SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.

Abstract

src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-y and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosinecontaining regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BlAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 X 107 to 40 x 107/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.Activation of receptors with tyrosine kinase activity by growth factors promotes tyrosine autophosphorylation and associations with proteins containing src homology 2 (SH2) domains (3,9,19,22,39,43), such as phospholipase C-y (PLC--y) (24,26,44), GTPase-activating protein (GAP) of ras (1,16,23,29), phosphatidylinositol (PI) 3'-kinase-associated p85 (6, 13-15, 32, 41), and GRB2 (21). Specific regions containing phosphorylated tyrosine within platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) receptors which are responsible for binding to SH2 domains of signaling molecules have been identified (13-15, 23, 28, 37). The functional significance of these interactions is not fully understood; however, the ubiquity of the interactions suggests that they serve to mediate phosphorylation events crucial for the control of activity of signaling molecules by both receptor-linked and cytoplasmic tyrosine kinases (3, 4, 9-12, 17, 20, 29-31, 36). Indeed, it has been recently shown that FGF-induced PI hydrolysis is eliminated by a point mutation of the FGF receptor at Y-766, which prevents the association with and tyrosine phosphorylation of PLC--y (27,28,33). Similarly, elimination of Y-740 and Y-751 of the PDGF receptor abolishes the binding of p85 and stimulation of PI 3'-kinase activity (13). Important in defining functional significance will be quantitative measures of the kinetics and strength of these interactions.We have used a new technique for real-time kinetics and affinit...

Phosphopeptide binding to the N‐terminal SH2 domain of the p85α subunit of PI 3′‐kinase: A heteronuclear NMR study

“…The kinetics of the interaction between SH2 domains and phosphotyrosine-containing sequences have been investigated in an earlier study employing a surface plasmon-resonance approach using a BIAcore system (Felder et al, 1993;Panayotou et al, 1993). In those experiments, phosphotyrosine-containing peptides were immobilized on a biosensor surface.…”

New insights into protein‐tyrosine kinase receptor signaling complexes

“…Following autophosphorylation of these sites, specific SH2 domain-containing proteins are recruited from the surrounding cytosol into complexes with the activated PTK receptor. This recruitment is mediated by phosphotyrosine-SH2 domain interactions that exhibit both high-affinity binding and rapid dissociation and exchange (Felder et al, 1993;. The critical requirement for the presence of phosphotyrosine has been demonstrated by mutation of key tyrosine residues to phenylalanine residues at the autophosphorylation sites on PTK receptors.…”

Tyrosine Phosphorylation of Nicotinic Acetylcholine Receptor Mediates Grb2 Binding

“…This approach, which allows real-time measurement of binding kinetics and affinities, has been used previously to study interactions between different SH2 domains and tyrosine autophosphorylation sites of growth factor receptors (Felder et al, 1993;Panayotou et al, 1993;Lombardo et al, 1995). Tyrosinephosphorylated peptides (pY 393 ) corresponding to amino acids 388 -401 of the Torpedo ␦ subunit were coupled to one cell of a sensor chip.…”