Stimulation of CTP:phosphocholine cytidylyltransferase by free cholesterol loading of macrophages involves signaling through protein dephosphorylation

Free cholesterol-loaded macrophages in atheromata synthesize excess phosphatidylcholine (PC), which may be an important adaptive response to the excess free cholesterol (FC) load. We have recently shown that FC loading of macrophages leads to 2-4-fold increases in PC mass and biosynthesis and to the post-translational activation of the membrane-bound form of CTP:phosphocholine cytidylyltransferase (CT), a key enzyme in PC biosynthesis. Herein, we explore further the mechanism of CT activation in FC-loaded macrophages. First, enrichment of membranes from control macrophages with FC in vitro did not increase CT activity, and PC biosynthesis in vivo is up-regulated by FC loading even when CT and FC appear to be mostly in different intracellular sites. These data imply that FC activates membrane-bound CT by a signaling mechanism. That the proposed signaling mechanism involves structural changes in the CT protein was suggested by data showing that two different antibodies against synthetic CT peptides showed increased recognition of membrane-bound CT from FC-loaded cells despite no increase in CT protein. Since CT is phosphorylated, two-dimensional maps of peptides from 32P-labeled control and FC-loaded macrophages were compared: six peptide spots from membrane-bound CT, but none from soluble CT, were dephosphorylated in the FC-loaded cells. Furthermore, incubation of FC-loaded macrophages with the phosphatase inhibitor, calyculin A, blocked increases in both PC biosynthesis and antipeptide-antibody recognition of CT. Last, treatment of membranes from control macrophages with lambda phage protein phosphatase in vitro increased both CT activity (2-fold) and antipeptide-antibody recognition of CT; soluble CT activity and antibody recognition were not substantially affected by phosphatase treatment. In summary, FC loading of macrophages leads to the partial dephosphorylation of membrane-bound CT, and possibly other cellular proteins, which appears to be important in CT activation. This novel regulatory action of FC may allow macrophages to adapt to FC loading in atheromata.