Vav1 Dephosphorylation by the Tyrosine Phosphatase SHP-1 as a Mechanism for Inhibition of Cellular Cytotoxicity

Abstract

Here, we present data suggesting a novel mechanism for regulation of natural killer (NK) cell cytotoxicity through inhibitory receptors. Interaction of activation receptors with their ligands on target cells induces cytotoxicity by NK cells. This activation is under negative control by inhibitory receptors that recruit tyrosine phosphatase SHP-1 upon binding major histocompatibility class I on target cells. How SHP-1 blocks the activation pathway is not known. To identify SHP-1 substrates, an HLA-C-specific inhibitory receptor fused to a substrate-trapping mutant of SHP-1 was expressed in NK cells. Phosphorylated Vav1, a regulator of actin cytoskeleton, was the only protein detectably associated with the catalytic site of SHP-1 during NK cell contact with target cells expressing HLA-C. Vav1 trapping was independent of actin polymerization, suggesting that inhibition of cellular cytotoxicity occurs through an early dephosphorylation of Vav1 by SHP-1, which blocks actin-dependent activation signals. Such a mechanism explains how inhibitory receptors can block activating signals induced by different receptors.In many cell types, activation induced by cell contact is controlled by inhibitory receptors that bind ligands on target cells (41, 48). The importance of this type of negative regulation is illustrated by the protection of normal cells from lysis by NK cells, of red blood cells from ingestion by macrophages (44), and of cardiomyocytes from immunoglobulin G (IgG)-mediated autoimmunity (43). A common feature of the receptors involved in these protective functions (i.e., CD158, SIRP-␣, and PD-1, respectively) and of many other receptors that inhibit cellular responses is the presence of immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic tail (12,48). Upon Tyr phosphorylation, ITIMs bind to the SH2 domains of protein tyrosine phosphatases (PTPases) SHP-1 and SHP-2, thereby releasing the catalytic site from autoinhibition (2, 12). Activation of human NK cell and T cell cytotoxicity is controlled by several inhibitory receptors, including the CD158 killer cell Ig-like receptors (KIR) and the lectin-like CD94/NKG2A, which bind to major histocompatibility complex (MHC) class I molecules expressed on target cells (5).Whereas the functional consequence of SHP-1 recruitment by ITIM-containing receptors in NK cells is well established, the specific point at which SHP-1 blocks activation signals has not been defined. Engagement of inhibitory receptors by ligands on target cells blocks conjugate formation (13), Ca 2ϩ flux (38, 52), polarization of lipid rafts in NK cells (42), and actin cytoskeleton rearrangement in T cells (26). As NK cell cytotoxicity depends on the activity of Tyr kinases (40), a number of Tyr-phosphorylated proteins are potential substrates for dephosphorylation by SHP-1. Two nonexclusive models, a "promiscuous" and a "selective" model, can be proposed for SHP-1-mediated inhibition. First, recruitment and activation of SHP-1 by inhibitory receptors at the NK-target cell interfac...

Vav1 Controls DAP10-Mediated Natural Cytotoxicity by Regulating Actin and Microtubule Dynamics

“…Given the widespread requirement for Vav proteins in NK cell activation, it follows that Vav would be a target for antagonism by NK inhibitory receptors. Indeed, recruitment of SHP-1 to ITIMs in inhibitory receptors appears to specifically target Vav1 and lead to its dephosphorylation (21). In this context, our findings suggest a possibility that dephosphorylation of Vav (30,31) may be functionally linked to inhibition of postconjugation events such as actin polymerization and MTOC polarization.…”

Regulating Vav1 phosphorylation by the SHP-1 tyrosine phosphatase is a fine-tuning mechanism for the negative regulation of DISC formation and Fas-mediated cell death signaling

“…17 We showed that Vav phosphorylation is negatively regulated upon FasL engagement and that this process partially depends on the presence of SHP-1. It is well documented that functions of Vav are tightly regulated by its tyrosine phosphorylation.…”

“…27 We hypothesized that the 95 kDa guanine nucleotide exchange factor Vav might correspond to the dephosphorylated protein of around 100 kDa, since it has been reported to be an SHP-1 substrate critical in the downregulation of NK cell killing. 17 We therefore analyzed the phosphorylation state of Vav (Figure 3b). Our results demonstrate a high constitutive phosphorylation of Vav, in accordance with data obtained from different B lymphocytes.…”

“…16 Vav1 dephosphorylation by SHP-1 is reported to be critical in the inhibition of natural killer cell cytotoxicity due to its ability to block the actin-dependent activation signal. 17 In addition, both actin 18 and myosin 19 have been identified as direct SHP-1 substrates in B cells. Cells transfected with a catalytically inactive form of SHP-1 displayed more prolonged actin polymerization.…”

Line of attack: NK cell specificity and integration of signals

“…Engagement of inhibitory receptors prevents actin cytoskeleton dynamics [47*,48], thereby preventing actin-dependent processes, such as coalescence of lipid rafts [49], recruitment and phosphorylation of co-activation receptors 2B4 and NKG2D to lipid rafts [50,51], and dephosphorylation of ezrin-radixin-moesin proteins, which connect actin filaments to membrane structures [48]. A direct substrate of SHP-1 during inhibition is Vav1, which is an essential regulator of actin dynamics [52]. An interesting imaging study in which phosphorylated inhibitory KIR was visualized, has shown that tyrosine phosphorylated KIR molecules are not evenly distributed over NK-target cell contact area but forms microclusters [53*].…”