A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein–Peptide Array Screening

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. 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. Immune receptor signaling, critical for proper immune response, involves signaling pathways mediated by specific tyrosine residues that are phosphorylated upon receptor activation (1.Guy C.S. Vignali K.M. Temirov J. Bettini M.L. Overacre A.E. Smeltzer M. Zhang H. Huppa J.B. Tsai Y.H. Lobry C. Xie J. Dempsey P.J. Crawford H.C. Aifantis I. Davis M.M. Vignali D.A. Distinct TCR signaling pathways drive proliferation and cytokine production in T cells.Nat. Immunol. 2013; 14: 262-270Crossref PubMed Scopus (150) Google Scholar). These tyrosine phosphorylation sites are frequently found in one of the three types of immunoreceptor tyrosine-based regulatory motifs (ITRMs) 1The The abbreviations used are:ITRMimmunoreceptor tyrosine-based regulatory motifsITAMimmunoreceptor tyrosine-based activation motifsITIMimmunoreceptor tyrosine-based inhibition motifsITSMimmunoreceptor tyrosine-based switch motifs.. The immunoreceptor tyrosine-based activation motifs (ITAMs) with the consensus sequence YxxI/Lx(6–12)YxxI/L, where x represents any amino acid, are typically associated with positive or activating immune response (2.van Leeuwen J.E. Samelson L.E. T cell antigen-receptor signal transduction.Current Opinion Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar). In contrast, the immune system elicits negative or inhibitory response through receptors bearing the immunoreceptor tyrosine-based inhibition motifs (ITIMs) with the degenerated sequence S/I/V/LxYxxI/V/L (3.Ravetch J.V. Lanier L.L. Immune inhibitory receptors.Science. 2000; 290: 84-89Crossref PubMed Scopus (1064) Google Scholar). Interestingly, SLAM/CD150 and related receptors of the CD2 subfamily contain a third type of signaling motif called Immunoreceptor Tyrosine-based Switch Motif (ITSM) with the consensus sequence TxYxx(V/I) (4.Dong Z. Veillette A. How do SAP family deficiencies compromise immunity?.Trends Immunol. 2010; 31: 295-302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). An ITSM can convey either an activating or inhibitory signal, depending on the type of immune cell, the receptor and the bound protein (5.Ostrakhovitch E.A. Li S.S. The role of SLAM family receptors in immune cell signaling.Biochem. Cell Biol. 2006; 84: 832-843Crossref PubMed Google Scholar). immunoreceptor tyrosine-based regulatory motifs immunoreceptor tyrosine-based activation motifs immunoreceptor tyrosine-based inhibition motifs immunoreceptor tyrosine-based switch motifs. In general, phosphorylated ITIMs recruit the SH2 domain-containing tyrosine phosphatase SHP-1 or SHP-2 (6.Steevels T.A. Meyaard L. Immune inhibitory receptors: essential regulators of phagocyte function.Eur. J. Immunol. 2011; 41: 575-587Crossref PubMed Scopus (55) Google Scholar), and phosphorylated ITAMs are recognized by protein tyrosine kinases such as ZAP70 in T cells and SYK in B cells (7.Bezbradica J.S. Medzhitov R. Role of ITAM signaling module in signal integration.Current Opinion Immunol. 2012; 24: 58-66Crossref PubMed Scopus (37) Google Scholar). Nevertheless, these distinctions are only relative and both motifs may be involved in either positive or negative immune functions. For example, ITIM-containing inhibitory receptors found on phagocytes can suppress or enhance inflammatory cytokine production depending on the downstream proteins that they recruit (6.Steevels T.A. Meyaard L. Immune inhibitory receptors: essential regulators of phagocyte function.Eur. J. Immunol. 2011; 41: 575-587Crossref PubMed Scopus (55) Google Scholar). Similarly, ITAM sequences have been found to mediate inhibitory signaling (8.Hamerman J.A. Lanier L.L. Inhibition of immune responses by ITAM-bearing receptors.Science's STKE. 2006; 2006: re1PubMed Google Scholar). Indeed, even within the same cell type, an ITAM-containing receptor may mediate functions as diverse as microbial killing, antigen presentation, cytokine production, T-Cell instruction, and tissue repair (2.van Leeuwen J.E. Samelson L.E. T cell antigen-receptor signal transduction.Current Opinion Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar). Moreover, exquisite binding specificities have been observed for different ITRMs. For example, the CD31 ITIMs recruit SHIP-1, SHP-1, and SHP-2 (9.Pumphrey N.J. Taylor V. Freeman S. Douglas M.R. Bradfield P.F. Young S.P. Lord J.M. Wakelam M.J. Bird I.N. Salmon M. Buckley C.D. Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31.FEBS Lett. 1999; 450: 77-83Crossref PubMed Scopus (92) Google Scholar). In contrast, the archetypal ITIM identified in the cytoplasmic domain of the inhibitory IgG Fc receptor FcγRIIB (10.Bonnerot C. Briken V. Amigorena S. Intracellular signaling and endosomal trafficking of immunoreceptors. Shared effectors underlying MHC class II-restricted antigen presentation.Immunology Lett. 1997; 57: 1-4Crossref PubMed Scopus (12) Google Scholar) recruits SHIP-1 and -2, not SHP-1 or the FcγRIIB ITIM an for the SH2 domain of either SHP-1 or -2, it not recruit these tyrosine as do other receptors M. S. L. Immunoreceptor tyrosine-based inhibition a in the and PubMed Scopus Google Scholar). Besides and other proteins have also been found associated with For example, through their ITIM the receptor recruits tyrosine A. Meyaard L. SH2 domain-containing and recruits Src J. Immunol. 2006; PubMed Scopus Google Scholar), with and M. Zhang of a novel a protein in J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and a with SHP-1 and S. R. is associated with the in the cell J. Immunol. 