Natural killer (NK) cells provide a unique opportunity to study integrin signaling in the absence of inside-out signals. In contrast to T cells where inside-out signaling is required to activate beta-2 integrin LFA-1, LFA-1 on primary NK cells binds directly to ICAM-1, which is sufficient to induce polarization of granules (aka secretory lysosomes) to the site of contact with target cells, but not degranulation. Conversely, binding of Fc receptor CD16 to IgG1 results in unpolarized degranulation. We took a proteomics approach to examine signaling by LFA-1. Primary human NK cells were stimulated on plates coated with ICAM-1. Tyrosine-phosphorylated proteins and their associated proteins were pulled down with mAb 4G10, eluted with phenyl-phosphate, and analyzed by mass spectrometry. Unique proteins enriched after stimulation by LFA-1, but not by CD16 were validated by immunoblotting. Through siRNA-mediated silencing, we showed that integrin-linked kinase (ILK), gamma-parvin, RhoGEF7, and Pyk2, all known to control cell polarity during migration, and that leupaxin were critical for LFA-1-dependent granule polarization. Proximity ligation assays to image protein complexes in cells revealed an enhanced association of ILK with LFA-1 upon ICAM-1 binding. Polarity in migrating cells is controlled also by a Cdc42-dependent pathway, which involves Par6, APC, and phosphorylation of kinase GSK3beta. Binding to ICAM-1 induced ILK-dependent GSK3beta phosphorylation in NK cells. Silencing of Cdc42, Par6 and APC blocked granule polarization to the NKtarget cell contact site. In addition, Pyk2, leupaxin, and CLIP-170 were required not only for MTOC polarization, but also for granule convergence to the MTOC. Our work has revealed that granule polarization induced by LFA-1 in NK cells uses a signaling pathway similar to that used to establish polarity during cell migration. Therefore, granule polarization induced by LFA-1 in NK cells uses a signaling pathway similar to that used to establish polarity during migration of adherent cells.
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