Abstract
Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)α is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. In this study we utilized siRNA silencing of caspases in HEK293 cells and caspase 3-deficient MCF7 cells to show that caspase 3 is required for CCTα proteolysis and release from the nucleus during apoptosis. CCTα-Δ28 (a caspase-cleaved mimic) expressed in CCTα-deficient Chinese hamster ovary cells was cytosolic and had increased in vitro activity. However, [³H]choline labeling experiments in camptothecin-treated MCF7 cells and MCF7 cells expressing caspase 3 (MCF7-C3) revealed a global suppression of the CDP-choline pathway that was consistent with inhibition of a step prior to CCTα. In camptothecin-treated MCF7 and MCF7-C3 cells, choline kinase activity was unaffected; however, choline transport into cells was reduced by 30 and 60%, respectively. We conclude that caspase 3-mediated removal of the CCTα NLS contributes minimally to the inhibition of PtdCho synthesis during DNA damage-induced apoptosis. Rather, the CDP-choline pathway is inhibited by caspase 3-independent and -dependent suppression of choline transport into cells.
Highlights
Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors
To address the possibility that individual knockdown of caspases 6 and 8 did not eliminate CTP:phosphocholine cytidylyltransferase (CCT)␣ processing due to redundancy, both caspases were simultaneously suppressed by RNA interference (RNAi), and CCT␣ processing was determined after induction of apoptosis in HEK293 cells for 4 h (Fig. 2E)
Caspase processing of CCT␣ and inhibition of choline/ethanolamine phosphotransferase (CEPT)/choline phosphotransferase (CPT) are implicated in the cessation of PtdCho synthesis but evidence is indirect and, based on radiolabeling experiments, the substrates for these enzymes (CDP-choline and phosphocholine) do not accumulate in apoptotic cells [28–30]
Summary
Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)␣ is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. [3H]choline labeling experiments in camptothecin-treated MCF7 cells and MCF7 cells expressing caspase 3 (MCF7-C3) revealed a global suppression of the CDP-choline pathway that was consistent with inhibition of a step prior to CCT␣. We conclude that caspase 3-mediated removal of the CCT␣ NLS contributes minimally to the inhibition of PtdCho synthesis during DNA damage-induced apoptosis. The CDP-choline pathway is inhibited by caspase 3-independent and -dependent suppression of choline transport into cells.—Morton, C. A mechanism for suppression of the CDP-choline pathway during apoptosis.
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