Structure and characterization of the genes for murine choline/ethanolamine kinase isozymes a and b

Choline/ethanolamine kinase (CK/EK) is the first enzyme in phosphatidylcholine/phosphatidylethanolamine biosynthesis in all animal cells. The highly purified CKs from mammalian sources and their recombinant gene products so far were all shown to have EK activity also, indicating that both activities reside on the same protein. CK/EK in most animal cells exists as several isoforms, for two of which ( a and b ) their cDNAs have been cloned from both the rat and mouse, and they are found to be separate gene products. The physiological significance for the existence of more than one CK/EK enzyme, however, remains to be clarified. In this study, we isolated mouse genes encoding both types of CK/EK isozyme and determined their entire structure. The 5 9 -flanking promoter regions were found to have quite different features from each other, indicating that their expression could be under distinct control. Comparison of the nucleotide sequence between the corresponding coding exons showed the best homology (75%) residing on exon VIII. A search of the database resulted in the possible existence of 17 different origins of eukaryotic CK and/or EK, each of which presumably contained the entire amino acid sequence. Multialignment of their putative amino acid sequences led to an identification of the novel consensus sequence possibly required for the expression of either CK or EK activity, which corresponded to the sequence within exons VII and VIII of CK/EKa and b genes from the mouse. This sequence was localized in close proximity to the C-terminal region of the general (Brenner’s) phosphotransferase concensus sequence which was also completely conserved in all of the putative eukaryotic CK/EK proteins. The results demonstrated that, while both CK/EKa and b genes were composed of 11 major exons, the size of their genes was quite different: 40 kb for CK/EKa , whereas it was only 3.5 kb for CK/EKb . —Aoyama, C., N. Yamazaki, H. Terada, and K. Ishidate. Structure and characterization of the genes for murine choline/ethanolamine kinase isozymes a and b . J. Lipid Res. 2000. 41: 452– 464. Supplementary key words choline kinase • ethanolamine kinase • gene structure • isozymes (isoforms) • carnitine palmitoyltransferase I (M-CPTI) • phosphatidylcholine biosynthesis • phosphatidylethanolamine biosynthesis Choline kinase (CK) (ATP: choline phosphotransferase, EC 2.7.1.32) and ethanolamine kinase (EK) (ATP: ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyze the phosphorylation of choline/ethanolamine by ATP yielding phosphocholine/phosphoethanolamine. This step commits choline/ethanolamine to the CDP-choline/ CDP-ethanolamine pathway for the biosynthesis of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) in all animal cells. Although the CTP: phosphocholine (phosphoethanolamine) cytidylyltransferase, the second enzyme in the pathway, is commonly referred to as the rate-limiting step in PC (PE) biosynthesis (1, 2), considerable evidence indicates that CK is also a slow step and can be regulatory for PC biosynthesis in a number of situations where increased (decreased) CK activity results in a similarly increased (decreased) rate of PC biosynthesis (2, 3). CK has been purified to apparent homogeneity from rat kidney (4), liver (5), and brain (6) and the purified preparations were all shown to have significant EK activities. These as well as other (7, 8) investigations suggested also that CK/EK does not exist in one particular active form but exists in several isoforms in rat tissues. Uchida and Yamashita (9) were the first to report a cloning of the mammalian CK cDNA from rat liver cDNA library. The cloned cDNA had an ORF encoding a protein of 435 amino acids with a calculated molecular size of 50 kDa Abbreviations: CK, choline kinase; EK, ethanolamine kinase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ORF, open reading frame; M-CPTI, carnitine palmitoyltransferase I (muscle type); DIG, digoxigenin; 5 9 -RACE, rapid amplification of cDNA 5 9 -end; XRE, xenobiotic responsive element; PAH, polycyclic aromatic hydrocarbon; CC1 4 , carbon tetrachloride; pfu, plaque-forming unit. 1 The nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases with the Accession Numbers of AB030616 (exon I), AB030617 (exon II), AB030618 (exon II 9 ), AB030619 (exon III), AB030620 (exon IV–VIII), AB030621 (exon IX–XI) for CK/EKa and AB030615 (complete cds.) for CK/EKb . 2 To whom correspondence should be addressed. by gest, on O cber 0, 2017 w w w .j.org D ow nladed fom