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

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf -1 on serine 338 (S338), a critical regulatory residue. The specificity req uirements for Pak-mediated phosphorylation of S338 were examined by substit ution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Pho sphorylation was found to be very sensitive to alterations in amino acid si de chains proximal to S338, Loss of N-terminal arginines resulted in decrea sed peptide phosphorylation while loss of these residues, as well as C-term inal glutamates and bulky C-terminal hydrophobic residues, decreased phosph orylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is s ignificant in epidermal growth factor- and Src-mediated signaling, suggesti ng 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 S3 38 by Pak was increased in the presence of Src, The complexity of this sign aling module could thus account for the different levels of Raf-1 activatio n required for fulfillment of different biological roles within the cell. ( C) 2001 Published by Elsevier Science B,V, on behalf of the Federation of E uropean Biochemical Societies.