Dephosphorylation of CTP-phosphocholine cytidylyltransferase is not required for binding to membranes.
The sequence of the reversible phosphorylation and activation of CTP:phosphocholine cytidylyltransferase was investigated. Treatment of primary rat hepatocytes with oleic acid or phospholipase C caused a significant increase in the activity and amount of particulate cytidylyltransferase which correlated with decreased cytidylyltransferase activity and protein in the cytosol. The increase in membrane-associated cytidylyltransferase is accompanied by a decrease in the phosphorylation of the enzyme. Reversal of membrane association resulted in an increased amount of phosphorylated cytidylyltransferase in the cytosol. We wished to determine if dephosphorylation of the enzyme were a prerequisite for its translocation from the cytosol to the membranes. In vitro studies with membranes from oleic acid- or phospholipase C-treated cells showed that phosphorylated cytosolic cytidylyltransferase associated with these membranes with negligible dephosphorylation. Incubation of hepatocytes with oleic acid for different periods of time demonstrated that cytidylyltransferase associated with membranes in an active, phosphorylated form and was subsequently dephosphorylated. This result was supported by comparison of phosphopeptide maps of 32P-labeled cytidylyltransferase obtained from cytosolic, as well as membrane fractions of control, oleic acid-treated, or phospholipase C-treated cells. These studies revealed dephosphorylation on some sites and phosphorylation on other sites. Our data strengthen the hypothesis that a change in the lipid composition of membranes can mediate the initial binding of cytidylyltransferase to the membrane and that subsequently the enzyme becomes dephosphorylated.
Houweling
M
M
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Jamil
H
H
Hatch
G M
GM
Vance
D E
DE
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019916:Choline-Phosphate Cytidylyltransferase; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D008297:Male; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000494:pharmacology
D009829:Oleic Acids; D010748:Phosphopeptides; D019301:Oleic Acid; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D010738:Type C Phospholipases