Oxysterols inhibit phosphatidylcholine synthesis via ERK docking and phosphorylation of CTP:phosphocholine cytidylyltransferase

Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCT alpha). Phosphorylation of CCT alpha decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCT alpha phosphorylation. Expression of truncated CCT alpha mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser(315) within CCT alpha was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCT alpha membrane-binding domain (residues 287-300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCT alpha that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.