A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

Tyrosine Kinase SYK Licenses MyD88 Adaptor Protein to Instigate IL-1α-Mediated Inflammatory Disease.

Mice carrying a hypomorphic point mutation in the Ptpn6 gene (Ptpn6spin mice) develop an inflammatory skin disease that resembles neutrophilic dermatosis in humans. Here, we demonstrated that interleukin-1α (IL-1α) signaling through IL-1R and MyD88 in both stromal and immune cells drive inflammation in Ptpn6spin mice. We further identified SYK as a critical kinase that phosphorylates MyD88, promoted MyD88-dependent signaling and mediates dermatosis in Ptpn6spin mice. Our studies further demonstrated that SHP1 encoded by Ptpn6 binds and suppresses SYK activation to inhibit MyD88 phosphorylation. Downstream of SHP1 and SYK-dependent counterregulation of MyD88 tyrosine phosphorylation, we have demonstrated that the scaffolding function of receptor interacting protein kinase 1 (RIPK1) and tumor growth factor-ß activated kinase 1 (TAK1)-mediating signaling were required to spur inflammatory disease. Overall, these studies identify SHP1 and SYK crosstalk as a critical regulator of MyD88 post-translational modifications and IL-1-driven inflammation.

Leishmania Uses Mincle to Target an Inhibitory ITAM Signaling Pathway in Dendritic Cells that Dampens Adaptive Immunity to Infection.

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c+ cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.

Protein tyrosine phosphatases in lymphocyte activation and autoimmunity.

Lymphocyte activation must be tightly regulated to ensure sufficient immunity to pathogens and prevent autoimmunity. Protein tyrosine phosphatases (PTPs) serve critical roles in this regulation by controlling the functions of key receptors and intracellular signaling molecules in lymphocytes. In some cases, PTPs inhibit lymphocyte activation, whereas in others they promote it. Here we discuss recent progress in elucidating the roles and mechanisms of action of PTPs in lymphocyte activation. We also review the accumulating evidence that genetic alterations in PTPs are involved in human autoimmunity.

Inhibition of TLR signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs.

The protein Tir (translocated intimin receptor) in enteric bacteria shares sequence similarity with the host cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs). Despite the importance of Tir in pedestal formation, relatively little is known about the role of Tir and its ITIMs in the regulation of the host immune response. Here we demonstrate that Tir from enteropathogenic Escherichia coli (EPEC) interacted with the host cellular tyrosine phosphatase SHP-1 in an ITIM phosphorylation-dependent manner. The association of Tir with SHP-1 facilitated the recruitment of SHP-1 to the adaptor TRAF6 and inhibited the ubiquitination of TRAF6. Moreover, the ITIMs of Tir suppressed EPEC-stimulated expression of proinflammatory cytokines and inhibited intestinal immunity to infection with Citrobacter rodentium. Our findings identify a previously unknown mechanism by which bacterial ITIM-containing proteins can inhibit innate immune responses.

The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45-/- as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1-mediated dephosphorylation of CD22 in CD22 clusters.

'Unlicensed' natural killer cells dominate the response to cytomegalovirus infection.

Natural killer (NK) cells expressing inhibitory receptors that bind to self major histocompatibility complex (MHC) class I are 'licensed', or rendered functionally more responsive to stimulation, whereas 'unlicensed' NK cells lacking receptors for self MHC class I are hyporesponsive. Here we show that contrary to the licensing hypothesis, unlicensed NK cells were the main mediators of NK cell-mediated control of mouse cytomegalovirus infection in vivo. Depletion of unlicensed NK cells impaired control of viral titers, but depletion of licensed NK cells did not. The transfer of unlicensed NK cells was more protective than was the transfer of licensed NK cells. Signaling by the tyrosine phosphatase SHP-1 limited the proliferation of licensed NK cells but not that of unlicensed NK cells during infection. Thus, unlicensed NK cells are critical for protection against viral infection.

