Adhesion stimulates direct PAK1/ERK2 association and leads to ERK-dependent PAK1 Thr212 phosphorylation.
The Rac1/Cdc42 effector p21-activated kinase (PAK) is activated by various signaling cascades including receptor-tyrosine kinases and integrins and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical Ras/Raf/MEK/ERK Map kinase signaling cascade, likely because of PAK co-activation of Raf and MEK. Herein, we found that adhesion signaling also stimulates an association between PAK1 and ERK1/2. PAK1 and ERK1/2 co-immunoprecipitated from rat aortic smooth muscle cells (SMC) plated on fibronectin, and the two proteins co-localized in membrane ruffles and adhesion complexes following PDGF-BB or sphingosine 1-phosphate treatment, respectively. Far Western analysis demonstrated a direct association between the two proteins, and peptide mapping identified an ERK2 binding site within the autoinhibitory domain of PAK1. Interestingly, deletion of a major ERK binding site in PAK attenuates activation of an ERK-dependent serum-responsive element (SRE)-luciferase reporter gene, indicating that association between PAK and ERK is required to facilitate ERK signaling. We also show that ERK2 phosphorylates PAK1 on Thr(212) in vitro and that Thr(212) is phosphorylated in smooth muscle cells following PDGF-BB treatment in an adhesion- and MEK/ERK-dependent fashion. Expression of a phosphomimic variant, PAK-T212E, does not alter ERK association, but markedly attenuates downstream ERK signaling. Taken together, these data suggest that PAK1 may facilitate ERK signaling by serving as a scaffold to recruit Raf, MEK, and ERK to adhesion complexes, and that subsequent growth factor-stimulated phosphorylation of PAK-Thr(212) by ERK may serve to provide a negative feedback signal to control coordinate activation of ERK by growth factor- and matrix-induced signals.
Sundberg-Smith
Liisa J
LJ
Department of Pathology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Doherty
Jason T
JT
Mack
Christopher P
CP
Taylor
Joan M
JM
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013912:Threonine / Q000378:metabolism*; D054462:p21-Activated Kinases
D017931:DNA Primers; D013912:Threonine; C515906:Pak1 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D019950:Mitogen-Activated Protein Kinase 1