A new class of mutations reveals a novel function for the original phosphatidylinositol 3-kinase binding site
Abstract
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.
Acknowledgments
We thank Drs. Joanne Chan, Ole Gjoerup, Helen McNamee, Romesh Subramanian, and Lei Zhang for critical reading of the manuscript and Meacie Fairfax for the manuscript preparation. This work was supported by National Institutes of Health Grants RO1 CA30002-21 and PO1 CA89021-02 (to T.M.R.) and CA34722 and CA50661 (to B.S.).
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Copyright © 2003, The National Academy of Sciences.
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Received: January 29, 2003
Published online: July 24, 2003
Published in issue: August 5, 2003
Acknowledgments
We thank Drs. Joanne Chan, Ole Gjoerup, Helen McNamee, Romesh Subramanian, and Lei Zhang for critical reading of the manuscript and Meacie Fairfax for the manuscript preparation. This work was supported by National Institutes of Health Grants RO1 CA30002-21 and PO1 CA89021-02 (to T.M.R.) and CA34722 and CA50661 (to B.S.).
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A new class of mutations reveals a novel function for the original
phosphatidylinositol 3-kinase binding site, Proc. Natl. Acad. Sci. U.S.A.
100 (16) 9434-9439,
https://doi.org/10.1073/pnas.1432964100
(2003).
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