Identification of the molecules dephosphorylated by SHP-1 during inhibition of natural cytotoxicity by an ITIM-containing receptor had the potential to not only illuminate the inhibitory mechanism but to also reveal important steps in the NK cell activation pathway. Furthermore, elucidating the mechanism by which KIR inhibits NK cells may provide relevant information about the many other ITIM-containing receptors that regulate various cell types.
Expression of a trapping mutant of SHP-1 in NK cells made it possible to study the interaction of SHP-1 with substrates encountered during inhibition by KIR when engaged by MHC class I on target cells. As receptors and signaling molecules segregate into discrete domains during the interaction of NK cells with target cells (16
), it was necessary to develop a trapping protocol that would be effective during NK-target cell interactions. The data obtained by our approach suggest that the inhibition of NK cell cytotoxicity is achieved by a selective dephosphorylation of Vav1 by the PTPase SHP-1. The trapping 2DL1/SHP-1 chimeric receptor bound specifically to Tyr-phosphorylated Vav1 upon engagement by HLA-Cw15 on target cells. Vav1 trapping was sensitive to vanadate being added either to cell lysates or to intact cells—implying that the SHP-1 catalytic site was essential for Vav1 binding. Vav1 trapping was resistant to cytochalasin D, placing it upstream of actin cytoskeletal rearrangement. The activation of the Vav1 GEF domain by Tyr phosphorylation (1
), the established importance of a Vav1-Rac1 signaling pathway in T cells for actin cytoskeleton-dependent membrane reorganization, receptor clustering, phosphatidylinositol 4-phosphate 5-kinase activation (27
), and the role of Vav1 in NK cell cytotoxicity (4
) all point to the dephosphorylation of Vav1 by SHP-1 as a viable mechanism for the inhibition of NK cell cytotoxicity.
It is possible that the use of a chimeric KIR/SHP-1 receptor in the cell line YTS led to the identification of a substrate that is not normally dephosphorylated during inhibition of NK cells by KIR. However, the inhibitory function of the chimeric KIR/SHP-1 receptor was comparable to that of a wild-type KIR. Dephosphorylation of Vav1 by SHP-1 can also explain how engagement of an ITIM-containing receptor blocked actin polymerization in T cells (26
) as well as the actin-dependent Tyr phosphorylation of the NK cell activation receptor 2B4 (57
). One of the primary regulatory tyrosines (Tyr174) in Vav1 is within a sequence context (DEIpYEDL) that closely matches two sequence motifs [(D/E)X(L/I/V)XpYXX(L/I/V) and (D/E)XpY, respectively] recently determined to be favored for dephosphorylation by SHP-1 in vitro (56
No change in overall tyrosine phosphorylation of Vav1 was detected in NK cells that were inhibited by KIR2DL1 or by the chimeric 2DL1/SHP-1 receptor when engaged by HLA-C on target cells (data not shown). Several reasons can account for this result. Target cells contribute a good amount of their own phosphorylated Vav1, which is not affected by SHP-1 in the NK cells. Furthermore, SHP-1 may dephosphorylate only a fraction of total cellular Vav1 during inhibition and only one out of the several phosphorylated tyrosines in each active Vav1. Full activation of Vav1 is known to require phosphorylation on multiple tyrosines (14
The intact SHP-1 inserted in place of the KIR cytoplasmic tail is probably still autoinhibited by its own SH2 domains, as occurs in wild-type SHP-1 (49
). A constitutively “open” SHP-1 carrying the trapping mutation would likely be blocked by constitutive occupancy of the catalytic site. Indeed, a chimeric KIR/SHP-1(DA) receptor that lacked the SH2 domains (and thereby lacked autoinhibitory regulation), which was expressed in YTS cells, failed to inducibly trap tyrosine-phosphorylated proteins upon binding to HLA-C on target cells under the same conditions that led to Vav1 trapping by 2DL1/SHP-1(DA), even though the KIR/SHP-1(wt) without SH2 domains was inhibitory (unpublished data). A possible mechanism for the activation of full-length SHP-1 in the 2DL1/SHP-1 chimeric receptor is through intramolecular binding of the N-terminal SH2 domain to a C-terminal tyrosine that is phosphorylated in SHP-1 (63
Our model of inhibition through Vav1 dephosphorylation differs from previous models of inhibition on the part of ITIM-containing receptors. Rather than promiscuous dephosphorylation of multiple components of signals from activation receptors, it is the selective dephosphorylation of a primary molecule not associated with any given activation receptor that blocks target cell killing. The reduced Tyr phosphorylation of several different proteins upon coligation of inhibitory receptors with activation receptors using antibodies had suggested that SHP-1 dephosphorylated multiple substrates. In the more physiological setting of NK cells or T cells incubated with target cells, Syk and ZAP70, respectively, showed reduced Tyr phosphorylation during inhibition (11
). Phosphorylation of LAT in NK cells was probably also reduced by KIR engagement, as it no longer bound Grb2 (52
). These data were consistent with an inhibition by KIR-associated SHP-1 that centered on early components of the ITAM activation pathway. Our data support a new model that can account for these earlier findings.
