Role that each NKG2A immunoreceptor tyrosine-based inhibitory motif plays in mediating the human CD94/NKG2A inhibitory signal

The human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6-11) and membrane-proximal (residues 38-43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcepsilonRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.     

Systematic identification of immunoreceptor tyrosine-based inhibitory motifs in the human proteome

Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are short sequences of the consensus (ILV)-x-x-Y-x-(LV) in the cytoplasmic tail of immune receptors. The phosphorylation of tyrosines in ITIMs is known to be an important signalling mechanism regulating the activation of immune cells. The shortness of the motif makes it difficult to predict ITIMs in large protein databases. Simple pattern searches find ITIMs in approximately 30% of the protein sequences in the RefSeq database. The majority are false positive predictions. We propose a new database search strategy for ITIM-bearing transmembrane receptors based on the use of sequence context, i.e. the predictions of signal peptides, transmembrane helices (TMs) and protein domains. Our new protocol allowed us to narrow down the number of potential human ITIM receptors to 109 proteins (0.7% of RefPep). Of these, 36 have been described as ITIM receptors in the literature before. Many ITIMs are conserved between orthologous human and mouse proteins which represent novel ITIM receptor candidates. Publicly available DNA array expression data revealed that ITIM receptors are not exclusively expressed in blood cells. We hypothesise that ITIM signalling is not restricted to immune cells, but also functions in diverse solid organs of mouse and man.     

Role of immunoreceptor tyrosine-based inhibitory motifs of PECAM-1 in PECAM-1-dependent cell migration

Platelet endothelial cell adhesion molecule (PECAM-1), a transmembrane glycoprotein, has been implicated in angiogenesis, with recent evidence indicating the involvement of PECAM-1 in endothelial cell motility. The cytoplasmic domain of PECAM-1 contains two tyrosine residues, Y663 and Y686, that each fall within an immunoreceptor tyrosine-based inhibitory motif (ITIM). When phosphorylated, these residues together mediate the binding of the protein tyrosine phosphatase SHP-2. Because SHP-2 has been shown to be involved in the turnover of focal adhesions, a phenomenon required for efficient cell motility, the association of this phosphatase with PECAM-1 via its ITIMs may represent a mechanism by which PECAM-1 might facilitate cell migration. Studies were therefore done with cell transfectants expressing wild-type PECAM or mutant PECAM-1 in which residues Y663 and Y686 were mutated. These mutations eliminated PECAM-1 tyrosine phosphorylation and the association of PECAM-1 with SHP-2 but did not impair the ability of the molecule to localize at intercellular junctions or to bind homophilically. However, in vitro cell motility and tube formation stimulated by the expression of wild-type PECAM-1 were abrogated by the mutation of these tyrosine residues. Importantly, during wound-induced migration, the number of focal adhesions as well as the level of tyrosine phosphorylated paxillin detected in cells expressing wild-type PECAM-1 were markedly reduced compared with control cells or transfectants with mutant PECAM-1. These data suggest that, in vivo, the binding of SHP-2 to PECAM-1, via PECAM-1’s ITIM domains, promotes the turnover of focal adhesions and, hence, endothelial cell motility. platelet endothelial cell adhesion molecule-1; endothelial cells; angiogenesis     

Dendritic cell immunoactivating receptor,a novel C-type lectin immunoreceptor,acts as an activating receptor through association with Fc receptor gamma chain

An increasing number of C-type lectin receptors are being discovered on dendritic cells, but their signaling abilities and underlying mechanisms require further definition. Among these, dendritic cell immunoreceptor (DCIR) induces negative signals through an inhibitory immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. Here we identify a novel C-type lectin receptor, dendritic cell immunoactivating receptor (DCAR), whose extracellular lectin domain is highly homologous to that of DCIR. DCAR is expressed similarly in tissues to DCIR, but its short cytoplasmic portion lacks signaling motifs like ITIM. However, a positively charged arginine residue is present in the transmembrane region of the DCAR, which may explain its association with Fc receptor gamma chain and its stable expression on the cell surface. Furthermore, cross-linking of DCAR in the presence of gamma chain activates calcium mobilization and tyrosine phosphorylation of cellular proteins. These signals are mediated by the immunoreceptor tyrosine-based activating motif (ITAM) of the gamma chain. Thus, DCAR is closely related to DCIR, but it introduces activating signals into antigen-presenting cells through its physical and functional association with ITAM-bearing gamma chain. The identification of this activating immunoreceptor provides an example of signaling via a dendritic cell-expressed C-type lectin receptor.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2

B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2.     

Differential roles of N- and C-terminal immunoreceptor tyrosine-based inhibition motifs during inhibition of cell activation by killer cell inhibitory receptors

Killer cell inhibitory receptors (KIRs) inhibit NK and T cell cytotoxicity when recognizing MHC class I molecules on target cells. They possess two tandem intracytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) that, when phosphorylated, each bind to the two Src homology 2 domain-bearing protein tyrosine phosphatases SHP-1 and SHP-2 in vitro. Using chimeric receptors having an intact intracytoplasmic KIR domain bearing both ITIMs (N + C-KIR), a deleted domain containing the N-terminal ITIM only (N-KIR), or a deleted domain containing the C-terminal ITIM only (C-KIR), we examined the respective contributions of the two ITIMs in the inhibition of cell activation in two experimental models (a rat mast cell and a mouse B cell line) that have been widely used to analyze KIR functions. We found that the two KIR ITIMs play distinct roles. When coaggregated with immunoreceptor tyrosine-based activation motif-bearing receptors such as high-affinity IgE receptors or B cell receptors, the N + C-KIR and the N-KIR chimeras, but not the C-KIR chimera, inhibited mast cell and B cell activation, became tyrosyl-phosphorylated, and recruited phosphatases in vivo. The N + C-KIR chimera recruited SHP-1 as expected, but also SHP-2. Surprisingly, the N-KIR chimera failed to recruit SHP-1; however, it did recruit SHP-2. Consequently, the N-terminal ITIM is sufficient to recruit SHP-2 and to inhibit cell activation, whereas the N-terminal and the C-terminal ITIMs are both necessary to recruit SHP-1. The two KIR ITIMs, therefore, are neither mandatory for inhibition nor redundant. Rather than simply amplifying inhibitory signals, they differentially contribute to the recruitment of distinct phosphatases that may cooperate to inhibit cell activation.     

SHP-1- and phosphotyrosine-independent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic domain in human NK cells

Killer cell Ig-like receptors (KIR) are MHC class I-binding immunoreceptors that can suppress activation of human NK cells through recruitment of the Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) to two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. KIR2DL4 (2DL4; CD158d) is a structurally distinct member of the KIR family, which is expressed on most, if not all, human NK cells. 2DL4 contains only one ITIM in its cytoplasmic domain and an arginine in its transmembrane region, suggesting both inhibitory and activating functions. While 2DL4 can activate IFN-gamma production, dependent upon the transmembrane arginine, the function of the single ITIM of 2DL4 remains unknown. In this study, tandem ITIMs of KIR3DL1 (3DL1) and the single ITIM of 2DL4 were directly compared in functional and biochemical assays. Using a retroviral transduction method, we show in human NK cell lines that 1) the single ITIM of 2DL4 efficiently inhibits natural cytotoxicity responses; 2) the phosphorylated single ITIM recruits SHP-2 protein tyrosine phosphatase, but not SHP-1 in NK cells; 3) expression of dominant-negative SHP-1 does not block the ability of 2DL4 to inhibit natural cytotoxicity; 4) surprisingly, mutation of the tyrosine within the single ITIM does not completely abolish inhibitory function; and 5) this correlates with weak SHP-2 binding to the mutant ITIM of 2DL4 in NK cells and a corresponding nonphosphorylated ITIM peptide in vitro. These results reveal new aspects of the KIR-inhibitory pathway in human NK cells, which are SHP-1 and phosphotyrosine independent.     

SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.     

Molecular basis of the recruitment of the SH2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 by fcgamma RIIB

FcgammaRIIB are single-chain low affinity receptors for IgG that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They bear one immunoreceptor tyrosine-based inhibition motif (ITIM) that becomes tyrosyl-phosphorylated upon coaggregation of FcgammaRIIB with immunoreceptor tyrosine-based activation motif-bearing receptors and that recruits SH2 domain-containing inositol 5-phosphatases (SHIPs) in vivo. Synthetic FcgammaRIIB ITIM phosphopeptides, however, also bind SH2 domain-containing protein-tyrosine phosphatases (SHPs) in vitro. To identify SHIP-binding sites, we exchanged residues between the FcgammaRIIB ITIM and the N-terminal ITIM of a killer cell Ig-like receptor that does not bind SHIPs. Loss of function and gain of function substitutions identified the Y+2 leucine, in the FcgammaRIIB ITIM, as determining the binding of both SHIP1 and SHIP2, but not the binding of SHP-1 or SHP-2. Conversely, the Y-2 isoleucine that determines the in vitro binding of SHP-1 and SHP-2 affected neither the binding nor the recruitment of SHIP1 or SHIP2. One hydrophobic residue, in the ITIM of FcgammaRIIB therefore determines the affinity for SHIPs. This residue is symmetrical to the hydrophobic residue that determines the affinity of all ITIMs for SHPs. It defines a SHIP-binding site, distinct from a SHP-binding site, that enables FcgammaRIIB to recruit SHIP1 and SHIP2 and that is preferentially used in vivo.     

KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.     

Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells

We have identified and characterized two mouse cDNAs in a mouse antigen-stimulated bone marrow-derived mast cell cDNA library, both of which encode type I transmembrane proteins. The genes were closely mapped in the distal region of mouse chromosome 11 and expressed not only in mast cells but also widely in leukocytes. The extracellular domains of their encoded proteins contain a single variable immunoglobulin (Ig) motif sharing about 90% identity with amino acids, showing that they comprise a pair of molecules and belong to the Ig superfamily. We named these molecules leukocyte mono-Ig-like receptor1 and 2 (LMIR1 and 2). The intracellular domain of LMIR1 contains several immunoreceptor tyrosine-based inhibition motifs (ITIMs). When cross-linked, the intracellular domain was tyrosine phosphorylated and capable of recruiting tyrosine phosphatases, SHP-1 and SHP-2 and inositol polyphosphate 5-phosphatase, SHIP. LMIR2, on the other hand, contains a short cytoplasmic tail and a characteristic transmembrane domain carrying two positively charged amino acids associated with three kinds of immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecules, DAP10, DAP12, and FcRgamma. These findings suggest that a new pair of ITIM/ITAM-bearing receptors, LMIR1 and 2, regulate mast cell-mediated inflammatory responses through yet to be defined ligand(s).     

KIR2DL4 (CD158d), an NK cell-activating receptor with inhibitory potential

KIR2DL4 (CD158d) is an unusual member of the killer cell Ig-like receptor family expressed in all NK cells and some T cells. KIR2DL4 activates the cytotoxicity of NK cells, despite the presence of an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail. The role of this ITIM on the activating function of KIR2DL4, and whether it can provide inhibitory signals, is not known. Mutated forms of KIR2DL4 were engineered that lacked either the tyrosine in the ITIM or an arginine-tyrosine motif in the transmembrane region that is required for the activation signal. The activity of the mutated KIR2DL4 molecules was tested in a redirected lysis assay. The ITIM was not necessary for activation of lysis by KIR2DL4. The activation signal of KIR2DL4 was sensitive to inhibition by another ITIM-containing receptor. The activation-deficient mutant of KIR2DL4 inhibited the signal delivered by the activating receptor CD16. In pull-down experiments with GST fusion proteins, the tyrosine-phosphorylated cytoplasmic tail of KIR2DL4 bound the Src homology 2-containing phosphatases 1 and 2, as did the tail of the inhibitory receptor KIR2DL1. Therefore, KIR2DL4 has inhibitory potential in addition to its activating function.     

IgSF13, a novel human inhibitory receptor of the immunoglobulin superfamily, is preferentially expressed in dendritic cells and monocytes

A novel inhibitory receptor of immunoglobin superfamily (IgSF), IgSF member 13 (IgSF13), has been identified from human dendritic cells (DC). IgSF13 is a type I transmembrane protein containing an N-terminal signal peptide, a extracellular region with a single Ig V-like domain, a transmembrane region, and a cytoplasmic tail with two classical immunoreceptor tyrosine-based inhibitory motifs (ITIM), suggesting its inhibitory function. IgSF13 shows significant homology to human CMRF35 and pIgR. IgSF13 gene is mapped to chromosome 17q25.2, very close to that of CMRF35. IgSF13 is preferentially expressed in myelo-monocytic cells, including monocytes, monocyte-derived DC, and monocyte-related cell lines. Upon pervanadate treatment, IgSF13 was hyper-phosphorylated and associated with Src homology-2 domain-containing phosphatases SHP-1 and SHIP, but not SHP-2. The identification of IgSF13 as a novel ITIM-bearing receptor selectively expressed by DC and monocytes suggests that it may be potentially involved in the negative regulation of specific leukocyte population.     

NKp44 triggers NK cell activation through DAP12 association that is not influenced by a putative cytoplasmic inhibitory sequence

NKp44 (NCR2) is a member of the natural cytotoxicity receptor (NCR) family that is expressed on activated human NK cells. We dissected structural attributes of NKp44 to determine their contributions to receptor function. Our results demonstrate that surface expression and NK cell activation by NKp44 is mediated through noncovalent association with the immunoreceptor tyrosine-based activation motif-containing protein, DAP12. Physical linkage to DAP12 requires lysine-183 in the NKp44 transmembrane domain. Intriguingly, the cytoplasmic domain of NKp44 also contains a sequence that matches the immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus. By expressing a chimeric receptor in an NK-like cell line, we found that this ITIM-like motif from NKp44 lacks inhibitory capacity in a redirected cytotoxicity assay. The NKp44 cytoplasmic tyrosine was efficiently phosphorylated in the chimeric receptor upon treating the cells with pervanadate, but it was unable to recruit ITIM-binding negative effector phosphatases. We also generated NK-like cell lines expressing epitope-tagged wild-type or tyrosine to phenylalanine mutant (Y238F) versions of NKp44 and compared their capacities to induce activation marker expression, promote IFN-gamma production, or stimulate target cell cytotoxicity. We did not detect any tyrosine-dependent reduction or enhancement of NK cell activation through wild-type vs. Y238F mutant NKp44. Finally, the cytoplasmic tyrosine-based sequence did not provide a docking site for the AP-2 clathrin adaptor, nor did it potentiate receptor internalization. In summary, all activating properties and surface expression of NKp44 are mediated through its association with DAP12, and the putative ITIM in the NKp44 cytoplasmic domain does not appear to attenuate activating function.     

