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

Abstract

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) ␣ and ␥ 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 PI5KI␣-and PI5KI␥-silenced cells. Our findings delineate a novel mechanism implicating PI5KI␣ and PI5KI␥ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca 2؉ IntroductionNatural killer (NK) cells and cytotoxic T lymphocytes are critical effectors in the defense against tumor and viral infections 1 ; they exert cytotoxic function through the polarized secretion of granules containing proteolytic molecules, such as perforin and granzymes. This process involves several steps, including the formation of a cytolytic synapse between cytolytic effector and target cell, the rapid reorientation of the microtubule-organizing center along with lytic granules toward the target contact area followed by granule docking and fusion at specialized secretory domains within the cytolytic synapse. 2,3 Several structurally distinct receptors have been implicated in the activation of NK-cell cytolytic machinery: when cross-linked by the corresponding ligands on target cell, they trigger multiple and intersecting signaling pathways responsible for functional activation. 4 A vast array of activating NK receptors belonging to different families are coupled to the lipid modifying enzymes phosphatidylinositol3-kinase (PI3K) and phospholipase C␥ (PLC␥), which provide signals critically required for the activation of the cytolytic machinery [5][6][7][8] ; notably, both enzymes use the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) as common substrate. 9 Besides its role as signaling intermediate, PIP2 also acts as critical regulator of various cellular processes, including actin remodeling, membrane and vesicle trafficking, adhesion, and ion transport. 10,11 Surprisingly, the role of PIP2 and its regulatory mechanisms in lymphocyte-mediated cytotoxicity remain completely undefined.The main cellular source of PIP2 are type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) family members which phosphorylate PI4P on the D5 position of the inositol ring. Three major PI5KI is...

Phosphatidylinositol 4–Phosphate 5–Kinase α and Vav1 Mutual Cooperation in CD28-Mediated Actin Remodeling and Signaling Functions

“…In different cell types and in response to several receptors, PIP5Ka promotes actin assembly and membrane ruffles (18,19), receptor-mediated phagocytosis, and endocytosis (20,21) as well as lytic granule exocytosis and retrieval (22,66). In T lymphocytes, the spatiotemporal analysis of both PIP2 distribution and turnover evidenced that PIP2 concentrates at the IS very early during Ag recognition (67) and that PIP2 synthesis occurs at the T-APC interface, where PIP5Ka accumulates in a sustained fashion (17).…”

“…Primary T cells express all three PIP5K isoforms, which are differentially enriched to the immunological synapse (IS) during T cell activation (17). PIP5Ka, for instance, is localized at the plasma membrane, where it promotes several actin-based processes (18)(19)(20)(21)(22). Despite much progress that has been made in elucidating the dynamic of PIP2 and PIP3 turnover in T lymphocytes, the mechanisms and surface receptors coupling PIP2 and PIP3 to cortical actin remodeling remain poorly understood.…”

Elucidation of the integrin LFA-1–mediated signaling pathway of actin polarization in natural killer cells

“…Moreover, interaction of a human NK cell with a sensitive, but not resistant target, results in the production and then consumption of cellular PIP 2 , and quenching of PIP 2 in these cells by competitive inhibition results in impaired cytotoxicity. 9 PIP 2 and PIPKI␥ are known to regulate cytoskeletal dynamics in adherent cells. Talin recruits PIPKI␥ to focal adhesions, where it is phosphorylated and activated by focal adhesion kinase, which both increases local production of PIP 2 and strengthens the association Figure 6.…”

“…Moreover, disruption of this PIP 2 enrichment impairs cytotoxicity. 9 The importance of the actin cytoskeleton in immune cell function has been well documented. 10 The formation of branched filaments by the actin nucleating Arp2/3 complex is crucial for immune synapse formation.…”

PIP2-dependent regulation of Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway

“…13 Primary cultured NK-cell populations were obtained as described previously. 14 NK cells were infected with the recombinant plasmid pWPT-GFP-PH or with shRNA-encoding DNA sequences targeting PIP5K␣, PIP5K␤, and PIP5K␥ as described previously 13,15 Cell microscopy To detect PIP2 distribution in membrane raft microdomains, green fluorescent protein-Pleckstrin homology (GFP-PH)-expressing live NK cells were stained with Alexa Fluor 594-conjugated CTxB (40 g/mL) for 40 minutes at 4°C and spun on microscope slides. Images were acquired at room temperature with a laser confocal inverted microscope (TSC-SP2; Leica) with Plan APO 40ϫ numerical aperture 1.25 oil objective.…”