A new class of mutations reveals a novel function for the original phosphatidylinositol 3-kinase binding site.
Previous studies have demonstrated that the specificity of Src homology 2 (SH2) and phosphotyrosine-binding domain interactions are mediated by phosphorylated tyrosines and their neighboring amino acids. Two of the first phosphotyrosine-based binding sites were found on middle T antigen of polyoma virus. Tyr-250 acts as a binding site for ShcA, whereas Tyr-315 forms a binding site for the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase. However, genetic analysis of a given phosphotyrosine's role in signaling can be complicated when it serves as a binding site for multiple proteins. The situation is particularly difficult when the phosphotyrosine serves as a secondary binding site for a protein with primary binding determinates elsewhere. Mutation of a tyrosine residue to phenylalanine blocks association of all bound proteins. Here we show that the mutation of the amino acids following the phosphorylated tyrosine to alanine can reveal phosphotyrosine function as a secondary binding site, while abrogating the phosphotyrosine motif's role as a primary binding site for SH2 domains. We tested this methodology by using middle T antigen. Our results suggest that Tyr-250 is a secondary binding site for phosphatidylinositol 3-kinase, whereas Tyr-315 is a secondary binding site for a yet-to-be-identified protein, which is critical for transformation.
Hong
Y Kate
YK
Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115, USA.
Mikami
Aki
A
Schaffhausen
Brian
B
Jun
Toni
T
Roberts
Thomas M
TM
D000409:Alanine / Q000737:chemistry; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012190:Retroviridae / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains
D014443:Tyrosine; D010649:Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D000409:Alanine