PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

Abstract

Although membrane phospholipid phos-phatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) α and γ isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphos- phate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5Kls, we found that lytic granule secretion but not polarization resulted in impaired PI5KIα-and PI5Klγ-silenced cells. Our findings delineate a novel mechanism implicating PI5Klα and PI5Kγ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca2+ response and lytic granule release. © 2008 by The American Society of Hematology.