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

Phosphatidylinositol transfer proteins (PI-TPs) consist of two isoforms (PI-TPalpha and PI-TPbeta), 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-TPalpha; Ser165 in PI-TPbeta). Only PI-TPbeta contains a major phosphorylation site at Ser262, which must be phosphorylated for PI-TPbeta 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-TPalpha die soon after birth and that embryonic stems cells lacking PI-TPbeta 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-TPalpha is present in all tissues investigated, with highest levels in brain (167 ng/100 microg total protein). The levels of PI-TPbeta are 50-100 times lower than those of PI-TPalpha, with relatively high levels found in liver and brain (1.2 and 1.8 ng/100 microg of total protein, respectively). In contrast to NIH3T3 cells overexpressing PI-TPalpha, cells overexpressing PI-TPbeta (SPIbeta 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-TPbeta being associated with the Golgi as cells overexpressing a mutant PI-TPbeta in which the major phosphorylation site is replaced (PI-TPbeta(S262A) behave as wild-type NIH3T3 cells. Since the SPIbeta 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 SPIbeta cells, but not the cells overexpressing PI-TPbeta(S262A), are very sensitive. We are currently investigating whether a relationship exists between PI-TPbeta being involved in maintaining plasma membrane sphingomyelin levels and the enhanced sensitivity towards apoptosis.