Expression of Wild-Type and Mutant Rat Liver CTP: Phosphocholine Cytidylyltransferase in a Cytidylyltransferase-Deficient Chinese Hamster Ovary Cell Line

Abstract

The strain 58 Chinese hamster ovary (CHO) mutant defective in CTP:phosphocholine cytidylyltransferase was characterized as an expression system for exogenous cytidylyltransferase. Strain 58 cells express less than 5% of the wild-type level of cytidylyltransferase protein at the permissive temperature even though the steady-state messenger RNA levels were found to be similar to those in the parental CHO-K1 cell line. A point mutation from arginine to histidine at amino acid 140 was identified in the strain 58 protein. Rat liver cytidylyltransferase was stably expressed in strain 58 cells and shown to be active, targeted to the nucleus, phosphorylated, and activated by methylethanolamine supplementation or phospholipase C treatment. Thus, the mechanisms by which cytidylyltransferase is processed and regulated in CHO-K1 cells are intact in strain 58 cells. The heterologously expressed protein complemented the strain 58 defects in both temperature-sensitive growth and phosphatidylcholine biosynthesis, consistent with a single lesion in the structural gene for cytidylyltransferase being responsible for both phenomena. Overexpression of cytidylyltransferase activity at levels up to eightfold higher than those in CHO-K1 cells did not appreciably affect phosphatidylcholine metabolism. A putative casein kinase II phosphorylation site was altered by site-directed mutagenesis and expressed in the strain 58 cells. Alteration of this site did not affect expression and regulation of cytidylyltransferase activity.