Overexpression of phosphatidylinositol transfer protein β in NIH3T3 cells has a stimulatory effect on sphingomyelin synthesis and apoptosis

• Published in: BBA - Molecular and Cell Biology of Lipids 2004-03, Vol.1636 (2), p.151-158
• Main Authors: van Tiel, Claudia M, Schenning, Martijn, Snoek, Gerry T, Wirtz, Karel W.A
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Carrier Proteins - chemistry; Carrier Proteins - physiology; Lipid Metabolism; Membrane Proteins - chemistry; Membrane Proteins - physiology; Mice; NIH 3T3 Cells; Phosphatidylinositol transfer protein; Phospholipase A 2 activation; Phospholipases A - metabolism; Phospholipid Transfer Proteins; Protein Isoforms - metabolism; Protein Kinase C - metabolism; Signal Transduction - physiology; Sphingomyelin metabolism; Sphingomyelins - chemical synthesis; Tissue distribution
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2003.08.009

Phosphatidylinositol transfer proteins (PI-TPs) consist of two isoforms (PI-TPα and PI-TPβ), which differ in phospholipid transfer properties and intracellular localization. Both PI-TP isoforms are substrates for protein kinase C and contain a minor phosphorylation site (Ser166 in PI-TPα; Ser165 in PI-TPβ). Only PI-TPβ contains a major phosphorylation site at Ser262, which must be phosphorylated for PI-TPβ to be associated with the Golgi. The PI-TP isoforms are completely conserved between mammals. Although their function is still not clear, their importance follows from knock-out studies, showing that mice lacking PI-TPα die soon after birth and that embryonic stems cells lacking PI-TPβ cannot be generated [Mol. Biol. Cell 13 (2002) 739]. We determined the levels of the PI-TP isoforms in various mouse tissues by immunoblotting. PI-TPα is present in all tissues investigated, with highest levels in brain (167 ng/100 μg total protein). The levels of PI-TPβ are 50–100 times lower than those of PI-TPα, with relatively high levels found in liver and brain (1.2 and 1.8 ng/100 μg of total protein, respectively). In contrast to NIH3T3 cells overexpressing PI-TPα, cells overexpressing PI-TPβ (SPIβ cells) were able to maintain steady-state levels of sphingomyelin in plasma membrane under conditions where this lipid is degraded by exogenous sphingomyelinase. This process of rapid sphingomyelin replenishment is dependent on PI-TPβ being associated with the Golgi as cells overexpressing a mutant PI-TPβ in which the major phosphorylation site is replaced (PI-TPβ(S262A) behave as wild-type NIH3T3 cells. Since the SPIβ cells display a decreased growth rate (35 h as compared to 21 h for wtNIH3T3 cells), we have investigated the sensitivity of these cells towards UV-induced apoptosis. We have found that the SPIβ cells, but not the cells overexpressing PI-TPβ(S262A), are very sensitive. We are currently investigating whether a relationship exists between PI-TPβ being involved in maintaining plasma membrane sphingomyelin levels and the enhanced sensitivity towards apoptosis.