In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.
We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
J A
JA
Cooper
A
A
Kashishian
D000818:Animals; D002417:Cattle; D004252:DNA Mutational Analysis; D020690:GTPase-Activating Proteins; D016391:Genes, src / Q000235:genetics*; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor