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     

Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric Fc gamma RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc gamma RIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1(-/-), SHP-2(-/-), SHIP(-/-), or SHP-1/2(-/-) and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type Fc gamma RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the Fc gamma RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc gamma RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.     

Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.     

Absence of platelet endothelial cell adhesion molecule-1 (CD31) leads to increased severity of local and systemic IgE-mediated anaphylaxis and modulation of mast cell activation

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1(+/+) and PECAM-1(-/-) mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1(+/+) and PECAM-1(-/-) mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1(-/-) mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1(+/+) mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1(-/-) mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1(+/+) mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1(-/-) bone marrow mast cells showed enhanced dense granule serotonin release after Fc epsilon RI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.     

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr⁸²² on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr⁸⁰⁷, a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr²⁹² on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.     

Counter-regulatory effects of incremental hypoxia on the transcription of a cardiac fatty acid oxidation enzyme-encoding gene

Platelet endothelial cell adhesion molecule (PECAM-1) is a member of the superfamily of immunoglobulins. This cell adhesion molecule has been implicated to mediate the adhesion and trans-endothelial migration of T lymphocytes/monocytes into the vascular wall, a critical step in the initiation of atherogenesis. Current thinking, however, posits that PECAM-1 by virtue of being a scaffolding molecule may well play a role in several signal transduction reactions. As a consequence, this cell adhesion molecule may be responsible for several biological and pathophysiological functions such as thrombosis, and inflammation. Evidence has also been put forward for a potential role of PECAM-1 in apoptosis and atherosclerosis. This article focuses on the structure of PECAM-1 and its role in intracellular signaling and implications in health and disease.     

Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages

Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes.     

A new role for platelet-endothelial cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduction.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3epsilon mAbs, anti-PECAM-1 mAbs, or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction.     

Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.     

Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration.

Cell migration is an important process in such phenomena as growth, development, and wound healing. The control of cell migration is orchestrated in part by cell surface adhesion molecules. These molecules fall into two major categories: those that bind to extracellular matrix and those that bind to adjacent cells. Here, we report on the role of a cell-cell adhesion molecule, platelet-endothelial cell adhesion molecule-1, (PECAM-1), a member of the lg superfamily, in the modulation of cell migration and cell-cell adhesion. PECAM-1 is a 120-130 kDa integral membrane protein that resides on endothelial cells and localizes at sites of cell-cell contact. Since endothelial cells express PECAM-1 constitutively, we studied the effects of PECAM-1 on cell-cell adhesion and migration in a null-cell population. Specifically, we transfected NIH/3T3 cells with the full length PECAM-1 molecule (two independent clones). Transfected cells containing only the neomycin resistance gene, cells expressing a construct coding for the extracellular domain of the molecule, and cells expressing the neu oncogene were used as controls. The PECAM-1 transfectants appeared smaller and more polygonal and tended to grow in clusters. Indirect immunofluorescence of PECAM-1 transfectants showed peripheral staining at sites of cell-cell contact, while the extracellular domain transfectants and the control cells did not. In two quantitative migration assays, the full-length PECAM-1 transfectants migrated more slowly than control cells. Thus, PECAM-1 transfected into a null cell appears to localize to sites of cell-cell contact, promote cell-cell adhesion, and diminish the rate of migration. These findings suggest a role for this cell-cell adhesion molecule in the process of endothelial cell migration.     

Pecam-1 is a modulator of stat family member phosphorylation and localization: lessons from a transgenic mouse

PECAM-1 (CD31) is a member of the immunoglobin (Ig) superfamily of cell adhesion molecules whose expression is restricted to hematopoietic and vascular cells. PECAM-1 can recruit adapter and signaling molecules via its immunoreceptor tyrosine activation motif (ITAM), suggesting that PECAM-1 plays a role in signal transduction pathways. To study the involvement of PECAM-1 in signaling cascades in vivo, we used the major histocompatibility (MHC) I gene promoter to target ectopic PECAM-1 expression in transgenic mice. We noted an attenuation of mammary gland development at early stages of virgin ductal branching morphogenesis. STAT5a, a modulator of milk protein gene expression during lactation, was localized to the nuclei of ductal epithelial cells of 6-week-old virgin PECAM-1 transgenics, but not in control mice. This correlated with decreases in ductal epithelial cell proliferation and induction of p21, an inhibitor of cell cycle progression. Using in vitro model systems we demonstrated PECAM-1/STAT5a association and found that residue Y701 in PECAM-1's cytoplasmic tail is important for PECAM-1/STAT5 association and that PECAM-1 modulates increases in STAT5a tyrosine phosphorylation levels. We suggest that by serving as a scaffolding, PECAM-1 can bring substrates (STAT5a) and enzymes (a kinase) into close proximity, thereby modulating phosphorylation levels of selected proteins, as previously noted for beta-catenin.     

The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31)

Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.     

Identification of a variant form of PZR lacking immunoreceptor tyrosine-based inhibitory motifs

PZR is an immunoglobulin superfamily protein that specifically binds tyrosine phosphatase SHP-2 through its intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Here we report a novel isoform of the protein designated PZR1b. PZR1b shares the same extracellular region with PZR, but it lacks intracellular ITIMs and thus the ability to recruit SHP-2. Genomic sequence analysis revealed that PZR1b is resulted from alternative gene splicing of the PZR gene localized at chromosome 1q24. Like PZR, PZR1b is widely expressed. However, the relative ratio of two forms varies in different human tissues and cells. More importantly, overexpression of PZR1b in human HT-1080 cells had a dominant negative effect by blocking concanavalin A-induced tyrosine phosphorylation of full-length PZR and recruitment of tyrosine phosphatase SHP-2. Therefore, PZR1b may have an important role in cell signaling by counteracting with PZR.     

Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.     

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.     

Platelet-endothelial cell adhesion molecule-1 modulates endothelial migration through its immunoreceptor tyrosine-based inhibitory motif

Coordinated migration of endothelial cells models the remodeling of existing endothelia as well as angiogenesis and vasculogenesis. Platelet-endothelial cell adhesion molecule-1, PECAM-1, a transmembrane endothelial adhesion protein, binds and activates the tyrosine phosphatase SHP-2 via phosphotyrosines 663 and 686. PECAM-1 phosphorylation and recruitment of SHP-2 are regulated by cell-cell and cell-substrate adhesion. We found that PECAM-1 is dephosphorylated on tyrosine 686 during endothelial migration, resulting in diffuse dispersal of PECAM-1 and SHP-2. Overexpression of native PECAM-1 slowed, and nonphosphorylatable PECAM-1 increased, endothelial migration, implying that the SHP-2-regulatory phosphotyrosines negatively regulate migration. Using differentially phosphorylated recombinant proteins we found that phosphotyrosine 686 preferentially mediates binding and 663 mediates activation of SHP-2 by PECAM-1. In PECAM-1-null endothelial cells, SHP-2 bound and dephosphorylated an alternative set of phosphoproteins and its distribution to the cytoskeletal fraction was significantly decreased. Tyrosine phosphorylation of beta-catenin and focal adhesion kinase was increased in endothelial cells overexpressing nonphosphorylatable PECAM-1. Thus homophilically engaged, tyrosine-phosphorylated PECAM-1 locally activates SHP-2 at cell-cell junctions; with disruption of the endothelial monolayer, selective dephosphorylation of PECAM-1 leads to redistribution of SHP-2 and pro-migratory changes in phosphorylation of cytoskeletal and focal contact components.