Distinct roles for neutrophils and dendritic cells in inflammation and autoimmunity in motheaten mice.

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.

The immune checkpoint receptor associated phosphatases SHP-1 and SHP-2 are not required for γδT17 cell development, activation, or skin inflammation.

IL-17-producing gamma delta (γδT17) cells are innate lymphocytes critical for antibacterial protection at barrier surfaces such as the skin but also highly pathogenic during inflammation. It is therefore important to understand the cellular and molecular mechanisms that could counter-balance overt γδT17 cell activation. Immune checkpoint receptors (ICRs) deliver inhibitory signals to activated lymphocytes and have been implicated as negative regulators of mouse γδT17 cells. In this report, we investigated the cytokine signals that induce ICR expression on γδT17 cells and studied the in vivo role of the Src-homology-2 phosphatases 1 and 2 (SHP-1 and SHP-2) in the context of γδT17-induced psoriasis. We found that surface expression of ICRs can be induced by cytokines; however, SHP-1 or SHP-2 could not inhibit γδT17 responses. In this regard, conditional deletion of SHP-1, SHP-2, or both did no impact γδT17 cell development, expansion, cytokine production, or skin pathology.

The Protein Phosphatase Shp1 Regulates Invariant NKT Cell Effector Differentiation Independently of TCR and Slam Signaling.

Invariant NKT (iNKT) cells are innate lipid-reactive T cells that develop and differentiate in the thymus into iNKT1/2/17 subsets, akin to TH1/2/17 conventional CD4 T cell subsets. The factors driving the central priming of iNKT cells remain obscure, although strong/prolonged TCR signals appear to favor iNKT2 cell development. The Src homology 2 domain-containing phosphatase 1 (Shp1) is a protein tyrosine phosphatase that has been identified as a negative regulator of TCR signaling. In this study, we found that mice with a T cell-specific deletion of Shp1 had normal iNKT cell numbers and peripheral distribution. However, iNKT cell differentiation was biased toward the iNKT2/17 subsets in the thymus but not in peripheral tissues. Shp1-deficient iNKT cells were also functionally biased toward the production of TH2 cytokines, such as IL-4 and IL-13. Surprisingly, we found no evidence that Shp1 regulates the TCR and Slamf6 signaling cascades, which have been suggested to promote iNKT2 differentiation. Rather, Shp1 dampened iNKT cell proliferation in response to IL-2, IL-7, and IL-15 but not following TCR engagement. Our findings suggest that Shp1 controls iNKT cell effector differentiation independently of positive selection through the modulation of cytokine responsiveness.

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

B Cell-Intrinsic SHP1 Expression Promotes the Gammaherpesvirus-Driven Germinal Center Response and the Establishment of Chronic Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the majority of adults worldwide. Chronic gammaherpesvirus infection has been implicated in both lymphomagenesis and, somewhat controversially, autoimmune disease development. Pathogenesis is largely associated with the unique ability of gammaherpesviruses to usurp B cell differentiation, specifically, the germinal center response, to establish long-term latency in memory B cells. The host tyrosine phosphatase SHP1 is known as a brake on immune cell activation and is downregulated in several gammaherpesvirus-driven malignancies. However, here we demonstrate that B cell- but not T cell-intrinsic SHP1 expression supports the gammaherpesvirus-driven germinal center response and the establishment of viral latency. Furthermore, B cell-intrinsic SHP1 deficiency cooperated with gammaherpesvirus infection to increase the levels of double-stranded DNA-reactive antibodies at the peak of viral latency. Thus, in spite of decreased SHP1 levels in gammaherpesvirus-driven B cell lymphomas, B cell-intrinsic SHP1 expression plays a proviral role during the establishment of chronic infection, suggesting that the gammaherpesvirus-SHP1 interaction is more nuanced and is modified by the stage of infection and pathogenesis.IMPORTANCE Gammaherpesviruses establish lifelong infection in a majority of adults worldwide and are associated with a number of malignancies, including B cell lymphomas. These viruses infect naive B cells and manipulate B cell differentiation to achieve a lifelong infection of memory B cells. The germinal center stage of B cell differentiation is important as both an amplifier of the viral latent reservoir and the target of malignant transformation. In this study, we demonstrate that expression of tyrosine phosphatase SHP1, a negative regulator that normally limits the activation and proliferation of hematopoietic cells, enhances the gammaherpesvirus-driven germinal center response and the establishment of chronic infection. The results of this study uncover an intriguing beneficial interaction between gammaherpesviruses that are presumed to profit from B cell activation and a cellular phosphatase that is traditionally perceived to be a negative regulator of the same processes.

