title: Sustained elevation in inositol 1,4,5-trisphosphate results in inhibition of phosphatidylinositol transfer protein activity and chronic depletion of the agonist-sensitive phosphoinositide pool.
abstract: The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) in a signal-terminating reaction. We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P(3) can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P(3), in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P(2). The depletion in cellular PtdIns(4,5)P(2) was confined to a Triton-insoluble cell compartment, enriched in caveolin. In resting cells with elevated Ins(1,4,5)P(3) concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P(2) by 61% in the caveolin-enriched Triton-insoluble compartment. Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolin-enriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P(3) concentrations. Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities. Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P(3) concentrations; however, no reduction in PITP(&agr;) protein expression was detected. These studies indicate that chronic elevation in cellular Ins(1,4,5)P(3) concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolin-enriched agonist-sensitive pool.
authors.affiliation: Monash University Department of Biochemistry and Molecular Biology, Clayton, 3168 Melbourne, Australia.
authors.forename: C J
authors.initials: CJ
authors.lastname: Speed
authors.forename: C A
authors.initials: CA
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D015870:Gene Expression / Q000235:genetics; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D000072181:Inositol Polyphosphate 5-Phosphatases; D008565:Membrane Proteins / Q000032:analysis; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000235:genetics; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014162:Transfection / Q000235:genetics
chemical_list: D002352:Carrier Proteins; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D015544:Inositol 1,4,5-Trisphosphate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases