Functional analysis of phosphatidylinositol transfer protein in vivo. (360G-Wellcome-080880_B_06_Z)

The hydrolysis of PI(4,5)P2 by phospholipase C isoenzymes is a conserved signalling mechanism used by animal cells to regulate a range of core cellular responses such as cell division, differentiation, changes in shape and migration. Despite the widespread use of PI(4,5)P2 as a signalling substrate, its levels in animal cells are stable and demonstrable changes during normal cell signalling are at best minimal. In order to tightly regulate PI(4,5)P2 levels at cellular membranes, it is essential for cells to closely match the rate of PI(4,5)P2 resynthesis to its consumption by PLC activity(Rana and Hokin, 1990; Rhee and Choi, 1992). A major mechanism that contributes to PI(4,5)P2 resynthesis is the sequential phosphorylation of phosphatidylinositol (PI) by PI and PIP kinases. In order for this mechanism to operate optimally, an adequate supply of PI needs to be available at cellular membranes for the sequential phosphorylations required to generate PI(4,5)P2. PI is synthesized in the endoplasmic reticulum (ER) by PI synthase and needs to be transferred from the ER to membranes where PI(4,5)P2 resynthesis occurs. One class of proteins that can perform this function are PITP (Wirtz, 1991). However their ability to transfer PI between membranes in vivo and its contribution to supporting PI(4,5)P2 resynthesis (Cunningham et al., 1995; Hardie et al., 2001; Kauffmann-Zeh et al., 1995) remains unknown. The proposed research will test the requirement of phosphatidylinositol (PI) transfer activity for the in vivo function of PITP in supporting PI(4,5)P2 resynthesis.