ym-201636 and phosphatidylinositol-5-phosphate

Neutrophils rapidly arrive at an infection site because of their unparalleled chemotactic ability, after which they unleash numerous attacks on pathogens through degranulation and reactive oxygen species (ROS) production, as well as by phagocytosis, which sequesters pathogens within phagosomes. Phagosomes then fuse with lysosomes and granules to kill the enclosed pathogens. A complex signaling network composed of kinases, GTPases, and lipids, such as phosphoinositides, helps to coordinate all of these processes. There are seven species of phosphoinositides that are interconverted by lipid kinases and phosphatases. PIKfyve is a lipid kinase that generates phosphatidylinositol-3,5-bisphosphate and, directly or indirectly, phosphatidylinositol-5-phosphate [PtdIns(5)P]. PIKfyve inactivation causes massive lysosome swelling, disrupts membrane recycling, and, in macrophages, blocks phagosome maturation. In this study, we explored for the first time, to our knowledge, the role of PIKfyve in human and mouse neutrophils. We show that PIKfyve inhibition in neutrophils does not affect granule morphology or degranulation, but it causes LAMP1

Topics: Aminopyridines; Animals; Cell Degranulation; Cells, Cultured; Chemotaxis; GTP Phosphohydrolases; Heterocyclic Compounds, 3-Ring; Humans; Hydrazones; Lysosomal-Associated Membrane Protein 1; Lysosomes; Membrane Fusion; Mice; Mice, Inbred Strains; Morpholines; Neutrophils; Phagocytosis; Phagosomes; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Reactive Oxygen Species; Transient Receptor Potential Channels; Triazines

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

Topics: 3T3-L1 Cells; Aminopyridines; Animals; CHO Cells; Cricetinae; DNA-Binding Proteins; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Insulin; Mice; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; RNA-Binding Proteins