Integrin 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. To study ligand-induced signaling by an integrin, without the complication of inside-out signals from other receptors, we took advantage of the ability of human NK cells to bind ICAM-1 directly via beta2 integrin. As this interaction controls polarization of lytic granules, we had an opportunity to determine signals required for this polarization. We performed an unbiased mass spectrometry analysis of proteins that were tyrosine phosphorylated or associated with tyrosine phosphorylated proteins in primary NK cells bound to ICAM-1. The impetus behind this approach was that beta2 integrin binding to ICAM-1 induces strong overall protein tyrosine phosphorylation in primary NK cells, similar to that induced by binding to human IgG1, a ligand for the Fc receptor CD16. This similarity was surprising, given that binding to IgG1 leads to degranulation but not granule polarization. To narrow the focus on proteins that are unique to beta2 integrin signaling, proteins identified in unstimulated NK cells, as well as in NK cells stimulated through CD16, were subtracted from the proteins identified in the ICAM-1 stimulated sample. A signaling network emerged from a bioinformatics analysis. 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. Overall, this work has revealed a greater signaling capacity of beta2 integrin than previously appreciated, and identified two connected signaling networks that control integrin-dependent granule polarization in NK cells. Our study has also contributed to the understanding of granule convergence to the MTOC, the requirements for which differ in NK cells and T cells. Stronger TCR signals are required for granule convergence than for MTOC relocation to the immunological synapse. In contrast, in NK cells, convergence occurs rapidly and precedes MTOC polarization. We have shown that Pyk2, leupaxin, and CLIP-170 are also required for integrin-dependent granule convergence.
| Zhang, Minggang; March, Michael E; Lane, William S et al. (2014) A signaling network stimulated by ?2 integrin promotes the polarization of lytic granules in cytotoxic cells. Sci Signal 7:ra96 |
| March, Michael E; Long, Eric O (2011) ?2 integrin induces TCR?-Syk-phospholipase C-? phosphorylation and paxillin-dependent granule polarization in human NK cells. J Immunol 186:2998-3005 |
| Long, Eric O (2011) ICAM-1: getting a grip on leukocyte adhesion. J Immunol 186:5021-3 |
| Gross, Catharina C; Brzostowski, Joseph A; Liu, Dongfang et al. (2010) Tethering of intercellular adhesion molecule on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization. J Immunol 185:2918-26 |
| Liu, Dongfang; Bryceson, Yenan T; Meckel, Tobias et al. (2009) Integrin-dependent organization and bidirectional vesicular traffic at cytotoxic immune synapses. Immunity 31:99-109 |
