AN INVESTIGATION OF PHOSPHO-REGULATION AND THE ROLE CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE α IN PROMOTING RAS-INDUCED MALIGNANT TRANSFORMATION OF INTESTINAL EPITHELIAL CELLS
McPhee, Michael Jordan
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CTP:phosphocholine cytidylyltransferase alpha (CCTα) catalyzes the rate-limiting step of PC synthesis via the CDP-choline pathway. This study investigated CCTα regulation by phosphorylation using phospho-specific antibodies raised against p-S315/S319 and p-Y359/S362. Both antibodies detected endogenous CCTα and CCTβ in human cells, but Y359/S362 did not detect overexpressed CCTα or CCTβ. Oleate/BSA treatments induced dephosphorylation of CCTα at S315/S319 but not Y359/S362 in multiple human and rat cell lines. This correlated with translocation of CCTα to the inner nuclear membrane (INM) and decreased nuclear p-S315/S319 signal. Choline depletion of H-ras-transformed rat intestinal epithelial cells (IEC-ras) also induced dephosphorylation of S315/S319. This study is the first to use phospho-specific antibodies for CCTα to monitor dephosphorylation in response to enzyme activation. The mechanism of CCTα promoting IEC-ras survival was also investigated, suggesting that autophagy, PERK-eIF2α-ATF4-CHOP, and Keap1-Nrf2 pathways are not induced by inhibiting PC synthesis in IEC-ras. How CCTα promotes malignancy remains unknown.