Regulation of UDP-N-acetylglucosamine: dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase by retinoic acid in P19 cells

UDP-N-acetylglucosamine :dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase (GPT) is the first enzyme in the dolichol pathway of protein N -glycosylation, and is implicated in the developmental programmes of a vari ety of eukaryotes. In the present study we describe the effects of all-tran s-retinoic acid (RA) on the levels of GPT protein and enzymic activity, and on the transcription rate of the GPT gene, in mouse P19 teratocarcinoma ce lls. RA caused a dose-dependent and protein-synthesis-dependent induction o f enzyme activity. The maximum induction of GPT activity (about 3-fold) req uired 2 days of exposure to 1 mu M RA. Induced GPT activity also resulted i n an increase in the rate of incorporation of [H-3]mannose into Glc(3)Man(9 )GlcNAc(2). Enzymic activities paralleled GPT gene expression. The GPT gene was induced (2-fold) after 7 h of RA treatment. An approx. 3-fold increase in a 48 kDa GPT protein and approx. 4-fold increases in the levels of thre e GPT transcripts (1.8, 2.0 and 2.2 kb) were observed after 2 days of RA tr eatment. The enhanced levels of GPT protein and mRNAs began to decline 3 da ys after the initiation of differentiation, and GPT expression was down-reg ulated during cellular differentiation. GPT activity decreased about 2.8-fo ld to a constant level in differentiated P19 cells. The results indicate th at the RA-induced enzyme activity was mainly determined by increased transc ription of the GPT gene. RA-treated P19 cells were about 4-fold more resist ant to tunicamycin, a fungal antibiotic which inhibits GPT, than were contr ol cells. In addition, GPT activity in membranes from RA-treated P19 cells exhibited approx. 4-fold increased resistance to tunicamycin compared with activity in membranes from untreated control cells, demonstrating that resi stance to tunicamycin is correlated with induced GPT activity. Furthermore, increased GPT activity had regulatory significance with regard to the rate of incorporation of [H-3]mannose into Glc(3)Man(9)GlcNAc(2)-P-P-dolichol a nd into glycoproteins. Together, the data provide additional insights into the hormonal regulation of GPT and present evidence that the RA-mediated in duction of GPT has a regulatory impact on the dolichol pathway.