PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway.
Although membrane phospholipid phosphatidylinositol-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) alpha and gamma 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-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KIalpha- and PI5KIgamma-silenced cells. Our findings delineate a novel mechanism implicating PI5KIalpha and PI5KIgamma isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca(2+) response and lytic granule release.
Micucci
Federica
F
Department of Experimental Medicine, Istituto Pasteur-Fondazione Cenci-Bolognetti, Rome, Italy.
Capuano
Cristina
C
Marchetti
Enzo
E
Piccoli
Mario
M
Frati
Luigi
L
Santoni
Angela
A
Galandrini
Ricciarda
R
D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004789:Enzyme Activation; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007694:Killer Cells, Natural / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D051966:Phospholipase C gamma