Role of Integrins in Natural Killer Cell Function

原代NK细胞上的整联蛋白LFA-1直接结合ICAM-1，这足以诱导颗粒（又名分泌性溶酶体）极化至与靶细胞接触的部位，但不脱粒。相反，Fc受体CD 16与IgG 1的结合导致非极化脱粒。为了研究整合素的配体诱导的信号传导，而没有来自其他受体的由内而外的信号的复杂化，我们利用了人NK细胞通过β 2整合素直接结合ICAM-1的能力。由于这种相互作用控制溶解颗粒的极化，我们有机会确定这种极化所需的信号。我们对与ICAM-1结合的原代NK细胞中酪氨酸磷酸化蛋白或与酪氨酸磷酸化蛋白相关的蛋白进行了无偏质谱分析。这种方法背后的推动力是β 2整合素与ICAM-1的结合在原代NK细胞中诱导强烈的整体蛋白酪氨酸磷酸化，类似于与Fc受体CD 16的配体人IgG 1结合诱导的磷酸化。这种相似性是令人惊讶的，因为与IgG 1的结合导致脱粒而不是颗粒极化。为了缩小对β 2整联蛋白信号传导特有的蛋白质的关注，从ICAM-1刺激的样品中鉴定的蛋白质中减去未刺激的NK细胞以及通过CD 16刺激的NK细胞中鉴定的蛋白质。一个信号网络出现在生物信息学分析中。 通过siRNA介导的沉默，我们发现整合素连接激酶（ILK），gamma-parvin，RhoGEF 7和Pyk 2，都已知在迁移过程中控制细胞极性，leupaxin是LFA-1依赖性颗粒极化的关键。对细胞中的蛋白质复合物进行成像的邻近连接测定揭示了在ICAM-1结合后ILK与LFA-1的增强的缔合。迁移细胞中的极性也由Cdc 42依赖性途径控制，该途径涉及Par 6、APC和激酶GSK 3 β的磷酸化。与ICAM-1结合诱导NK细胞中ILK依赖性GSK 3 β磷酸化。Cdc 42、Par 6和APC的沉默阻断了颗粒向NK靶细胞接触位点的极化。此外，Pyk 2，leupaxin，和CLIP-170所需的不仅是MTOC极化，而且颗粒收敛到MTOC。我们的工作已经揭示了在NK细胞中由LFA-1诱导的颗粒极化使用与用于在细胞迁移期间建立极性的信号传导途径类似的信号传导途径。因此，在NK细胞中由LFA-1诱导的颗粒极化使用与用于在粘附细胞的迁移期间建立极性的信号传导途径类似的信号传导途径。总的来说，这项工作揭示了β 2整合素比以前认识到的更大的信号传导能力，并确定了两个连接的信号传导网络，控制NK细胞中的整合素依赖性颗粒极化。 我们的研究也有助于理解MTOC的颗粒收敛，NK细胞和T细胞的要求不同。更强的TCR信号所需的颗粒收敛比MTOC搬迁到免疫突触。相比之下，在NK细胞中，会聚迅速发生并且先于MTOC极化。我们已经表明，Pyk 2，leupaxin和CLIP-170也需要整合素依赖性颗粒收敛。

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.