Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.
Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.
Yoakim
M
M
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111.
Hou
W
W
Songyang
Z
Z
Liu
Y
Y
Cantley
L
L
Schaffhausen
B
B
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)