Rac-PAK Signaling Stimulates Extracellular Signal-Regulated Kinase (ERK) Activation by Regulating Formation of MEK1-ERK Complexes

Abstract

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-Δ19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-Δ19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

We thank the members of the Weber lab and PWP for helpful discussions. We also thank Scott Weed and Tom Parsons for Rac plasmids, Channing Der for Ras V12, Debbie Morrison for Raf-1 CAAX and Raf-1 SAAX, Alexis Rahal for technical assistance and Cliff Martin for assistance in preparing the manuscript.

This work was supported by Public Health Service grants GM47332, CA40042, and CA39076 from the National Institutes of Health. S. T. Eblen was supported by National Research Service Award 5F32 GM18672-02. J. K. Slack was supported by NIH grants CA76465 and CA40042 to J. T. Parsons.