Expression of rat liver CTP:phosphocholine cytidylyltransferase in a mutant Chinese hamster ovary cell line

Thomas D Sweitzer, Purdue University

Abstract

CTP:phosphocholine cytidylyltransferase catalyzes the major regulatory step in the biosynthesis of phosphatidylcholine in higher eukaryotes. Cytidylyltransferase was purified to homogeneity from rat liver and subjected to proteolytic digestion with trypsin. The resultant peptides were purified by reversed-phase chromatography, sequenced by Edman degradation, and used to design oligonucleotide probes. A cDNA encoding the Chinese hamster ovary enzyme was isolated using the polymerase chain reaction. A mutant Chinese hamster ovary cell line, strain 58, was characterized. Strain 58 cells have less than 5% of the cytidylyltransferase protein found in wild-type cells. The strain 58 cytidylyltransferase has a point mutation altering an arginine to a histidine at position 140 in the putative catalytic domain of the enzyme. The mutation apparently alters the stability and the activity of the encoded protein. Overexpression of rat liver cytidylyltransferase in strain 58 cells did not appreciably affect phosphatidylcholine metabolism. The cells apparently were able to modulate the activity of the excess cytidylyltransferase so that a dynamic homeostasis was maintained. A potential casein kinase II site at the carboxy terminus of the protein was altered by oligonucleotide-directed mutagenesis, and the altered proteins were expressed in strain 58 cells. Altered proteins were active, appropriately targeted to the nucleus, and translocated to the nuclear envelope upon activation. Alteration of the putative casein kinase II site changed the two-dimensional phosphopeptide map of expressed cytidylyltransferase in a complex fashion. Cells expressing the altered enzyme were not able to activate a phospholipase A activity when stimulated with phospholipase C.

Degree

Ph.D.

Advisors

Kent, Purdue University.

Subject Area

Biochemistry|Genetics

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