Regulation of CTP:phosphocholine cytidylyltransferase activity and subcellular location by phosphorylation in Chinese hamster ovary cells. The effect of phospholipase C treatment.
The phosphorylation state of cytidylyltransferase in Chinese hamster ovary (CHO) cells was correlated with its subcellular distribution and activity in vivo. Western blot analysis of soluble and particulate fractions from control and phospholipase C-treated cells revealed slower migrating forms of cytidylyltransferase present only in the soluble fraction of control cells. These were abolished by incubating the soluble fraction at 37 degrees C in the presence of 5 mM Mg2+ but persisted if 135 mM NaF was present in the incubation. CHO cells were labeled with 32Pi, and cytidylyltransferase was immunoprecipitated from soluble and particulate fractions from control and phospholipase C-treated cells. The slower migrating forms of cytidylyltransferase, present in the soluble fraction of control cells, were phosphorylated at multiple sites. Although an equivalent amount of cytidylyltransferase was immunoprecipitated from the particulate fraction of phospholipase C-treated cells, it contained little 32P. Pretreatment of the CHO cells with okadaic acid, an inhibitor of type 1 and 2A phosphatases, prevented the stimulation of cytidylyltransferase in vivo by phospholipase C. These results demonstrate that dephosphorylation of soluble cytidylyltransferase is required for the phospholipase C-mediated translocation of cytidylyltransferase in CHO cells.
Watkins
J D
JD
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Kent
C
C
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002451:Cell Compartmentation / Q000187:drug effects; D019916:Choline-Phosphate Cytidylyltransferase; D006224:Cricetinae; D004789:Enzyme Activation; D004988:Ethers, Cyclic / Q000494:pharmacology; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019319:Okadaic Acid; D010449:Peptide Mapping; D010766:Phosphorylation; D010738:Type C Phospholipases / Q000494:pharmacology*
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D010738:Type C Phospholipases