Src homology 2 (SH2) domains are found in a variety of cytoplasmic proteins involved in mediating signals from cell surface receptors to various intracellular pathways. They fold as modular units and are capable of recognizing and binding to short linear peptide sequences containing a phosphorylated tyrosine residue. Here we show that each of the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase selects phage displayed peptide sequences containing the core (L/I)-A-(R/K)-I-R. The serine/threonine kinase A-Raf, containing the sequence LQRIRS, is associated with the p85 protein in both quiescent and growth factor stimulated cells. This suggests that p85 and 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 least one of four distinct basic-X-basic sequence motifs within A-Raf for binding. This is the first description of a phosphotyrosine-independent SH2 domain interaction that requires basic residues on the SH2 ligand.Phosphatidylinositol 3-kinase (PI 3-kinase) 1 consists of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110), the latter of which is responsible for the phosphorylation of phosphatidylinositol lipids at the D3 position and serine phosphorylation of proteins (1-3). The p85 subunit contains a Src homology 3 (SH3) domain capable of binding to proline-rich sequences, a region homologous to the breakpoint cluster region (BCR) gene product, a p110 binding domain (110), and two SH2 domains. PI 3-kinase activity increases in response to platelet-derived growth factor (PDGF) binding to its receptor, in large part because the p85⅐p110 complex is relocalized from the cytosol to the lipids at the plasma membrane, by p85 SH2 domains binding directly to tyrosine phosphorylated sites on the receptor (4, 5).The SH2 domains of p85 recognize and bind to proteins such as the PDGF receptor at sites that contain a pY-X-X-M sequence (pY ϭ phosphotyrosine, X ϭ any amino acid, M ϭ methionine) in a phosphorylation-dependent manner. The residues within the p85 SH2 domain responsible for binding this sequence include a critical arginine residue that coordinates twice with the phosphate group of the phosphotyrosine residue and a hydrophobic pocket involved in methionine binding (6 -8).Over the past several years, there have also been reports of SH2 domains binding to proteins via a phosphotyrosine-independent mechanism. These reports include several phosphoserine/phosphothreonine-dependent interactions (9 -14). In addition, there have been a few reports that concluded that the SH2-mediated interaction was phosphotyrosine-independent but did not determine whether or not the interaction was instead dependent upon phosphoserine or phosphothreonine (15-18). In each instance, the precise amino acid residues involved in mediating these SH2 domain interactio...