PHOSPHORYLATION OF ETHANOLAMINE, METHYLETHANOLAMINE, AND DIMETHYLETHANOLAMINE BG OVEREXPRESSED ETHANOLAMINE KINASE IN NIH 3T3 CELLS DECREASES THE CO-MITOGENIC EFFECTS OF ETHANOLAMINES AND PROMOTES CELL-SURVIVAL

Ethanolamine (Etn). as well as its N-methyl (MeEtn) and N,N-dimethyl ( Me(2)Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum-starved NIH 3T3 fibroblas ts. In the present work we assessed the impact of the co-mitogenic eff ects of Etn and its methyl analogues on cell proliferation and cell su rvival, and examined whether the cell growth regulatory effects of the se ethanolamines involve an Etn-kinase-mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Et n kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me(2)Etn, methylamine (MeNH2) and dimethylamine (Me2NH) were studied. P-31-NMR analysis of the water-soluble cell met abolites revealed that both MeEtn and Me(2)Etn, but not choline, are e xcellent substrates for the expressed Etn kinase. The methylated ethan olamines (MeEtn and Me(2)Etn) and methylamines (MeNH2, Me2NH) were use d as Etn models that can or cannot be phosphorylated, respectively. In serum-starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM ) were more effective than Etn in enhancing insulin-induced DNA synthe sis, and both were almost as effective as MeEtn and Me(2)Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn , MeEtn and Me(2)Etn, but not those of MeNH2 and Me2NH, were significa ntly reduced. Moreover, in the overexpressor cells, lower concentratio ns of Etn (50-200 mu M) inhibited the combined mitogenic effects of Me 2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non-phosphorylated ethanolamines are nega tive and positive regulators of insulin-induced mitogenesis, respectiv ely. After incubating the cells for 13 days in serum-free medium in 96 -well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6-13 days, 0.1-1 mM MeEtn and, particula rly, Me(2)Etn provided significant protection against cell death in th e Etn kinase overexpressor cells. In vector control cells, only Me(2)E tn in combination with insulin had similar effects on cell survival. T he data suggest that phosphorylated ethanolamines may function as prom oters of cell survival.