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| - Lipid Activation of CTP:Phosphocholine Cytidylyltransferase α: Characterizationand Identification of a Second Activation Domain
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| - 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 helicaldomain is recognized as a site of lipid interaction, and all three tandem α-helical repeats from residues257 through 290 were found to be required for regulation of enzymatic activity by this domain. Truncationof the carboxy-terminus to remove one or more of the α-helical repeats yielded catalytically compromisedproteins that were not responsive to lipids but retained sufficient activity to accelerate PtdCho biosynthesiswhen overexpressed in vivo. The role of the helical region in lipid-activation was tested further by excisingresidues 257 through 309 to yield a protein that retained a 57-residue carboxy terminal domain fusedto the catalytic core. This construct tested the hypothesis that the helical region inhibits activity in theabsence of lipid rather than activates the enzyme in the presence of lipid. This hypothesis predictsconstitutive activity for CCTα[Δ257−309]; however, this protein was tightly regulated by lipid withactivities comparable to the full-length CCTα, in both the absence and presence of lipid. Activation ofCCTα[Δ257−309] was dependent exclusively on anionic lipids, whereas full-length CCTα responded toeither anionic or neutral lipids. Phosphatidic acid delivered in Triton X-100 micelles was the preferredactivator of the second lipid-activation domain. These data demonstrate that CCTα can be regulated bylipids by two independent domains: (i) the three amphipathic α-helical repeats that interact with bothneutral and anionic lipid mixtures and (ii) the last 57 residues that interact with anionic lipids. The resultsshow 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. Thedata also demonstrate for the first time that the 57-residue carboxy-terminal domain in CCTα participatesin lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.
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