A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that pla! essential roles in signal transduction. Gab? becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular completes,with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K). the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. interaction with p85-PI3K is mediated by tyrosine residues Y-452 Y-476 and Y-584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y-614. CrkL interaction is mediated by its SH2 domain recognizing, Y-266 and Y-293, despite the latter being in a nonconsensus (YTFK) environment. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.