Role of protein glycosylation in the cAMP-mediated induction of alkaline phosphatase in mouse L-cells.

As illustrated in the preceding paper (Firestone, G. L., and Heath, E. C. (1981) J. Biol. Chem. 256, 13961403), the de novo synthesis of a cell surface-localized glycoprotein that exhibits alkaline phosphatase activity was induced by treatment of mouse L-cell cultures with dibutyryl cyclic AMP. When the exposure of L-cell cultures to dibutyryl cyclic AMP was carried out in the presence of inhibitors of protein glycosylation, the induction of alkaline phosphatase activity was markedly inhibited. Thus, when 2-deoxyglucose (2 m ~ ) or tunicamycin (25 pg/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 molecular weight, 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 cyclic AMP and 2-deoxyglucose indicated that functional alkaline phosphatase mRNA is produced in quantities proportional to that produced in cells treated only with dibutyryl cyclic AMP. 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