In NK cells, eta2 integrin binding to ICAM family members is sufficient to promote not only adhesion to target cells, but also the polarization of intracellular lytic granules towards the target. This feature enabled us to study the polarization of lytic granules in the absence of their release through degranulation, as well as 2 integrin signaling independently of inside-out signals from other receptors. Using a proteomics approach, we identified a signaling network centered on an integrin-linked kinase (ILK)Pyk2Paxillin core that was required for granule polarization. Downstream of ILK, the conserved Cdc42Par6 signaling pathway, which controls cell polarity, was activated and required for granule polarization. Polarization of NK lytic granules toward target cells occurs in two distinct steps: granule convergence to the MTOC, followed by reorientation with the MTOC toward the site of contact with the target cell. By measuring each step separately, we have found three signaling components that are required for the convergence step. This work has revealed a greater signaling capacity of eta2 integrins than was previously appreciated, and delineates two connected signaling networks that are integrated to control integrin-dependent granule polarization in NK cells. The unbiased mass spectrometry and bioinformatics analysis we performed with NK cells that had been stimulated through integrin LFA-1 resulted in the discovery of a complete signaling pathway that controls granule polarization toward the target cell. In addition to a network of 11 proteins tested for a role in granule polarization, there were 12 orphan proteins with no known interactions. The orphan protein with the highest score in the mass spectrometry analysis was cytohesin 4. The four members of the cytohesin family carry a PH domain and a Sec7 domain with guanine exchange factor activity for members of the small GTPase Arf family. While cytohesin 1 plays a role in inside-out signaling for eta2 integrin LFA-1, the function of cytohesin 4 is unknown. Silencing of cytohesin 4 but not cytohesin 1 resulted in defective granule polarization. To our knowledge, this is the first time a function has been attributed to cytohesin 4.
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