The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo.

Src homology 2 (SH2) domains are the largest family of interaction modules encoded by the human genome to recognize tyrosine-phosphorylated sequences and thereby play pivotal roles in transducing and controlling cellular signals emanating from protein-tyrosine kinases. Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motif recognized by an SH2 domain is the key to understanding its cellular function. Here we cloned all 120 SH2 domains identified in the human genome and determined the phosphotyrosyl peptide binding properties of 76 SH2 domains by screening an oriented peptide array library. Of these 76, we defined the selectivity for 43 SH2 domains and refined the binding motifs for another 33 SH2 domains. We identified a number of novel binding motifs, which are exemplified by the BRDG1 SH2 domain that selects specifically for a bulky, hydrophobic residue at P ؉ 4 relative to the Tyr(P) residue. Based on the oriented peptide array library data, we developed scoring matrix-assisted ligand identification (or SMALI), a Web-based program for predicting binding partners for SH2-containing proteins. When applied to SH2D1A/SAP (SLAM-associated protein), a protein whose mutation or deletion underlies the Xlinked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this diseaseassociated protein. SMALI also identified a number of potential interactors for BRDG1, a protein whose function is largely unknown. Peptide in-solution binding analysis demonstrated that a SMALI score correlates well with the binding energy of a peptide to a given SH2 domain. The definition of the specificity space of the human SH2 domain provides both the necessary molecular basis and a platform for future exploration of the functions for SH2-containing proteins in cells.

Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes.

SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on ∼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.

Background: WWOX encodes a 46-kDa tumor suppressor. Results: WW1 domain of WWOX mediates its protein-protein interaction with PY motifs that are involved in molecular processes, including transcription, RNA processing, and metabolism. Conclusion: The WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks. Significance: This study provides a better understanding of WWOX biology in normal and disease states.