STIMULATION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE BY FREE-CHOLESTEROL LOADING OF MACROPHAGES INVOLVES SIGNALING THROUGH PROTEIN DEPHOSPHORYLATION

Free cholesterol-loaded macrophages in atheromata synthesize excess ph osphatidylcholine (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 bio synthesis and to the post-translational activation of the membrane-bou nd form of CTP:phosphocholine cytidylyltransferase (CT), a key enzyme in PC biosynthesis, Herein, we explore further the mechanism of CT act ivation 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 an d FC appear to be mostly in different intracellular sites, These data imply that FC activates membrane-bound CT by a signaling mechanism, Th at the proposed signaling mechanism involves structural changes in the CT protein was suggested by data showing that two different antibodie s against synthetic CT peptides showed increased recognition of membra ne-bound CT from FC-loaded cells despite no increase in CT protein. Si nce CT is phosphorylated, two-dimensional maps of peptides from P-32-l abeled control and FC-loaded macrophages were compared: six peptide sp ots from membrane-bound CT, but none from soluble CT, were dephosphory lated in the FC-loaded cells, Furthermore, incubation of FC-loaded mac rophages with the phosphatase inhibitor, calyculin A, blocked increase s in both PC biosynthesis and antipeptide-antibody recognition of CT, Last, treatment of membranes from control macrophages with lambda phag e protein phosphatase in vitro increased both CT activity (Q-fold) and antipeptide-antibody recognition of CT; soluble CT activity and antib ody recognition were not substantially affected by phosphatase treatme nt, In summary, FC loading of macrophages leads to the partial dephosp horylation 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 atheroma ta.