Molecular Basis of the Recruitment of the SH2 Domain-containing Inositol 5-Phosphatases SHIP1 and SHIP2 by FcγRIIB

Abstract

Immunoreceptor tyrosine-based inhibition motifs (ITIMs) 1 are present in the intracytoplasmic (IC) domains of a large group of transmembrane molecules that negatively regulate cell activation induced by receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs) (1). Fc␥RIIB, a subgroup of Fc receptors that bind IgG complexes (2), and killer cell Ig-like receptors with a long IC domain (KIRLs), which bind major histocompatibility complex class I molecules (3), are two prototypes of ITIM-bearing receptors. Fc␥RIIB exist as two (Fc␥RIIB1 and B2 in humans) or three (Fc␥RIIB1, B1Ј, and B2 in mice) alternatively spliced products of a single gene. Fc␥RIIB were shown to negatively regulate cell activation induced by all ITAM-bearing immunoreceptors (4) and to control the magnitude of both antibody responses and anaphylactic reactions (5). Fc␥RIIB were also recently found to negatively regulate cell proliferation induced by growth factor receptors with an intrinsic protein-tyrosine kinase activity (6). KIRLs are polymorphic molecules with two (KIR2DLs) or three (KIR3DLs) Ig-like extracellular (EC) domains encoded by related but distinct genes. KIRLs inhibit NK and T cell-mediated cytotoxicity (7,8). Negative regulation exerted by Fc␥RIIB (9) and KIR2DL3 (10) was shown to require their coaggregation with ITAM-bearing receptors by extracellular ligands. Fc␥RIIB possess one ITIM, while KIR2DL3 possess two ITIMs.ITIMs are constituted by a tyrosine, preceded by an isoleucine, valine, or leucine at position YϪ2, and followed by a valine or leucine at position Yϩ3 (1, 11). Upon coaggregation of ITIMbearing receptors with ITAM-bearing receptors, ITIMs are tyrosyl-phosphorylated by Src family protein-tyrosine kinases (12, 13). Phosphorylated ITIMs (pITIMs) then recruit cytoplasmic phosphatases containing Src homology 2 (SH2) domains that interfere with signals transduced by ITAM-bearing receptors (14 -16). These include the two-SH2 domain-containing protein-tyrosine phosphatases SHP-1 (14, 17) and SHP-2 (18) and the single-SH2 domain-containing inositol 5-phosphatases SHIP1 (19) and SHIP2 (20). SHP-1 is thought to dephosphorylate tyrosines in ITAMs, protein-tyrosine kinases, and/or adapter proteins whose phosphorylation is critical for activation signals, thereby stopping the initial steps of transduction. SHP-1 was recently found to inhibit the redistribution of cholesterol/sphyngolipid-rich membrane lipid microdomains following the engagement of a KIR3DL, on NK cells, by major histocompatibility complex class I molecules on target cells (21). The possible role of SHP-2 is not clear, because both positive and negative effects have been assigned to this phosphatase (22-24). SHIP1 and SHIP2 remove 5-phosphate groups in inositol phosphates and phosphatidylinositol phosphates (25). The preferred substrate of SHIP1 is phosphatidylinositol 3,4,5-trisphosphate, which enables the membrane recruitment of the Bruton's tyrosine kinase via its pleckstrin homology domain (15, 16). Bruton's tyrosine kinase is manda-

Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering

“…In a cytotoxicity assay with 721.221͞HLA-Cw4-GFP cells as targets, effector cells YTS͞KIR2DL1(Y3775), YTS͞ KIR2DL1(Y3775,Y4075), or YTS͞KIR2DL1(K346*), termed signal incompetent mutants, were unable to inhibit NK cytotoxicity as reported (20). YTS͞KIR2DL1(Y4075) remained inhibitory signal competent (Fig.…”

“…The membrane-distal ITIM (amino acids 407-410) canAbbreviations: NK, natural killer; KIR, killer immunoglobulin-like receptor; ITIM, immunoreceptor tyrosine-based inhibition motif; GFP, green fluorescent protein; CTX ␤, cholera toxin ␤ subunit; TCR, T cell receptor. not recruit either phosphatase alone but acts in concert with the membrane-proximal ITIM to recruit SHP-2, which then amplifies the signal from SHP-1 (20). SHP-1 and SHP-2 binding leads to the propagation of a downstream signaling cascade that inhibits NK cytotoxicity, sparing the HLA-Cw4-expressing target cell (17).…”

Insufficient Phosphorylation Prevents FcγRIIB from Recruiting the SH2 Domain-containing Protein-tyrosine Phosphatase SHP-1

“…cDNA encoding Fc␥RIIB1, inserted in a NT-neo vector (39), was stably transfected in K46 cells by electroporation. Transfectants were selected with neomycin (Cayla, Toulouse, France) and by cell sorting using a FACSCalibur (Becton Dickinson, Mountain View, CA).…”

“…was recently found to bind also to phosphorylated Fc␥RIIB ITIM (27,29). It follows that phosphorylated Fc␥RIIB ITIM peptides can bind all four known SH2 domain-containing phosphatases in vitro.…”

“…Remarkably, the Fc␥RIIB ITIM also bound SHIP1 (26) and SHIP2 (27). We previously identified two hydrophobic residues, at positions Y-2 and Yϩ2, that determine the binding of SHPs (28) and SHIPs (29), respectively. The in vivo recruitment of phosphatases by ITIM-bearing receptors was analyzed by coprecipitation, following their tyrosyl phosphorylation upon coaggregation with ITAM-bearing receptors.…”