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 requirements for Pak-mediated 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.
King
A J
AJ
Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202, USA.
Wireman
R S
RS
Hamilton
M
M
Marshall
M S
MS
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016177:Macrophage-1 Antigen / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology; D054462:p21-Activated Kinases; D019061:src-Family Kinases / Q000378:metabolism* / Q000494:pharmacology
D016177:Macrophage-1 Antigen; D010446:Peptide Fragments; D019061:src-Family Kinases; C515911:PAK3 protein, human; C515913:Pak3 protein, rat; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D054462:p21-Activated Kinases