Tandem SH2 Domains Confer High Specificity in Tyrosine Kinase Signaling

Abstract

SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors.Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-␥1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/ SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000-to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the ϳ20 -50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase receptors (1). The SH2 domains bind phosphorylated receptor tyrosines, and, since many SH2 domain proteins also contain or associate with catalytic subunits, these interactions recruit the effector enzymes to activated receptors. Tyrosine kinase signaling pathways thus gain specificity from the intrinsic binding preferences of SH2 domains for short sequences that flank phosphotyrosine.A great deal has been learned by studying isolated, individual SH2 domains. Common mechanisms are used for phosphotyrosine recognition (2-12). Most notably, SH2 domain residues Arg␣A2 and Arg␤B5 chelate the phosphotyrosine phosphate. The latter is within the conserved FLVRES sequence. Binding site selectivity is conferred by interactions between two variable loops within SH2 domains (EF and BG) and peptide residues COOH-terminal to phosphotyrosine. The degree of selectivity varies, but phosphopeptides derived from biologically relevant sites typically bind with 20 -50-fold higher affinity than irrelevant or randomized sequences (e.g. Refs. 13-18). Although it is true that a small subset of SH2 domains shows greater selectivity (e.g. 1,000-fold for Grb2), 1 these are exceptions and not the rule.Nevertheless, biological specificity in intact cells is substantially greater than 50-fold, suggesting that more is involved than individual SH2 domain interactions. In fact, all SH2 domain proteins contain additional binding modules (e.g. SH2, SH3, PTB, and PH domains) or motifs. A fundamental concept may have been overlooked by the common tendency to evaluate specificity using isolated, individual domains. Simultaneous binding to multiple domains could enhance specificity through combinatorial effects, as 1) each ...

SHP-2 binds to Tyr763 and Tyr1009 in the PDGF β-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis

“…One possible explanation is that the two SH2 domains of SHP-2 have di erent binding speci®city. When comparing the sequences surrounding naturally occuring association sites for SHP-2, Tyr763 is quite similar to the association sites found in a number of other molecules (Table 1) (Case et al, 1994;Fujioka et al, 1996;Jackson et al, 1997;Ohnishi et al, 1996;Ottinger et al, 1998;Sugimoto et al, 1994). Many of these molecules have an alanine residue in position +1, an acidic residue in position +2, followed by either leucine or isoleucine in position +3.…”

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

“…[24,25] Grucza et al [26] determined binding at different temperatures by fluorescence titration and found a somewhat lower affinity for an ITAM peptide derived from the CD3e-chain of the T cell receptor (35 nM at 25 8C). We performed a van't Hoff analysis of the Grucza data and the outcome compares very well with our results, especially with respect to the DH 0 and DC p values (Table 1).…”

Differential Roles of the Src Homology 2 Domains of Phospholipase C-γ1 (PLC-γ1) in Platelet-derived Growth Factor-induced Activation of PLC-γ1 in Intact Cells

“…These domains share an ability to bind to phosphorylated tyrosine residues, but they also possess independent binding sites for the residues surrounding the target phosphotyrosine (1)(2)(3). Each SH2 domain thus recognizes specific sequence patterns with a high selectivity and thereby promotes specific protein-protein interactions (4,5). The specificity of such interactions is further increased through an avidity effect when the participating proteins possess additional complementary binding sites.…”

“…The specificity of such interactions is further increased through an avidity effect when the participating proteins possess additional complementary binding sites. Proteins such as phospholipase C-␥ (PLC-␥1 and PLC-␥2), the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase), Ras GTPase-activating protein, the SH2 domain-containing protein-tyrosine phosphatase (SH-PTP), and members of the Syk family of protein-tyrosine kinases (PTKs) contain two SH2 domains (5)(6)(7)(8). The tandem SH2 domains of Syk PTKs interact with the biphosphorylated tyrosine-based activation motif that is present in various immune receptors (5).…”