Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

Abstract

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein ␣ (PI-TP␣), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TP␣. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2-and 3-fold increase, respectively, in the level of PI-TP␣. Overexpression of PI-TP␣ resulted in a decrease in the duration of the cell cycle from 21 h for the wild type Upon equilibrium labeling of the cells with myo-[ 3 H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TP␣ to a total lysate of myo-[3 H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca 2؉ further increased this formation. Based on these observations, we propose that PI-TP␣ is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (plateletderived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.Phospholipid transfer proteins are proteins that are able to transfer phospholipids between membranes in vitro. A major phospholipid transfer protein in mammalian tissues is the phosphatidylinositol transfer protein (PI-TP) 1 (1). Recently, two isoforms of PI-TP have been identified (i.e. PI-TP␣ and PI-TP␤) that demonstrate differences in cellular localization and in specific lipid transfer activity (2-5).PI-TP␣ has been purified from both rat and bovine brain (6, 7). Cloning of the cDNA encoding rat brain PI-TP␣ showed that the protein consists of 271 amino acid residues (8). The subsequent isolation of the cDNAs encoding mouse and human PI-TP␣ revealed a high homology between the different mammalian PI-TPs (about 99% amino acid sequence identity) (9, 10). Furthermore, the cross-reactivity of the antibodies raised against bovine PI-TP␣ with a 35-kDa protein from other animals (e.g. rat, mouse, chicken, frog, and lizard) indicates an extensive conservation of the amino acid sequence between species (11). An exception is PI-TP from yeast (i.e. SEC14p) that has the same molecular weight as mammalian PI-TP and comparable phospholipid transfer activities yet shows no homology in the amino acid sequence (12)(13)(14).So far, very little is known about the precise cellular role of mammalian PI-TP␣. Since PI-TP␣ is able to transfer in vitro PtdIns between membrane...

Phosphatidylinositol Transfer Protein Expression Altered by Aging and Parkinson Disease

“…These proteins are involved in phospholipids degradation, phospholipids transport, as well as in dynamic cytoskeleton changes and in function of Golgi system (Wirtz, 1991(Wirtz, , 1997Snoek et al, 1999). It seems that the decrease of PI-TP protein level may be one of the important factors that lead to disturbing of phospholipids signaling and also may be involved in neurodegeneration of CNS.…”

“…Recent studies have shown that the cellular functions of PI-TP are not only connected with phospholipid transfer per se from its site of synthesis in the endoplasmic reticulum to the other membranes but also with other important cell functions as vesicle transport, dynamics of cytoskeleton proteins, and PI metabolism (Thomas et al, 1993b;Liscovitch and Cantley, 1995;Monaco et al, 1998;Snoek et al, 1999). PI-TP is an essential for agonist stimulated synthesis of polyphosphoinositide (Thomas et al, 1993b;Kauffmann-Zeh et al, 1995;Kearns et al, 1998).…”

“…The high affinity of PI-TP for PIP and PIP 2 impose that these lipids remain bound to PI-TP after their formation. The PI-TP may also deliver these lipids as a substrates for phospholipase C (PLC) and phospholipase A 2 (PLA 2 ) (Thomas et al, 1993b;Kauffmann-Zeh et al, 1995;Snoek et al, 1999). A remarkable evidence indicates that membrane traffic events are connected with the activity of PI3 -kinase and with the formation of different class of lipids as phosphatidylinositol (3) phosphate PI(3)P, PI(3,4)P 2 , and PI(3,4)P 3 (Kular et al, 1997;Panaretou et al, 1997).…”

Phosphatidylinositol transfer proteins: From closed for transport to open for exchange

“…As yet, it is not at all clear how PI-TPa interacts with PI 4-kinase for PI to become available for phosphorylation or with phospholipase A 2 for PI to become hydrolyzed Snoek et al, 1999). In the open structure of PI-TPa the hydroxyl groups at positions 3, 4 and 5 of the phosphorylinositol moiety are located in front of the hydrophilic hole of the lipid-binding channel (Schouten et al, 2002).…”

“…Both isoforms act as carriers of PI and phosphatidylcholine (PC) between membranes in vitro, yet PI-TPb can also transfer sphingomyelin (SPM) (Van Paridon et al, 1987b;De Vries et al, 1995). At this point it is still questionable whether these proteins actually transfer these phospholipids between membranes within cells or whether they interact directly with enzymes like PI 4-kinases and PI-specific phospholipase A 2 thereby providing these enzymes with substrates Snoek et al, 1999). In analogy with observations in yeast it has also been proposed that by binding either PI, PC or SPM, PI-TPs act as regulators of phospholipid metabolism at distinct sites in the cell thereby fulfilling the roles of biosensors (McGee et al, 1994;Bankaitis, 2002).…”