Differential dephosphorylation of CTP:phosphocholine cytidylyltransferase upon translocation to nuclear membranes and lipid droplets.

CTP:phosphocholine cytidylyltransferase-alpha (CCT alpha) and CCT beta catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCT alpha is activated by association of its alpha-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCT alpha translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCT alpha from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCT alpha translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCT alpha-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCT alpha-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCT alpha recruitment to nuclear membranes.