The SH2-Containing 5 -Inositol Phosphatase (SHIP) Is Tyrosine Phosphorylated after Fc Receptor Clustering in Monocytes 1 Diane L. Maresco, 2 * Jeanne M. Osborne, 2 * Damon Cooney, ² K. Mark Coggeshall, ² and Clark L. Anderson 3 * Current models of FcR signal transduction in monocytes describe a molecular cascade that begins upon clustering of FcR with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of FcRIIa or the -chain subunit of FcRI and FcRIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ulti- mately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after FcR clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either FcRIIa or FcRI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of FcRIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that FcRIIb and FcRIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits FcR-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif- containing cytoplasmic domains of FcRIIa and FcRI-associated -chain. The Journal of Immunology, 1999, 162: 6458 – 6465. I mmunoreceptors for Ag and immune complexes that mediate positive biological responses are composed of three parts; namely, a receptor proper that binds ligand, cytoplasmic ty- rosines in a specific sequence called an immunoreceptor tyrosine- based activation motif (ITAM), 4 and protein tyrosine kinases (PTK) of the Src family (1). Most of the Fc receptors for IgG (FcR), specifically those that produce a positive biological re- sponse, conform to this schema (reviewed in Ref. 2). Thus, clus- tering of these FcR by immune complexes or by anti-FcR Abs stimulates Src family PTK activity and results in the phosphory- lation of ITAM tyrosines situated either in the cytoplasmic tail of FcRIIa or in the -chain subunit of FcRI and FcRIIIa. These phosphotyrosines along with the three amino acid residues imme- diately downstream comprise docking sites for specific Src homol- ogy 2 (SH2) domain-containing enzymes and adapter proteins, which, in turn, propagate positive signals (3, 4). Critical to all downstream events is the PTK Syk, which binds to phosphorylated ITAM (phospho-ITAM) residues and becomes activated. A variety of subsequent major signaling pathways, either downstream of Syk or dependent upon Syk, are then activated or assembled. These subsequent pathways include one dependent upon phospholipase C that generates the second messengers inositol trisphosphate and diacylglycerol, one dependent upon Shc that cascades via Grb2 and SOS to involve additional downstream elements of the Ras activation pathway, and one dependent upon phosphatidylinositol 3-kinase (PI3K) that leads to phagocytosis and superoxide gener- ation (reviewed in Ref. 2). Recent progress suggests that the PI3K pathway to phagocytosis requires recruitment of the p110 catalytic subunit of PI3K to the plasma membrane and activation of mem- bers of the Rho family of small GTPases (5–7). While ITAM-associated receptors lead to a positive biological response, a different group of receptors has recently been described that, when clustered by Abs or ligand, act to inhibit cellular re- sponses (8, 9). Termed negative signaling receptors, these function somewhat like positive signaling receptors by undergoing phos- phorylation on cytoplasmic tyrosine residues and subsequently re- cruiting SH2 domain-containing enzymes, but the enzymes are of a specific sort that includes the phosphotyrosine phosphatases SHP-1 and -2 and the inositol 5-phosphatase, SHIP (9 –11). The cytoplasmic tyrosine of negative signaling receptors is situated in an immunoreceptor tyrosine-based inhibitory motif (ITIM) (8, 9) that is structurally similar to the activating ITAM. SH2 domain- containing phosphatases recruited to phospho-ITIM of these re- ceptors act to reverse biochemical events elicited by positive sig- naling receptors and so reduce or inhibit the biological responses. Included in this group of negative signaling receptors are killer cell inhibitory receptors, pg49B1, PIR-B, CTLA4, and others, includ- ing a single FcR (FcRIIb) (8, 9). The difference in the primary sequence between the activating ITAM and the inhibiting ITIM appears minor. The ITAM contains the sequence YxxI/L-x (6 –12)-YxxI/L, where x is any amino acid. The residues in the +1 and/or +2 position relative to the tyrosine of the ITAM are generally acidic and therefore form an optimal recognition sequence for phosphorylation by the Src family PTKs Departments of *Internal Medicine and ² Microbiology, Ohio State University, Co- lumbus, OH 43210 Received for publication July 22, 1998. Accepted for publication March 17, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by awards from the U.S. Public Health Service (RO1-CA44983 and P30-CA16058). K.M.C. is a Scholar of the Leukemia Society of America. 2 D.L.M. and J.M.O. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Clark L. Anderson, 2054 Davis Research Center, 480 West Ninth Ave., Columbus, OH 43210. E-mail address: anderson.48@osu.edu 4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-mediated signal activation motif; PTK, protein tyrosine kinase; FcR, Fc receptors for IgG; SH2, Src homology 2; phospho-ITAM, phosphorylated ITAM; PI3K, phosphatidylinositol 3-kinase; SHIP, SH2-containing 5'-inositol phosphatase; ITIM, immunoreceptor tyrosine-based inhibitory motif; phospho-ITIM, phosphorylated ITIM; GAM, goat F(ab') 2 anti-murine IgG; TLB, Triton lysis buffer; ECL, enhanced chemiluminescence. 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