Raf-1 serine 338 phosphorylation plays a key role in adhesion-dependent activation of extracellular signal-regulated kinase by epidermal growth factor.
Activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade by polypeptide growth factors is tightly coupled to adhesion to extracellular matrix in nontransformed cells. Raf-1, the initial kinase in this cascade, is intricately regulated by phosphorylation, localization, and molecular interactions. We investigated the complex interactions between Raf-1, protein kinase A (PKA), and p21-activated kinase (PAK) to determine their roles in the adhesion dependence of signaling from epidermal growth factor (EGF) to ERK. We conclude that Raf-1 phosphorylation on serine 338 (S338) is a critical step that is inhibited in suspended cells. Restoration of phosphorylation at S338, either by expression of highly active PAK or by expression of an S338 phospho-mimetic Raf-1 mutation, led to a partial rescue of ERK activation in suspended cells. Raf-1 inhibition in suspension was not due to excessive negative regulation on inhibitory sites S43 and S259, as these serines were largely dephosphorylated in suspended cells. Finally, strong phosphorylation of Raf-1 S338 provided resistance to PKA-mediated inhibition of ERK activation. Phosphorylation at Raf-1 S43 and S259 by PKA only weakly inhibited EGF activation of Raf-1 and ERK when cells maintained high Raf-1 S338 phosphorylation.
Edin
Matthew L
ML
Department of Pharmacology, University of North Carolina, Chapel Hill, 1017 Mary Ellen Jones Building, CB 7365, Chapel Hill, NC 27599-7365, USA.
Juliano
Rudy L
RL
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology / Q000502:physiology*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D034741:RNA, Small Interfering; D012694:Serine; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3