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 phosphotyrosinecontaining 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 104-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 ofthe 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.Src homology 2 (SH2) domains are regions of approximately 100 amino acids that bind phosphotyrosine-containing peptide sequences (20,29,33). SH2 domains are implicated in the formation of signaling complexes, such as those that occur upon activation of the platelet-derived growth factor receptor (PDGF-R) and epidermal growth factor receptor. These receptors are autophosphorylated on several tyrosine residue...

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site

“…The application of a phosphopeptide competition binding assay by Alonso et al (1995) underscored the importance of an aspartic acid residue at position +4. A similar study demonstrated the relevance of a glutamic acid at position 73 or 74 with respect to phosphotyrosine (Bibbins et al, 1993). In fact, in the case of the Ufo RTK both amino acids are found at these positions.…”

c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism.

“…This mutation has been shown to abolish phosphotyrosine binding in vitro (Bibbins et al 1993) but is still able to associate with focal adhesions (Kaplan et al 1994). Despite its ability to associate with focal adhesions, Src251 R175L did not enhance cell spreading on fibronectin (Fig.…”

Src SH3/2 Domain-mediated Peripheral Accumulation of Src and Phospho-myosin Is Linked to Deregulation of E-cadherin and the Epithelial-Mesenchymal Transition

“…When required, cells were serum-starved in serum-free MEM for 20 h and stimulated with MEM containing 10% FBS for 10 min. Src527F containing mutations that disrupt either the Src SH2 or SH3 domain binding capacity were generated with the QuickChange mutagenesis kit (Stratagene, Amsterdam, The Netherlands) using the mutant sense oligonucleotide 5Ј-CCT TCT TGG TCC TGG AGA GCG AGA CG-3Ј to create the SH2 domain mutant (Arg to Leu at position 175 of chicken Src; Mayer et al, 1992 andBibbins et al, 1993) or 5Ј-GAA GGT GAC GCG TGG CTG GCT CA-3Ј to create the SH3 domain mutant (Trp to Ala at position 118 of chicken Src; Erpel et al, 1995). The resulting Src527F/R175L and Src527F/W118A were cloned into the retroviral pBabe Puro vector.…”