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

Abstract

Immune receptor signaling, critical for proper immune response, involves signaling pathways mediated by specific tyrosine residues that are phosphorylated upon receptor activation (1). These tyrosine phosphorylation sites are frequently found in one of the three types of immunoreceptor tyrosine-based regulatory motifs (ITRMs)1 . 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). 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). 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). An ITSM can convey either an activating or inhibitory signal, depending on the type of immune cell, the receptor and the bound protein (5).In general, 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). 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). Similarly, ITAM sequences have been found to mediate inhibitory signaling (8). Indeed, even within the same cell type, an ITAM-containing receptor may mediate From the ‡Department of Biochemistry and the Siebens-Drake

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“…Peptide synthesis, array constructions and assay conditions were essentially as described elsewhere [34]. …”

“…Screening of SH2 domain specificity of the various phosphopeptides was performed by Dr. Shawn Li's Laboratory, University of Ontario, as a service. Peptide synthesis, array constructions and assay conditions were essentially as described elsewhere [ 34 ].…”

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“…Proteomic array-based analysis can also be used for the identification of a large set of proteins or multiprotein complexes (Liu et al 2015). In this array based on analysis, purified ligands are separated individually.…”

“…Conflict of interest The authors declare no conflict of interest. Liu et al (2015) Sugarcane Highly demanded for ethanol production…”

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“…Downstream the receptor, immune signaling involves specific tyrosine residues that are phosphorylated upon receptor activation. These phosphorylation sites are frequently found in one of the three types of Immunoreceptor tyrosine-based regulatory motifs (ITRM), including IT activation M (ITAM), IT inhibition M (ITIM), and IT switch M (ITSM) for SLAM/CD150 and related receptors of the CD2 subfamily [ 26 ]. Generally, ITIMs recruit the SH2 domain-containing tyrosine phosphatase SHP-1 or SHP-2, and phosphorylated ITAMs are recognized by SYK in B-cells [ 27 , 28 ].…”