Phosphorylation site specificity of the Pak‐mediated regulation of Raf‐1 and cooperativity with Src

Abstract

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pakmediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor-and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Mitogen-Activated Protein Kinase Feedback Phosphorylation Regulates MEK1 Complex Formation and Activation during Cellular Adhesion

“…Transfection of constitutively active PAK into cells increased not only S298 phosphorylation but also T292 phosphorylation (12), although only S298 was phosphorylated by PAK in vitro (12,23). Investigation of the PAK phosphorylation of Raf-1 on S338 revealed a consensus sequence containing a tyrosine at the ϩ2 position and arginine residues at the Ϫ2 and Ϫ5 positions (33). The sequence surrounding S298 of MEK1 loosely conforms to this consensus, with a tyrosine at the ϩ2 position and arginines at the Ϫ3 and Ϫ7 positions.…”

The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation

“…However, in studies examining the ability of PAK to phosphorylate Raf-1 in vitro, this was not the case (58). Another explanation is that Y341D mutants relocalize Raf-1 to sites of Ser-338 phosphorylation.…”

Characterization of Ser338 Phosphorylation for Raf-1 Activation

“…In response to mitogens, phosphorylation of both Ser 338 and Tyr 341 occurs in the junction region between the aminoterminal negative domain and the carboxyl-terminal kinase domain (23)(24)(25)(26)(27)(28)(29). The Src family has been implicated in the phosphorylation of Tyr 340 (in insect Sf9 cells) and Tyr 341 (in mammalian cells) (23,24,(27)(28)(29).…”

“…The Src family has been implicated in the phosphorylation of Tyr 340 (in insect Sf9 cells) and Tyr 341 (in mammalian cells) (23,24,(27)(28)(29). Interestingly, phosphorylation of Tyr 341 is barely detectable unless v-Src is coexpressed with Raf-1.…”