Binding affinities of tyrosine-phosphorylated peptides to the COOH-terminal SH2 and NH2-terminal phosphotyrosine binding domains of Shc

Ming Ming Zhou, John E. Harlan, Warren S. Wade, Seth Crosby, Kodimangalam S. Ravichandran, Steven J. Burakoff, Stephen W. Fesik

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Abstract

The adaptor protein Shc has been implicated in Ras signaling via association with many tyrosine-phosphorylated receptors, including growth factor receptors, antigen receptors on T and B cells, and cytokine receptors. Shc could interact with the activated receptors through the carboxyl-terminal Src homology 2 (SH2) domain or the structurally unrelated amino-terminal phosphotyrosine binding (PTB) domain. Using NMR and surface plasmon resonance techniques, we have measured the binding affinities of the SH2 and the PTB domains of Shc to a series of phosphotyrosine-containing peptides derived front known Shc binding sites. Tyrosine-phosphorylated peptides derived from Trk (pY490), polyoma virus middle T-antigen (pY250), ErbB3 (pY1309), and epidermal growth factor receptor (pY1086, pY1148, and pY1114) that contain NPXpY sequences bind preferentially to the PTB domain of Shc with Kd values of 0.02-5.3 μM. The binding affinities of these peptides to the Shc SH2 domain were in the range of 220-1290 μM. In contrast, tyrosine-phosphorylated peptides from epidermal growth factor receptor (pY992, pY1173) and the ζ chain of the T-cell receptor bind preferentially to the SH2 domain (Kd = 50-130 μM) versus the PTB domain (Kd > 680 μM). From these studies, the relative contribution of the individual domains of Shc for binding to the phosphotyrosine-containing portions of these proteins was determined. In addition, our data indicate that the high affinity binding of the PTB domain to the NPXpY-containing peptides results from a very high association rate and a rapid dissociation rate, which is similar to previous results observed for the SH2-phosphopeptide complexes.

Original languageEnglish
Pages (from-to)31119-31123
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number52
StatePublished - Dec 29 1995
Externally publishedYes

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    Zhou, M. M., Harlan, J. E., Wade, W. S., Crosby, S., Ravichandran, K. S., Burakoff, S. J., & Fesik, S. W. (1995). Binding affinities of tyrosine-phosphorylated peptides to the COOH-terminal SH2 and NH2-terminal phosphotyrosine binding domains of Shc. Journal of Biological Chemistry, 270(52), 31119-31123.