Regulation of Programmed Cell Death and Lipid Droplet Formation by CTP:Phosphocholine Cytidylyltransferase Alpha (CCTα)

Abstract

Phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes, is synthesized by the CDP-choline pathway under the control of the rate-limiting nuclear enzyme CTP:phosphocholine cytidylyltransferase (CCT)α. Increased production of PtdCho is often observed in tumours and during the formation of lipid droplets (LDs). To understand how CCTα regulates PtdCho synthesis during LD formation and apoptosis, we examined its activity, cellular localization, phosphorylation and association with membranes. Using caspase 3-replete MCF7 cells, we showed that CCTα is cleaved by caspase 3 and released from the nucleus during apoptosis. However, inhibition of high-affinity choline transport was the mechanism for inhibition of PtdCho synthesis during apoptosis in MCF7 cells and during anoikis in intestinal epithelial cells (IEC-18). During detachment of anoikis-resistant Ras-transformed IEC-18 (IEC-Ras) from an extracellular matrix (ECM), CCTα activity and PtdCho synthesis were increased, with no change in enzyme localization. PtdCho synthesis is also required for LD biogenesis which involves targeting CCTα to the phospholipid surface. CCTα-GFP was previously shown to localize to the surface of expanding LDs in Drosophila S2 cells, but localization of endogenous CCTα in mammalian cells was not reported. In oleate-treated cells and differentiating 3T3-L1 preadipocytes, endogenous and V5-tagged CCTα localized to the nuclear envelope and cytoplasm, whereas CCTα-GFP localized to the surface of LDs in IEC-18. Silencing CCTα resulted in larger LDs and sensitized IEC-18 to oleate-induced toxicity, indicating an important role for cytoplasmic and nuclear CCTα in LD formation. I conclude that CCTα activity is regulated through changes in nuclear and cytosolic distribution that control PtdCho synthesis during apoptosis and LD formation.