Role of Integrins in Natural Killer Cell Function

原代NK细胞上的整合素LFA-1直接与ICAM-1结合，这足以诱导颗粒(又名分泌型溶酶体)极化到与靶细胞接触的部位，但不会导致脱颗粒。相反，Fc受体CD16与IgG1结合可导致非极化脱颗粒。为了研究配体诱导的整合素信号，而不需要其他受体的内向外信号，我们利用了人NK细胞通过β2整合素直接与ICAM-1结合的能力。由于这种相互作用控制着溶解颗粒的偏振，我们有机会确定这种偏振所需的信号。我们对ICAM-1结合的原代NK细胞中酪氨酸磷酸化的蛋白质或与之相关的蛋白质进行了无偏倚的质谱分析。这种方法背后的动机是，β2整合素与ICAM-1结合在原代NK细胞中诱导强烈的总蛋白酪氨酸磷酸化，类似于与人IgG1结合，人IgG1是Fc受体CD16的配体。这种相似性令人惊讶，因为与IgG1结合会导致脱颗粒，而不是颗粒极化。为了缩小对Beta2整合素信号独特蛋白质的关注范围，从ICAM-1刺激样本中鉴定的蛋白质中减去在未刺激的NK细胞以及通过CD16刺激的NK细胞中鉴定的蛋白质。通过生物信息学分析，出现了一个信号网络。 通过siRNA介导的沉默，我们发现整合素连接的激酶(ILK)、伽马-Parvin、RhoGEF7和Pyk2都已知在迁移过程中控制细胞的极性，而亮蛋白对LFA-1依赖的颗粒极化至关重要。邻近连接分析显示ILK与ICAM-1结合后与LFA-1的结合增强。迁移细胞的极性也受一条依赖于CDC42的通路控制，该通路涉及Par6、APC和磷酸化的GSK3β。与ICAM-1结合诱导NK细胞ILK依赖的GSK3β磷酸化。沉默CDC42、PAR6和APC可阻断NK靶细胞接触部位的颗粒极化。此外，PYK2、亮蛋白和CLIP-170不仅是MTOC极化所必需的，也是颗粒向MTOC会聚所必需的。我们的工作揭示了LFA-1在NK细胞中诱导的颗粒极化使用的信号通路类似于在细胞迁移过程中建立极性的信号通路。因此，LFA-1在NK细胞中诱导的颗粒极化使用的信号通路类似于在贴壁细胞迁移过程中建立极性的信号通路。总体而言，这项工作揭示了比先前所认识到的更大的Beta2整合素的信号能力，并发现了两个控制NK细胞中整合素依赖的颗粒极化的相连的信号网络。 我们的研究也有助于理解颗粒向MTOC的会聚，NK细胞和T细胞对MTOC的要求不同。颗粒会聚需要更强的TCR信号，而MTOC重新定位到免疫突触则需要更强的TCR信号。相反，在NK细胞中，收敛发生得很快，并且先于MTOC极化。我们已经证明，PYK2、亮蛋白和CLIP-170也是整合素依赖的颗粒收敛所必需的。

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.