Phosphatidylinositol 3-Kinase p85 SH2 Domain Specificity Defined by Direct Phosphopeptide/SH2 Domain Binding

Elizabeth Piccione, Randi D. Case, Susan M. Domchek, Hu Patrick, Manas Chaudhuri, Jonathan M. Backer, Joseph Schlessinger, Steven E. Sholson

Research output: Contribution to journalArticle

128 Scopus citations

Abstract

We have developed a competition binding assay to quantify relative affinities of isolated Srchomology 2 (SH2) domains for phosphopeptide sequences. Eleven synthetic 11-12-amino acid phosphopeptides containing YMYM or YVXM recognition motifs bound to a PI 3-kinase p85 SH2 domain with highest affinities, including sequences surrounding phosphorylated tyrosines of the PDGF, CSF-1/c-Fms, and kit-encoded receptors, IRS-1, and polyoma middle T antigens; matched, unphosphorylated sequences did not bind. A scrambled YMYM phosphopeptide or sequences corresponding to the GAP or PLC-7 SH2 domain binding motifs of the PDGF, FGF, and EGF receptors bound to the p85 SH2 domain with 30- 100-fold reduced affinity, indicating that this affinity range confers specificity. Binding specificity was appropriately reversed with an SH2 domain from PLC-γ: a phosphopeptide corresponding to the site surrounding PDGF receptor Tyrl021 binds with ≈40-fold higher affinity than a YMYM-phosphopeptide. We conclude that essential features of specific phosphoprotein/SH2 domain interactions can be reconstituted using truncated versions of both the phosphoprotein (a phosphopeptide) and cognate SH2 domain-containing protein (the SH2 domain). SH2 domain binding specificity results from differences in affinity conferred by the linear sequence surrounding phosphotyrosine.

Original languageEnglish (US)
Pages (from-to)3197-3202
Number of pages6
JournalBiochemistry
Volume32
Issue number13
DOIs
Publication statusPublished - Apr 1 1993
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Piccione, E., Case, R. D., Domchek, S. M., Patrick, H., Chaudhuri, M., Backer, J. M., ... Sholson, S. E. (1993). Phosphatidylinositol 3-Kinase p85 SH2 Domain Specificity Defined by Direct Phosphopeptide/SH2 Domain Binding. Biochemistry, 32(13), 3197-3202. https://doi.org/10.1021/bi00064a001