Effects of altered phosphorylation sites on the properties of XXXX : @entity2945 cytidylyltransferase.

@entity20029 @entity73195 @entity93 @entity73193 @entity2434 @entity73194 @entity367 @entity2945 @entity3569 @entity52828
To investigate the role of phosphorylation and dephosphorylation in modulating the activity and location of @entity93 : @entity2945 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 @entity52828 ; @entity73193 , in which all of the seven Ser-Pro sequences converted to @entity52828 ; @entity73194 , in which all sixteen Ser residues that can be phosphorylated in wild type cytidylyltransferase were converted to Ala; and @entity73195 , in which all sixteen Ser residues were converted to @entity367 . The mutant enzymes were expressed in the strain 58 @entity3569 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 @entity20029 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 @entity73194 mutant remained soluble, however, indicating that dephosphorylation alone does not trigger membrane association. Although the amount of membrane-associated enzyme in the @entity73194 mutant was about 10-fold greater than that of wild type, @entity2434 synthesis was increased by only about 75%, suggesting that membrane association does not necessarily cause full activation. All mutant forms, including the @entity73195 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 @entity2434 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.
