Using a phage display library to identify basic residues in A-Raf requiredto mediate binding to the Src homology 2 domains of the p85 subunit of phosphatidylinositol 3 '-kinase

Src homology 2 (SH2) domains are found in a variety of cytoplasmic proteins involved in mediating signals from cell surface receptors to various intra cellular pathways. They fold as modular units and are capable of recognizin g and binding to short linear peptide sequences containing a phosphorylated tyrosine residue. Here we show that each of the SH2 domains of the p85 sub unit of phosphatidylinositol 3-kinase selects phage displayed peptide seque nces containing the core (L/I)-A-(R/K)-I-R. The serine/threonine kinase A-R af, containing the sequence LQRIRS, is associated with the p85 protein in b oth quiescent and growth factor stimulated cells. This suggests:that p85 an d A-Raf exist in a protein complex in cells and that complex formation does not require growth factor stimulation. We also show that p85 and A-Raf can bind directly to each other in vitro and that this interaction is mediated in part by the p85 SH2 domains. Further, the p85 SH2 domains require at le ast one df four distinct basic-X-basic sequence motifs within A-Raf for bin ding. This is the first description of a phosphotyrosine-independent SH2 do main interaction that requires basic residues on the SH2 ligand.