Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene

Abstract

Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.