Role of protein glycosylation in the cAMP-mediated induction of alkaline phosphatase in mouse L-cells
Firestone, G.L.; Heath, E.C.
Journal of Biological Chemistry 256(3): 1404-1411
1981
ISSN/ISBN: 0021-9258
PMID: 6161133
Accession: 068628632
The de novo synthesis of a cell surface-localized glycoprotein exhibiting alkaline phosphatase activity was induced by treatment of mouse [fibroblast] L-cell cultures with dibutyryl cAMP. When the exposure of L-cell cultures to dibutyryl cAMP was carried out in the presence of inhibitors of protein glycosylation, the induction of alkaline phosphatase activity was markedly inhibited. When 2-deoxyglucose (2 mM) or tunicamycin (25 .mu.g/ml) was included in the induction medium, the incorporation of either [6-3H]glucosamine or [2-3H]mannose was inhibited greater than 90% and no detectable increase in alkaline phosphatase activity was observed. These inhibitors of protein glycosylation exhibit little or no effect on total protein synthesis under these conditions. Immunotitration of extracts of inhibited cells indicated that exposure to the protein glycosylation inhibitors resulted in a suppression of the net synthesis of alkaline phosphatase. Inhibition of the appearance of immunologically reactive protein is proportional to the suppression of induction of alkaline phosphatase activity. Analysis by gel electrophoresis of antialkaline phosphatase immunoadsorbed material from L-cells incubated with [35S]methionine substantiated that the net production of alkaline phosphatase protein was suppressed when protein glycosylation was inhibited. A lower MW, nonglycosylated form of alkaline phosphatase was not detected under these conditions. In vitro translation of L-cell mRNA isolated from cells simultaneously exposed to dibutyryl cAMP and 2-deoxyglucose indicated that functional alkaline phosphatase mRNA is produced in quantitites proportional to that produced in cells treated only with dibutyryl cAMP. Also, inhibition of protein glycosylation did not affect the amount of RNA associated with the rough endoplasmic reticulum or the levels of de novo synthesized rough endoplasmic reticulum-associated protein. In vitro studies indicated that the nonglycosylated form of alkaline phosphatase, isolated from cell-free translation mixtures, is intrinsically more sensitive to endogenous rough endoplasmic reticulum-associated proteolytic enzymes than native glycosylated alkaline phosphatase isolated from dibutyryl cAMP-treated cells. These results suggest that the presence of carbohydrate in native alkaline phosphatase renders the enzyme less sensitive to proteolytic degradation. Apparently the normal incorporation of the carbohydrate constituents of alkaline phosphatase is essential for the induction of this enzyme by dibutyryl cAMP and/or for its normal intercalation into the plasma membrane of cultured L-cells.