In this paper we describe an NKG2D-mediated signaling pathway for the regulation of conjugate formation, MTOC polarization and granule secretion. Integrins have been shown to play a critical role in lymphocyte function regulated by ITAM-containing receptors such as the TCR, but little is know about the regulation of integrin by non-ITAM containing receptor such as NKG2D. We establish that ligation of FcR and NKG2D increase adhesion to β1 and β2 integrin ligands, VCAM-1 and ICAM-1. Using blocking antibodies we have established a role for the β2 integrin in NKG2D-mediated NK – tumor target formation and cytotoxicity. This is the consistent with the observation that patients with leukocyte adhesion deficiencies display impaired NK cell function (10
The results presented herein indicate that NKG2D signaling regulates NK cell adhesion and that LFA1 is required for optimal killing through NKG2D. Surprisingly, previous studies aimed at dissecting NKG2D-mediated signaling in both human and mouse NK cells suggest that neither the PI3K nor Grb2/Vav1 pathways regulate NKG2D-mediated cellular adhesion (8
). Taken at face value, these studies would seem to indicate that neither of the two known signaling pathways engaged by NKG2D are important in stimulating adhesion. However, the amount of conjugate formation between NK cells and NKG2D-sensitive targets in both studies is only slightly, if at all, increased over control targets (8
), suggesting the possibility that NKG2D-engagement does not trigger integrin activation, similar to known ITAM-containing receptors. However, our data provide evidence that NKG2D ligation does in fact increase integrin-mediated adhesion to plate-bound substrates, as well as conjugate formation to an NKG2D-ligand-bearing target cells. Moreover, our data identify PI3K signaling as an important mediator of NKG2D-stimulated adhesion. Thus, our work adds to the understanding of the NKG2D-mediated signaling pathways by establishing that NKG2D-mediated conjugate formation is PI3K-dependent. Future studies will be important to determine if the Grb2/Vav1 pathway also participates in NKG2D-stimulated adhesion.
The Crk family of adaptor proteins, Crk and CrkL, are important regulators of cell motility and adhesion in multiple cell types, and in the regulation of the oncogenic BCR-Abl mutation (19
). The lymphocyte Crk family member, CrkL, has been clearly established as an important adapter protein in T-lymphocyte biology (16
) and it has been suggested that CrkL plays a role in NK cell activation, but it has not been established if CrkL plays a role in the activation of NK cells by non-ITAM containing receptors such as NKG2D (46
). In this study we establish that CrkL regulates NKG2D-mediated NK cell-mediated cytotoxicity, in part through its effects on cell adhesion and establishing cell polarity. These results are significant since a recent study has shown that engagement of an inhibitory KIR on NK cells results in the inactivation of Crk through Abl-mediated phosphorylation at a known inhibitory site (20
). Interestingly, the KIR-generated inhibitory signal could be overcome by expression of a Crk protein lacking the inhibitory phosphorylation site. Our data implicate CrkL in both ITAM-dependent and ITAM-independent signaling pathways leading to granule release and cellular cytotoxicity. Whether CrkL is phosphorylated by KIR engagement and serves a functionally redundant role with Crk in the regulation of NK cell cytotoxicity remains to be determined.
PI3K has been shown to regulate cell polarity and adhesion in multiple cell types (48
). Additionally, the regulatory subunit of PI3K, p85, has also been shown to interact with CrkL in transformed cell lines (38
). These observations are consistent with our study using normal human NK cells, and we add the observation that PI3K interacts with the N-terminal SH3 domain of CrkL in primary human NK clones and this interaction is important for NKG2D-stimulated killing. Moreover, our data indicate that CrkL is involved in establishing cell polarity downstream of NKG2D, affecting both MTOC polarization and granule release. Bryceson et. al. has indicated that in resting NK cells, granule polarization is integrin-regulated whereas degranulation is controlled by FcR ligation (50
). In our study, beads that are coated with only anti-NKG2D stimulate recruitment of the MTOC to the NK – bead interface in a CrkL-dependent manner, suggesting that in IL-2 expanded human NK clones, integrins are not required for MTOC polarization.
How an activation signal passes from NKG2D-PI3K-CrkL to Rap1 remains to be determined. Indeed, PI3K has been shown to regulate Rap1-dependent adhesion (48
). Rap1 is an integral protein involved in the activation of integrins in T cells, where it has also been shown to regulate cell motility and polarity (51
). Recently, Rap1 activation following TCR ligation has been shown to occur via a complex of proteins including CrkL and C3G (16
). Interestingly, although CrkL is required for NKG2D-mediated conjugate formation and cytotoxicity, C3G does not appear to participate in NKG2D-mediated cellular cytotoxicity (Supplemental Figure 4
). Still, our data suggest that Rap1 is activated in a PI3K- and CrkL-dependent manner following NKG2D ligation and mediates NK cell cytotoxicity through regulation of both NKG2D-mediated adhesion and MTOC polarization in human NK cells. Given that C3G suppression does not effect NKG2D-mediated cytotoxicity, it is likely that the NKG2D-PI3K-CrkL signaling cascade signals through another Rap1 activator such as CalDAG-GEF, PDZ-GEF, or DOCK4 (53
). Additionally, the Rap1 effector pathways required for NKG2D-initiated cell killing remain to be determined.
Taken together, we have identified that PI3K activity regulates cell adhesion stimulated through engagement of NKG2D. Moreover, we have shown that the adaptor protein CrkL and the small GTPase Rap1 play important roles downstream of PI3K controlling not only integrin activation, but also establishing NKG2D-dependent cell polarity leading to granule exocytosis and optimal cellular cytotoxicity. A further understanding of the NKG2D-PI3K-CrkL-Rap1 signaling pathway, as well as other signaling pathways engaged by this activating receptor, will likely provide important insight into the causes of leukocyte adhesion deficiencies as well as other disorders of NK cell function.