YEAST 2-HYBRID IN-VIVO ASSOCIATION OF THE SRC KINASE LYN WITH THE PROTOONCOGENE PRODUCT CBL BUT NOT WITH THE P85 SUBUNIT OF PI 3-KINASE

Ligand binding of multi-chain antigen receptors and hematopoietin/cyto kine receptors results in rapid activation of protein tyrosine kinase (PTK)-dependent signalling molecules such as phosphatidylinositol 3-ki nase (PI 3-kinase). Go-precipitation studies have shown that Src-relat ed PTK, such as Lyn, associates with the p85 regulatory subunit of PI 3-kinase via SH2 and SH3 domain binding with their cognate ligands, Mo re recent studies have shown that the proto-oncogene product Cbl co-pr ecipitates with p85 following engagement of cytokine and antigen recep tors, As opposed to in vitro co-precipitation studies, the yeast two-h ybrid screen reveals lit vivo protein-protein interactions, Using the yeast two-hybrid screen, we demonstrate an in vivo association of Lyn' s SH3 and SH2 domains with the proline-rich domain of Cbl, Lyn's SH3 a nd SH2 domains do not interact with p85 in the yeast two-hybrid screen , as would be predicted from glutathione-S-transferase (GST) fusion pr otein pull-down or co-immunoprecipitation studies from whole cell lysa tes, However, the SH3 domain of p85 interacts with the proline-rich do main of Cbl, When yeast were transformed with catalytic Lyn, an intera ction between p85's SH2 domain and Cbl occurred, From the data, we pro pose the following three step process of PI 3-kinase activation: (1) c omplexes of Lyn-Cbl and Cbl-p85 exist without ligand stimulation, (2) upon ligand binding, Lyn becomes active and phosphorylates Cbl, and (3 ) Cbl's tyrosine phosphorylated residue serves as a docking site for t he SH2 domains of p85 - thereby stabilizing the complex and activating PI 3-kinase, The yeast two-hybrid system can be used to dissect the p recise mechanisms of in vivo protein-protein interactions, including t hose between phosphotyrosine and SH2-containing proteins.