Lipid Activation of CTP:Phosphocholine Cytidylyltransferase α: Characterization and Identification of a Second Activation Domain

Abstract

The CTP:phosphocholine cytidylyltransferase (CCT) governs the rate of phosphatidylcholine (PtdCho) biosynthesis, and its activity is governed by interaction with membrane lipids. The carboxy-terminus was dissected to delineate the minimum sequences required for lipid responsiveness. The helical domain is recognized as a site of lipid interaction, and all three tandem α-helical repeats from residues 257 through 290 were found to be required for regulation of enzymatic activity by this domain. Truncation of the carboxy-terminus to remove one or more of the α-helical repeats yielded catalytically compromised proteins that were not responsive to lipids but retained sufficient activity to accelerate PtdCho biosynthesis when overexpressed in vivo. The role of the helical region in lipid-activation was tested further by excising residues 257 through 309 to yield a protein that retained a 57-residue carboxy terminal domain fused to the catalytic core. This construct tested the hypothesis that the helical region inhibits activity in the absence of lipid rather than activates the enzyme in the presence of lipid. This hypothesis predicts constitutive activity for CCTα[Δ257−309]; however, this protein was tightly regulated by lipid with activities comparable to the full-length CCTα, in both the absence and presence of lipid. Activation of CCTα[Δ257−309] was dependent exclusively on anionic lipids, whereas full-length CCTα responded to either anionic or neutral lipids. Phosphatidic acid delivered in Triton X-100 micelles was the preferred activator of the second lipid-activation domain. These data demonstrate that CCTα can be regulated by lipids by two independent domains: (i) the three amphipathic α-helical repeats that interact with both neutral and anionic lipid mixtures and (ii) the last 57 residues that interact with anionic lipids. The results show that both domains are inhibitory in the absence of lipid and activating in the presence of lipid. Removal of both domains results in a nonresponsive, dysregulated enzyme with reduced activity. The data also demonstrate for the first time that the 57-residue carboxy-terminal domain in CCTα participates in lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.