Abstract
Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein-protein interactions in a systematic and reliable manner.
Highlights
From the ‡Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1; §Department of Chemistry, University of Waterloo, Waterloo, ON, N2L 3G1, Canada
Interactions driven by post-translational modifications such as phosphorylation, which comprises a significant part of the Protein–protein interaction (PPI) network, have proven difficult to decipher systematically by high throughput methods such as yeast two-hybrid [15] and affinity purification coupled to mass spectrometry (AP-MS) [16]
Systematic yeast two-hybrid screen has been applied to human open-reading frames (ORFs) [15], it is not suitable for deciphering interactions mediated by phosphorylation
Summary
From the ‡Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1; §Department of Chemistry, University of Waterloo, Waterloo, ON, N2L 3G1, Canada. Phosphorylated ITIMs recruit the SH2 domaincontaining tyrosine phosphatase SHP-1 or SHP-2 [6], and phosphorylated ITAMs are recognized by protein tyrosine kinases such as ZAP70 in T cells and SYK in B cells [7] These distinctions are only relative and both motifs may be involved in either positive or negative immune functions. Through their respective ITIM sequences, the LAIR-1 receptor recruits tyrosine kinase CSK [12], Lax interacts with GRB2, PIK3R1 and GRAP2 [13], and CD72 forms a complex with SHP-1 and GRB2 [14] These findings indicate that immunoreceptor signaling is context-dependent and that the precise pairing of an ITRM sequence with an SH2-containing protein plays a critical role in dictating the signaling and biological outcome. The interactome has provided a systematic view of immunoreceptor signaling network, and generated unique insights into the signaling specificity for the different immune signaling motifs
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