2000; PubMed Scopus Google Scholar). These that immunoreceptor signaling is and that the of an ITRM sequence with an protein a critical role in the signaling and the critical of SH2 interaction in immune signaling, we to identify SH2 interactions. A of different have been developed for the identification of protein–protein or protein-peptide interactions to These Li S. C. Z. J. S. S. A. H. J. I. M. a of human human PubMed Scopus Google Scholar), to J. of interactions, and in Biol. 2012; PubMed Scopus (12) Google Scholar), H. S.S. an system for or 2012; PubMed Scopus Google Scholar), and protein L. in PubMed Scopus Google Scholar) and H. Li L. C. S. Li C. Z. Li S.S. the of the human 2 Full Text Full Text PDF PubMed Scopus Google Scholar). We protein and in our study of their and to we SH2 domain array with array to an interactome for immunoreceptors. 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The of the B and in to Full Text Full Text PDF PubMed Scopus Google Scholar), and to by a in the A. the activation and of Src 2000; PubMed Scopus Google Scholar). The of different binding by the ITIM including kinases and kinases and and and proteins and that the of the ITIM inhibitory is that CD22 of binding to both and negative the interaction in we to the Indeed, to to the to the is in with their by for and for Moreover, with CD22 cells upon B cell receptor activation these that is the binding in CD22 for is to that the ITIM-containing receptor through both the family and P.J. Immunoreceptor tyrosine-based inhibitory motif inhibitory signaling is by phosphorylation mediated by distinct tyrosine a study 2013; PubMed Scopus Google Scholar). is that a signal transduction by We applied the bidirectional array approach to identify a comprehensive interaction network mediated by ITRMs. this we the R. M.J. C. M. R. M. L. R. A. R. J. R. J. M. J. S. S. M. S. H. M. M. I. L. A. A. J. L. L. A. 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Role of ITAM signaling module in signal integration.Current Opinion Immunol. 2012; 24: 58-66Crossref PubMed Scopus (37) Google Scholar), the relative for In contrast, the SHP-1 and SH2 domains the for Interestingly, ITAM and ITSM sequences binding the SH2 domains. These are in with the that SHP-1 and SHP-2 are involved in inhibitory immune response (6.Steevels T.A. Meyaard L. Immune inhibitory receptors: essential regulators of phagocyte function.Eur. J. Immunol. 2011; 41: 575-587Crossref PubMed Scopus (55) Google Scholar). the and SH2 domains showed the same for ITIM as the SH2 that these proteins are associated with inhibitory for ITSM observed for the and and to a for the Fyn and SH2 domains. the three have been to critical in signaling by the SLAM and related receptors through ITSM (4.Dong Z. Veillette A. How do SAP family deficiencies compromise immunity?.Trends Immunol. 2010; 31: 295-302Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), the of in immune signaling further be that the of an SH2 domain for a motif type is not SH2 domains are of binding to one type of For example, the ZAP70 and SYK SH2 domains have a for they can also to ITIM and ITSM an with our on CD22 SH2 including and are of binding to three types of a In to interactions the reciprocal array we the bidirectional for and the array the of in binding in as high-confidence binding We used the of interactions within as to potential interactions that within and the same approach as described for the interactions by with the the bidirectional array screens identified 1137 interactions between SH2 domains and ITRM interactome interactions that are in the for protein–protein interaction A. S. M. M. A. J. 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L. protein as a of cellular signalling and Scholar). regulatory motifs and the associated SH2 domains a critical role in immune the three types of ITIM is to inhibitory ITAM to immune this is For ITAMs recruit SHP-1, a phosphatase found associated with ITIMs in inhibitory immune response S. M. J. M. ITAM signaling activating receptors with the phosphatase SHP-1 to 2011; PubMed Scopus Google Scholar). ITIMs have been to activation J. ITAM and the the of immunoreceptor J. Immunol. 2006; PubMed Scopus Google Scholar). to the that a ITRM can immunoreceptor signal transduction depending on the specific SH2 domain proteins that it the by a of SH2 SH2 domains to ITRM sequences with a of SH2 domains and interactome that it is that ITIMs may mediate positive and ITAM negative immune signaling For in to binding SHP-1 L. CD22 and inhibitory receptors with distinct PubMed Scopus Google Scholar), the ITIM found to SH2 domains of the tyrosine kinases and In contrast, ITAMs found to associate with tyrosine including and of binding to tyrosine SHP-1 or SHP-2 cells to an immune is on the activation of specific may contain Motif in cell Biol. 2012; PubMed Scopus Google Scholar), the interactome identified herein a for understanding signal in immune that the a specific ITRM convey an activating or inhibitory signal is not only on the residues the it is by the downstream SH2 domain-containing effectors by the the of in the interaction network a of immune signaling may be by the activation of specific receptors in different the of specific protein by proteins through and 1997; PubMed Scopus Google Scholar), or by such as found in or V. M. in immunoreceptor Lett. PubMed Scopus Google Scholar). In we have that of and protein the of the identified network. Using the bidirectional array we identified the immunoreceptor signaling network mediated by to novel interactions have been a for the cell signaling Moreover, our interactome the of to the of immune and the of for We that the bidirectional array to an in the identification of the human protein–protein interactions mediated by peptide-binding domains. We for with

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