The selective dephosphorylation of Vav1 by SHP-1 during inhibition by KIR leads to some predictions about the mechanism of NK cell activation as well (Fig. ). Upon initial contact with a target cell, early signals lead to Tyr phosphorylation and activation of Vav1. The GEF activity of Vav1 promotes a Rac1-dependent rearrangement of the actin cytoskeleton. Engagement of KIR by MHC class I on target cells leads to early ITIM phosphorylation and SHP-1 recruitment (29
). Vav1 and KIR have both been reported to be phosphorylated by the Src-family kinase p56-Lck (8
). Only after formation of a tight actin-dependent NK-target cell interface do receptors cluster, thereby leading to amplification of early signals and binding of additional receptors to their ligand on target cells. Vav1 dephosphorylation by SHP-1 is independent of actin polymerization, as its trapping by the 2DL1/SHP-1(DA) receptor was not sensitive to cytochalasin D. In contrast, Tyr phosphorylation of the activation receptor 2B4 was inhibited by cytochalasin D and by a dominant-negative Rac1 to the same extent as it was by KIR binding to HLA-C on target cells. Therefore, the block of 2B4 phosphorylation by KIR (58
) occurs upstream of receptor phosphorylation and not by direct dephosphorylation of 2B4 by SHP-1. Consistent with this interpretation, we have recently shown that engagement of inhibitory KIR by HLA-C on target cells prevents the movement of 2B4 to lipid rafts, thereby blocking 2B4 phosphorylation (57
The early clustering and phosphorylation of KIR predicted by our model are consistent with recent data. The striking propensity of KIR to cluster at the site of contact with target cells expressing a KIR ligand (23
), which occurs independently of actin polymerization (28
), may contribute to an early phosphorylation of the ITIMs. Indeed, we have recently shown that ITIM phosphorylation induced by the binding of KIR to HLA-C on target cells occurs independently of actin polymerization (29
). Specific localization of KIR within the “NK immune synapse” and its proximity to other molecules may contribute to the selectivity in Vav1 dephosphorylation by KIR-associated SHP-1.
Upon contact of T cells with antigen-presenting cells, early signals from the T-cell receptor precede adhesion through LFA-1, Vav1-dependent actin polymerization, and formation of the immune synapse (10
). Recruitment to the membrane and phosphorylation of Vav1 constitute a very proximal event in T-cell activation, as Vav1-dependent actin polymerization precedes Ca2+
flux induced by contact with an antigen-presenting cell (24
). Upon T-cell receptor stimulation, Vav1 is recruited to a signaling complex through a LAT-Gad-SLP76-Vav1 set of interactions (9
). However, as LAT and SLP76 are dispensable for NK cytotoxicity (47
), and as overexpression of a PH domain-deleted Vav1 still resulted in enhanced lysis of K562 cells by NK cells (6
), what signals and what molecular interactions recruit Vav1 during NK cell activation are unclear. The early activation signal(s) in NK cells that leads to Vav1 phosphorylation may be delivered by specialized receptors or, alternatively, by the same activation receptors that induce cytotoxicity once signal amplification through Vav1-dependent receptor clustering has taken place.
A test of our model would be to express a Tyr phosphorylation-independent form of Vav1 in NK cells and to show that it can overcome inhibition by KIR. Such a regulated, Tyr phosphorylation-independent Vav1 has not been developed yet. Constitutively active forms of Vav1 and Rac1 are no substitute, because actin polymerization is under stringent temporal and spatial regulation (50
). The model suggests that ITIM-based inhibitory signals in NK cells could be bypassed by a Vav1-independent activation of actin polymerization. It will be interesting to see if the lysis of MHC class I-positive tumor cell lines by NK cells expressing the NKG2D/DAP10 receptor, which is apparently resistant to inhibition by ITIM-containing receptors (3
), could be explained by a DAP10/phosphatidylinositol 3-kinase-dependent (59
) but Vav1-independent signal.
The main conclusion from our work is that an early, actin polymerization-independent dephosphorylation of Vav1 by SHP-1 during engagement of KIR by MHC class I on target cells may block activation signals that depend on actin cytoskeleton rearrangements. Such a mechanism is possible because of the unique ability of KIR to cluster and become Tyr phosphorylated in the absence of actin polymerization and of cell adhesion (29
). Whether Vav1 dephosphorylation alone can account for the full inhibitory effect of KIR and if this proposed mechanism accounts for inhibition of normal NK cells by a wild-type KIR remains to be seen. A selective dephosphorylation of Vav1 by SHP-1 is consistent with the known inhibition of early NK activation signals upon KIR binding to its ligand on target cells and suggests that other ITIM-based inhibitory pathways may also rely on a selective dephosphorylation of substrates by SHP-1.