Phosphatase SHP-1 promotes TLR- and RIG-I-activated production of type I interferon by inhibiting the kinase IRAK1.

Unbalanced production of proinflammatory cytokines and type I interferons in immune responses may lead to immunopathology; thus, the mechanisms that ensure the beneficial production of proinflammatory cytokines and type I interferons are of particular importance. Here we demonstrate that the phosphatase SHP-1 negatively regulated Toll-like receptor-mediated production of proinflammatory cytokines by inhibiting activation of the transcription factor NF-kappaB and mitogen-activated protein kinase. Simultaneously, SHP-1 increased the production of type I interferon mediated by Toll-like receptors and the helicase RIG-I by directly binding to and inhibiting activation of the kinase IRAK1. Our data demonstrate that SHP-1 contributes to immune homeostasis by balancing the production of proinflammatory cytokines and type I interferons in the innate immune response.

Phospholipase C-ß3 regulates FcɛRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1.

Mast cells are major effectors in high-affinity IgE receptor (FcɛRI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-ß3 is crucial for FcɛRI-mediated mast cell activation. Plcb3(-/-) mice showed blunted FcɛRI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, FcɛRI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-ß3 constitutively interacts with FcɛRI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-ß3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, FcɛRI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-ß3- and SHP-1-mediated signaling pathway for FcɛRI-mediated cytokine production.

SHP1 tyrosine phosphatase gets involved in host defense against Streptococcus agalactiae infection and BCR signaling pathway in Nile tilapia (Oreochromis niloticus).

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a kind of protein tyrosine phosphatases (PTPs), is a critical regulator of antigen receptor signal transduction. Signal transduction of BCR is regulated by phosphatases in teleost as in mammals. In this study, SHP1 from Nile tilapia (Oreochromis niloticus) (OnSHP1) was identified and characterized, including the expression pattern against bacterial infection and regulation function in BCR signaling pathway. The open reading frame of OnSHP1 contains 1749 bp of nucleotide sequence, encoding a protein of 582 amino acids. The OnSHP1 protein was highly conversed compared to that of other species, including two amino-terminal SH2 domains at the N terminus and a PTP catalytic domain. Transcriptional expression analysis revealed that OnSHP1 was detected in all examined tissues and highly expressed in spleen. The up-regulated OnSHP1 expression was observed in peripheral blood, spleen and anterior kidney following challenge with Streptococcus agalactiae or lipopolysaccharide (LPS) in vivo, as well as that displayed in leukocytes stimulated with S. agalactiae or LPS in vitro. Further, after induction with mouse anti-tilapia IgM monoclonal antibody in vitro, OnSHP1 was significantly up-regulated in leukocytes. When spleen leukocytes treated with PTP Inhibitor II in vitro, the phosphorylation level of OnSHP1 at the phosphorylation sites (Y535 and Y557) and the cytoplasmic free Ca2+ concentration were up-regulated significantly. Overall, the findings of this study indicate that SHP1 gets involved in host defense against bacterial infection and BCR signaling pathway in Nile tilapia.

Cutting Edge: Dysregulated CARD9 Signaling in Neutrophils Drives Inflammation in a Mouse Model of Neutrophilic Dermatoses.

