Identification of phosphorylation sites in rat liver CTP: phosphocholine cytidylyltransferase.
CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine biosynthesis. The enzyme exists as a soluble, inactive form that is highly phosphorylated; activation of the enzyme is accompanied by dephosphorylation and translocation to the membrane. We have used a recombinant baculovirus clone to obtain CT labeled in vivo with 32PO4. The tryptic phosphopeptide pattern of the baculovirus-expressed CT was the same as for CT expressed in mammalian cells, indicating that insect cells modify the same phosphorylation sites as do mammalian cells. 32PO4-labeled, baculovirus-expressed CT was digested with trypsin and the peptides purified by reversed-phase high performance liquid chromatography. Phosphoamino acid analysis of the complete protein as well as individual peptides revealed that only serine residues were phosphorylated. Sequence analysis of purified radioactive peptides revealed that phosphorylation of CT was confined to the carboxyl-terminal region and that all or nearly all Ser residues from Ser315 to the carboxyl terminus were labeled. Ser315, Ser319, Ser329, Ser323, Ser331, Ser343, and Ser347 all reside in potential sites for proline-directed kinases. Two other phosphorylated serine residues, Ser315 and Ser333, are found within protein kinase C consensus phosphorylation sites. Ser321, Ser322, Ser333, Ser345, Ser346, Ser350, Ser352, and Ser362 were also found to be phosphorylated. Serine362 resides within a putative casein kinase II phosphorylation site, and there are five potential sites for phosphorylation by glycogen synthase kinase 3. Identification of these sites will allow investigations that focus on the establishment of the physiological function of phosphorylation at each site.
MacDonald
J I
JI
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Kent
C
C
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D016466:CHO Cells; D002478:Cells, Cultured; D019916:Choline-Phosphate Cytidylyltransferase; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007313:Insecta; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats
D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase