ERK2 in vitro and ERK activation in vivo

An ERK2-binding site at the N terminus of MEK1 was reported to mediate thei r stable association. We examined the importance of this binding site in th e feedback phosphorylation of MEK1 on Thr(292) and Thr(386) by ERK2, the ph osphorylation and activation of ERK2 by MEK1, and the interaction of MEK1 w ith ERK2 and Raf-1, Deletion of the binding site from MEK1 reduced its phos phorylation by ERK2, but had no effect on its phosphorylation by p21-activa ted protein kinase-1 (PAK1). A MEK1 N-terminal peptide containing the bindi ng site inhibited MEK1 phosphorylation by ERK2, However, it did not affect MEK1 phosphorylation by p21-activated protein kinase or myelin basic protei n phosphorylation by ERK2, Deletion of the N-terminal ERK-binding domain of MEK1 also reduced its ability to phosphorylate ERK2 in vitro, to co-immuno precipitate with ERK2, and to stimulate ERK2 activation in transfected cell s, but it did not alter the association with endogenous Raf-1. Using ERK2-p 38 chimeras and an ERK2 deletion mutant, a MEK1-binding site of ERK2 was lo calized to its N terminus.