Binding of the Src SH2 Domain to Phosphopeptides is Determined by Residues in both the SH2 Domain and the Phosphopeptides

Abstract

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

Intramolecular Regulation of Protein Tyrosine Phosphatase SH-PTP1: A New Function for Src Homology 2 Domains

“…These results also suggest that pY628 may be the in vivo binding site on the IL-3 receptor for SH-PTP1, although additional site(s) on the receptor may also be important for high-affinity association. One point worth mentioning is that the above Ko values are 1-3 orders of magnitude higher than those reported for pY peptides binding to other SH2 domains, such as those of Src (Payne et al, 1993;Bibbins et al, 1993), Lck (Payne et al, 1993), PI3 kinase (Felder et al, 1993), and SH-PTP2 (Sugimoto et al, 1994). We believe that pY peptides with higher affinity to SH-PTP1 SH2 domains might yet be discovered.…”

“…Further experimentation is necessary to understand this phenomenon. Bibbins et al (1993) have previously reported that a R175L mutant of Src SH2 domain is still capable of high-affinity binding to a PDGFR pY751 peptide.…”

A Divalent Metal Ion Binding Site in the Kinase Insert Domain of the .alpha.-Platelet-Derived Growth Factor Receptor Regulates Its Association with SH2 Domains

“…Phosphotyrosine-dependent (Yu et al, 1991;Fantl et al, 1992;Kashishian et al, 1992) and -independent binding of SH2 domains has previously been demonstrated (Cooper & Kashishian, 1993;Pendergast et al, 1991). Recently, the wild-type Src SH2 domain was shown to bind a 13-residue peptide from the /3-PDGFR KI domain incorporating Y751 (PD 751) (Bibbins et al, 1993). Further, the mutant R175L SH2 domain displayed higher affinity for this nonphosphorylated peptide than other phosphopeptides (Bibbins et al, 1993).…”

“…Recently, the wild-type Src SH2 domain was shown to bind a 13-residue peptide from the /3-PDGFR KI domain incorporating Y751 (PD 751) (Bibbins et al, 1993). Further, the mutant R175L SH2 domain displayed higher affinity for this nonphosphorylated peptide than other phosphopeptides (Bibbins et al, 1993). We are also able to show that the nonphosphorylated KI domain is able to associate with the p85 N-SH2 domain in the absence of calcium and this effect is markedly enhanced in the presence of calcium ions (Mahadevan et al, unpublished results).…”

Reverse Binding Mode of Phosphotyrosine Peptides with SH2 Protein

“…The relative importance of different amino acids at a given position was determined by replacing the Z with a defined residue. The LCK SH2 domain showed a strong preference for aliphatic/hydrophobic residues (e.g., Val and Ile) at positions pY+3 and pY+4, but no apparent selectivity for N-terminal residues 54,55 (Figure 5A). This result is in accordance with the reported specificity of the LCK SH2 domain.…”