Phosphatidylinositol 5-phosphate biosynthesis is linked to PIKfyve and is involved in osmotic response pathway in mammalian cells.
The cellular functions, regulation and enzymology of phosphatidylinositol (PtdIns) 5-P, the newest addition to the family of phosphoinositides (PI), are still elusive. Whereas a kinase that uses PtdIns-5-P as an intracellular substrate has been assigned, a kinase that produces it remained to be identified. Here we report that PIKfyve, the enzyme found to synthesize PtdIns-5-P in vitro and PtdIns-3,5-P(2) in vitro and in vivo, is responsible for PtdIns-5-P production in a cellular context. Evidence is based on examination of two groups of cell types by two independent approaches. First, [(32)P]orthophosphate-labeled cells (Sf9, 3T3-L1 fibroblasts, and 3T3-L1 adipocytes) that show a high pressure liquid chromatography (HPLC)-detectable peak of the PtdIns-5-P head group at basal conditions demonstrated a 20-50% increase in radioactive PtdIns-5-P amounts upon expression of PIKfyve(WT). Second, cell types (HEK293), in which the basal levels of radioactive PtdIns-5-P were undetectable by HPLC head group analysis, demonstrated higher in vitro type II PIP kinase-directed conversion of the endogenous PtdIns-5-P pool into PtdIns-4,5-P(2), when induced to express PIKfyve(WT). Conversely, a decrease by 60% in the conversion of PtdIns-5-P to PtdIns-4,5-P(2) was associated with induced expression of the dominant-negative kinase-deficient PIKfyve(K1831E) mutant in HEK293 cells. When 3T3-L1 fibroblasts and 3T3-L1 adipocytes were subjected to osmotic shock, levels of PtdIns-5-P measured by both approaches were found to decrease profoundly upon a hypo-osmotic stimulus. Together, these results identify PIKfyve as an enzyme responsible for PtdIns-5-P biosynthesis and indicate a role for PtdIns-5-P in osmotic response pathways in mammalian cells.
Sbrissa
Diego
D
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Ikonomov
Ognian C
OC
Deeb
Robert
R
Shisheva
Assia
A
D016475:3T3 Cells; D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D007313:Insecta; D051379:Mice; D009995:Osmosis; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D013997:Time Factors; D014162:Transfection
D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; C113567:phosphatidylinositol 5-phosphate; D019869:Phosphatidylinositol 3-Kinases; C492139:PIKFYVE protein, human; C116930:Pikfyve protein, mouse