Effects of altered phosphorylation sites on the properties of CTP:phosphocholine cytidylyltransferase.

To investigate the role of phosphorylation and dephosphorylation in modulating the activity and location of CTP:phosphocholine cytidylyltransferase, we used site-directed mutagenesis to construct four mutant forms of cytidylyltransferase. These forms were 5SP-->AP, in which five of the seven Ser-Pro sequences were converted to Ala-Pro; 7SP-->AP, in which all of the seven Ser-Pro sequences converted to Ala-Pro; 16S-->A, in which all sixteen Ser residues that can be phosphorylated in wild type cytidylyltransferase were converted to Ala; and 16S-->E, in which all sixteen Ser residues were converted to Glu. The mutant enzymes were expressed in the strain 58 Chinese hamster ovary cell line, which is temperature-sensitive for growth and cytidylyltransferase activity. All mutant enzyme forms were enzymatically as active as the wild type when assayed under optimal conditions. In untreated cells, more of the Ser-->Ala mutants were membrane-associated than in cells expressing wild type enzyme, consistent with the phosphorylation state of the enzyme affecting its affinity for membranes. About half of the 16S-->A mutant remained soluble, however, indicating that dephosphorylation alone does not trigger membrane association. Although the amount of membrane-associated enzyme in the 16S-->A mutant was about 10-fold greater than that of wild type, phosphatidylcholine synthesis was increased by only about 75%, suggesting that membrane association does not necessarily cause full activation. All mutant forms, including the 16S-->E mutant, translocated to the particulate fraction upon oleate treatment, indicating that a high negative charge in the phosphorylation region does not preclude association of cytidylyltransferase with membranes. All mutant enzymes were able to support growth of strain 58 at 40 degrees C, and the rate of phosphatidylcholine synthesis was not greatly altered in the cell lines expressing mutant cytidylyltransferase forms. These results are consistent with a role for phosphorylation in the equilibrium distribution of cytidylyltransferase but suggest that changes in enzyme activity and location are not triggered exclusively by changes in the phosphorylation state.