CD22 regulates B cell receptor-mediated signals via two domains that independently recruit Grb2 and SHP-1

Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate.     

Enteropathogenic Escherichia coli Tir recruits cellular SHP-2 through ITIM motifs to suppress host immune response

Immune responses to pathogens are regulated by immune receptors containing either an immunoreceptor tyrosine-based activation motif (ITAM) or an immunoreceptor tyrosine-based inhibitory motif (ITIM). The important diarrheal pathogen enteropathogenic Escherichia coli (EPEC) require delivery and insertion of the bacterial translocated intimin receptor (Tir) into the host plasma membrane for pedestal formation. The C-terminal region of Tir, encompassing Y483 and Y511, shares sequence similarity with cellular ITIMs. Here, we show that EPEC Tir suppresses the production of inflammatory cytokines by recruitment of SHP-2 and subsequent deubiquitination of TRAF6 in an ITIM dependent manner. Our findings revealed a novel mechanism by which the EPEC utilize its ITIM motifs to suppress and evade the host innate immune response, which could lead to the development of novel therapeutics to prevent bacterial infection.     

CD5-negative regulation of B cell receptor signaling pathways originates from tyrosine residue Y429 outside an immunoreceptor tyrosine-based inhibitory motif.

CD5 is a cell surface receptor that negatively regulates B cell function, but whose relationship to the immunoreceptor tyrosine-based inhibitory motif (ITIM) family of B cell inhibitory receptors is unclear. Using Fcgamma type IIB receptor-CD5 chimeras encompassing the cytoplasmic domain of CD5, we previously showed that a particular region of the molecule containing two tyrosine residues, Y429 and Y441, in an amino acid stretch similar to the Src autophosphorylation motif and a putative ITIM, respectively, antagonized early signaling events triggered through the B cell receptor (BCR). In this study, we provide evidences that only Y429 is mandatory for the inhibition by CD5 of the calcium response activated via the BCR. This residue also efficiently controls inhibition of the Ras/extracellular signal-related kinase-2 pathway. Analyzing the membrane translocation of the AKT protooncogene using its 3'-phosphoinositide-specific pleckstrin homology domain fused to the green fluorescent protein as a probe, we also show that CD5 strongly impairs its cellular redistribution and demonstrate the role played by Y429 in this process. We finally report that Y429 controls almost exclusively CD5 phosphorylation as well as inhibition of BCR-triggered IL-2 production upon coaggregation of the two receptors. Thus, CD5 uses an ITIM-independent strategy, centered on Y429, the major tyrosine-phosphorylated residue in its cytoplasmic domain, to inhibit BCR activation.     

Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor CD85j

The CD85j inhibitory receptor (also termed ILT2 or LIR-1) is a type-I transmembrane protein that belongs to the Ig superfamily and is expressed by different leukocyte lineages. The extracellular region of CD85j binds HLA class I molecules and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs (ITIM). Upon tyrosine phosphorylation CD85j recruits the SHP-1 tyrosine phosphatase, involved in negative signaling. In order to identify other molecules to which CD85j might interact with in a phosphotyrosine-dependent manner, a cDNA B-cell library was screened in a three-hybrid system in yeast using the CD85j cytoplasmic tail as bait in the presence of the Src-kinase c-fyn420, 531Y-F, 176R-Q mutant. In this system, the C-terminal Src kinase (Csk) was shown to interact with CD85j. Phosphorylation-dependent recruitment of Csk to the CD85j cytoplasmic tail was confirmed in CD85j-transfected mammalian cells by immunoprecipitation and Western blot analysis. Mutational analyses and phospho-peptide mapping suggested that the SH2 domain of Csk may preferentially bind to ITIM Y562 of CD85j; yet, mutation to phenylalanine of Y533, Y614, and Y644 also significantly reduced Csk recruitment by CD85j. Even though CD85j was detected in both anti-SHP1 and CSK immunoprecipitates, these two molecules did not co-precipitate together with CD85j. Our data support the possibility that Csk regulates the function of CD85j.