Mice homozygous for the Y208N amino acid substitution in the carboxy terminus of SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe inflammatory disease resembling neutrophilic dermatosis in humans. Disease in Ptpn6spin mice is characterized by persistent footpad swelling and suppurative inflammation. Recently, in addition to IL-1α and IL-1R signaling, we demonstrated a pivotal role for RIPK1, TAK1, and ASK1 in promoting inflammatory disease in Ptpn6spin mice. In the current study we have identified a previously unknown role for CARD9 signaling as a critical regulator for Ptpn6spin-mediated footpad inflammation. Genetic deletion of CARD9 significantly rescued the Ptpn6spin-mediated footpad inflammation. Mechanistically, enhanced IL-1α-mediated signaling in Ptpn6spin mice neutrophils was dampened in Ptpn6spinCard9-/- mice. Collectively, this study identifies SHP-1 and CARD9 cross-talk as a novel regulator of IL-1α-driven inflammation and opens future avenues for finding novel drug targets to treat neutrophilic dermatosis in humans.

cIAP1/2-TRAF2-SHP-1-Src-MyD88 Complex Regulates Lipopolysaccharide-Induced IL-27 Production through NF-κB Activation in Human Macrophages.

The inhibitors of apoptosis (IAP) proteins, initially described in the context of apoptosis regulation as promoting cell survival, have recently emerged as key regulators of innate immune signaling. As a result, downregulation of IAP via Smac mimetics (SMM) has both survival and immunoregulatory effects. IAPs modulate cytokine production in murine models either as a single agent or in response to LPS. However, the role of SMM and the involvement of IAPs in primary human cells and in particular macrophages with respect to cytokine production and innate immune responses remain largely unknown. IL-27, a member of the IL-12 cytokine family produced by APCs such as macrophages, has broad immunoregulatory properties in both innate and adaptive immune responses. Herein, we show that cellular IAPs (cIAPs) positively regulate LPS-induced IL-27 production in both primary human monocytes and macrophages. Investigations for the signaling mechanism of cIAPs involvement in IL-27 production in human macrophages revealed that LPS-induced IL-27 production is regulated by a novel signaling complex comprising cIAP1/2, TNFR-associated factor 2 (TRAF2), SHP-1, Src, and MyD88 leading to p38, c-Jun N-terminal kinases (JNK) and Akt activation and NF-κB signaling. In cancer cells, SMM induce the production of cytokines by activating the noncanonical alternate NF-κB pathway. However, in human macrophages, SMM do not induce the production of TNF-α and other cytokines while inhibiting LPS-induced IL-27 production by inhibiting the classical NF-κB pathway. These signaling pathways may constitute novel therapeutic avenues for immune modulation of IL-27 and provide insight into the modulatory immune effects of SMM.

The Ly49Q receptor plays a crucial role in neutrophil polarization and migration by regulating raft trafficking.

Neutrophils rapidly undergo polarization and directional movement to infiltrate the sites of infection and inflammation. Here, we show that an inhibitory MHC I receptor, Ly49Q, was crucial for the swift polarization of and tissue infiltration by neutrophils. During the steady state, Ly49Q inhibited neutrophil adhesion by preventing focal-complex formation, likely by inhibiting Src and PI3 kinases. However, in the presence of inflammatory stimuli, Ly49Q mediated rapid neutrophil polarization and tissue infiltration in an ITIM-domain-dependent manner. These opposite functions appeared to be mediated by distinct use of effector phosphatase SHP-1 and SHP-2. Ly49Q-dependent polarization and migration were affected by Ly49Q regulation of membrane raft functions. We propose that Ly49Q is pivotal in switching neutrophils to their polarized morphology and rapid migration upon inflammation, through its spatiotemporal regulation of membrane rafts and raft-associated signaling molecules.

Inhibitory receptor signaling via tyrosine phosphorylation of the adaptor Crk.

Many cellular responses, such as autoimmunity and cytotoxicity, are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here, we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk, concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore, Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity, providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex, observed here with two types of inhibitory receptors, expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism.