Caspase Processing and Nuclear Export of CTP:Phosphocholine Cytidylyltransferase α during Farnesol-Induced Apoptosis

Abstract

CTP:phosphocholine cytidylyltransferase alpha (CCTα) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP-choline pathway, the primary route for synthesis of phosphatidylcholine (PtdCho) in eukaryotic cells. Induction of apoptosis by farnesol (FOH) and other cytotoxic drugs has been shown to alter PtdCho synthesis via the CDP-choline pathway. Here we report that FOH-induced apoptosis in CHO cells caused a dose-dependent activation of CCTα and inhibition of the final step in the pathway, resulting in a biphasic effect on PtdCho synthesis. Activation of CCTα was accompanied by enzyme translocation to the nuclear envelope within 30 min of FOH addition to cells. Following translocation to membranes, CCTα was exported from the nucleus and underwent caspase-mediated proteolysis that coincided with poly(ADP-ribose) polymerase cleavage. Site-directed mutagenesis and in vivo and in vitro expression studies mapped a caspase 6 and/or 8 cleavage site to TEED^28↓G, the final residue in the CCTα nuclear localization signal. Nuclear export of CCTα appeared to be an active process in FOH-treated CHO cells that was independent of caspase removal of the nuclear localization signal. Caspase cleavage of CCTα occurred during UV or chelerythrine-induced apoptosis; however, nuclear membrane translocation and nuclear export were not evident under these conditions. Thus, caspase cleavage of CCTα was a late feature of several apoptotic programs that occurred in the nucleus or at the nuclear envelope. Activation and nuclear export of CCTα were early events in FOH-induced apoptosis that contributed to altered PtdCho synthesis and, in conjunction with caspase cleavage, excluded CCTα from the nucleus.

We thank Robert Zwicker and Gladys Keddy for maintaining and culturing cells.

This work was supported by a Canadian Institutes of Health grant to N.D.R. and Cancer Care Nova Scotia trainee awards to T.A.L. and J.R.M.