Phosphorylation and localization of CTP:phosphocholine cytidylyltransferase in mammalian cells
Abstract
In mammalian cells the rate of phosphatidylcholine biosynthesis is often regulated by the activity of CTP:phosphocholine cytidylyltransferase. Evidence obtained in vitro suggests that cytidylyltransferase activity is regulated both by its relative distribution between soluble and particulate fractions and by its phosphorylation state. To better understand the mechanism of activation of cytidylyltransferase the role of reversible phosphorylation in the regulation of cytidylyltransferase activity was examined. Furthermore, the localization of activated cytidylyltransferase in intact cells was determined with indirect immunofluorescence. A polyclonal antibody to rat liver cytidylyltransferase was produced in chickens and characterized. This antibody quantitatively precipitated cytidylyltransferase activity from rat liver and HeLa cell cytosol and was used to demonstrate the phosphorylation of cytidylyltransferase in HeLa cells in vivo. Cytidylyltransferase was phosphorylated on serine residues only. Activation of the CDP-choline pathway by phorbol ester treatment of HeLa cells resulted in a 2-fold increase in cytidylyltransferase activity in vitro and an enhancement of the V$\sb{\rm max}$ of choline transport. The increased cytidylyltransferase activity was not accompanied by a change in the phosphorylation state of the enzyme as demonstrated by ($\sp{32}$P) phosphopeptide mapping. Furthermore, no correlation between the phosphorylation state of cytidylyltransferase and its distribution between particulate and soluble fractions was observed. The cAMP levels of freshly isolated rat hepatocytes were elevated by treatment with cholera toxin. In contrast to previous studies, no effect on the CDP-choline pathway, cytidylyltransferase activity, or the phosphorylation state of cytidylyltransferase was detected. However, when the hepatocytes were maintained in culture for 24 hours, as in the previous studies, a modest decrease in ($\sp3$H) choline incorporation into phosphatidylcholine was observed. Polyclonal antibodies raised against synthetic peptides corresponding to the amino- and carboxy-terminal regions of cytidylyltransferase were produced and characterized. Use of these antibodies for indirect immunofluorescence in Chinese hamster ovary (CHO) cells suggested that cytidylyltransferase might be associated with the endoplasmic reticulum. However, following activation of cytidylyltransferase by phospholipase C treatment the fluorescent staining pattern was consistent with cytidylyltransferase being associated with the nuclear envelope.
Degree
Ph.D.
Advisors
Kent, Purdue University.
Subject Area
Biochemistry|Cellular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.