Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo. Lack of effect of phorbol ester treatment in HeLa cells.
The production and characterization of an antibody to rat liver CTP:phosphocholine cytidylyltransferase is described. This antibody quantitatively precipitated cytidylyltransferase from both rat liver and HeLa cell cytosol. Following affinity purification, the antibody was used to demonstrate, for the first time, the phosphorylation of cytidylyltransferase in vivo. Following the immunoprecipitation of cytidylyltransferase from HeLa cells, acid hydrolysis, and thin layer electrophoresis of the amino acids, only [32P]phosphoserine was detected. The phosphorylation state of cytidylyltransferase in HeLa cells was examined following treatment with phorbol ester for 1 h. In agreement with previous studies, the incorporation of [3H]choline into phosphatidylcholine via the CDP-choline pathway was stimulated 5-fold in cultures of HeLa cells following treatment with phorbol ester for 1 h. However, no appreciable translocation of cytidylyltransferase was detected, despite the utilization of two different methods of cell lysis. Furthermore, the inclusion of phosphatase inhibitors and chelators of divalent cations in the homogenization buffers had no effect on the observed distribution or activity of the enzyme. Immunoprecipitated cytidylyltransferase was phosphorylated to the same extent, and on serine residues only, in both control and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated cells. Measurement of the pool sizes of the aqueous intermediates of the CDP-choline pathway, following TPA treatment, revealed a modest decrease in the phosphocholine pool only, consistent with an activation of cytidylyltransferase.
Watkins
J D
JD
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Kent
C
C
D000818:Animals; D000906:Antibodies; D002463:Cell Membrane Permeability; D019916:Choline-Phosphate Cytidylyltransferase; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004072:Digitonin; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000906:Antibodies; D010768:Phosphoserine; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate