Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding

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

Research output: Contribution to journalArticle

127 Citations (Scopus)

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 YMXM 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 YMXM phosphopeptide or sequences corresponding to the GAP or PLC-γ 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 Tyr1021 binds with ≈40-fold higher affinity than a YMXM-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
StatePublished - Jan 1 1993
Externally publishedYes

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Phosphatidylinositol 3-Kinase
Phosphopeptides
Phosphoproteins
Programmable logic controllers
Platelet-Derived Growth Factor Receptors
Phosphotyrosine
Macrophage Colony-Stimulating Factor
Viral Tumor Antigens
Phosphatidylinositol 3-Kinases
Tyrosine
Assays
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Piccione, E., Randi D, R. D., Domchek, S. M., Hu, P., Chaudhuri, M., Backer, J. M., ... Shoelson, S. E. (1993). Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding. Biochemistry, 32(13), 3197-3202.

Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding. / Piccione, Elizabeth; Randi D, Randi D.; Domchek, Susan M.; Hu, Patrick; Chaudhuri, Manas; Backer, Jonathan M.; Schlessinger, Joseph; Shoelson, Steven E.

In: Biochemistry, Vol. 32, No. 13, 01.01.1993, p. 3197-3202.

Research output: Contribution to journalArticle

Piccione, E, Randi D, RD, Domchek, SM, Hu, P, Chaudhuri, M, Backer, JM, Schlessinger, J & Shoelson, SE 1993, 'Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding', Biochemistry, vol. 32, no. 13, pp. 3197-3202.
Piccione E, Randi D RD, Domchek SM, Hu P, Chaudhuri M, Backer JM et al. Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding. Biochemistry. 1993 Jan 1;32(13):3197-3202.
Piccione, Elizabeth ; Randi D, Randi D. ; Domchek, Susan M. ; Hu, Patrick ; Chaudhuri, Manas ; Backer, Jonathan M. ; Schlessinger, Joseph ; Shoelson, Steven E. / Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding. In: Biochemistry. 1993 ; Vol. 32, No. 13. pp. 3197-3202.
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