Martyn C. Botfield

Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries.

Many proteins involved in intracellular signal transduction contain a small, 50‐60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein‐protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P‐X‐X‐P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage‐displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain‐mediated processes and form the basis for the design of SH3‐specific inhibitors of disease pathways.

Tandem SH2 Domains Confer High Specificity in Tyrosine Kinase Signaling

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-γ1. 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 ∼20–50-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.

Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide

BACKGROUND Recruitment of the intracellular tyrosine kinase Syk to activated immune-response receptors is a critical early step in intracellular signaling. In mast cells, Syk specifically associates with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within the IgE receptor. The mechanism by which Syk recognizes these motifs is not fully understood. Both Syk SH2 (Src homology 2) domains are required for high-affinity binding to these motifs, but the C-terminal SH2 domain (Syk-C) can function independently and can bind, in isolation, to the tyrosine-phosphorylated IgE receptor in vitro. In order to improve understanding of the cellular function of Syk, we have determined the solution structure of Syk-C complexed with a phosphotyrosine peptide derived from the gamma subunit of the IgE receptor. RESULTS The Syk-C:peptide structure is compared with liganded structures of both the SH2 domain of Src and the C-terminal SH2 domain of ZAP-70 (the 70 kDa zeta-associated protein). The topologies of these domains are similar, although significant differences occur in the loop regions. In the Syk-C structure, the phosphotyrosine and leucine residues of the peptide ligand interact with pockets on the protein, and the intervening residues are extended. CONCLUSIONS Syk-C resembles other SH2 domains in its peptide-binding interactions and overall topology, a result that is consistent with its ability to function as an independent SH2 domain in vitro. This result suggests that Syk-C plays a unique role in the intact Syk protein. The determinants of the binding affinity and selectivity of Syk-C may reside in the least-conserved structural elements that comprise the phosphotyrosine- and leucine-binding sites. These structural features can be exploited for the design of Syk-selective SH2 antagonists for the treatment of allergic disorders and asthma.

Chapter 24. SH2 and SH3 Domains: Choreographers of Multiple Signaling Pathways

Publisher Summary To a large degree cellular signal transduction pathways are choreographed by modular Src homology 2 (SH 2 ) and Src homology 3 (SH 3 ) domains that mediate well-specific protein: protein interactions. SH 2 domains are modules of ∼100 amino acids that specifically bind phosphotyrosine-containing proteins and peptides. SH 3 domains are modules of ∼60 amino acids that bind to the proline-rich sequences. This chapter includes a list of selected therapeutic targets, possessing SH 2 or SH 3 domains. The design of specific antagonists to these domains holds the promise of targeted treatment of a broad range of pathologies. The role of SH 2 and SH 3 domains in signal transduction has been extensively studied. In growth factor, cytokine and antigen signaling, occupancy of a receptor by agonist results in receptor dimerization and the phosphorylation of regulatory tyrosines on the cytoplasmic surface. Phosphorylation is catalyzed by kinases that are a part of the receptor (receptor tyrosine kinases) or recruited to the receptor from the cytoplasm (non-receptor tyrosine kinases). The resulting phosphotyrosines permit binding of specific SH 2 -containing proteins and initiate a cascade of the sequential protein interactions. SH 3 domains are independently folded protein modules of 55-70 amino acids that selectively bind proline-rich protein sequences. Although generally of lower affinity than SH 2 -mediated interactions, SH 3 interactions are essential for multiple signaling cascades.