Secretion, Glycosylation, and Phosphorylation of Rat-specific, Transformation- associated Proteins in Moloney Murine Sarcoma Virus-transformed Rat Cells1

Previously, we detected, by monoclonal antibody, three Moloney mu rine sarcoma virus (Mo-MSV)-activated intraccllular transformationassociated proteins (TAP), (P66, P63, and P60) in several Mo-MSVtransformed rat cell lines (Gian et al., Biochem. Biophys. Res. Commun., 134:1223-1230, 1986) and found the release of TAP into the extracel lular medium with a change from three intraccllular polypeptides to two extracellular polypeptides (P68 and P64) (Li et al., Virology, 156: 91100, 1987). Since then, we have further analyzed TAP in terms of their secretion, glycosylation, and phosphorylation in a temperature-sensitive Mo-MSV-transformed normal rat kidney (NRK) cell line, the 6M2 Une, and found these results. Extracellular TAP were detected by imrnunoblotting and immunoprecipitation techniques as two polypeptides (P68 and P64). The secretion of TAP was rapid, with a 50% secretion rate of 78 min. Both intracellular (except for P63) and extracellular TAP were glycosylated. As a result of inhibition of glycosylation by the antibiotic tunicamycin, a fourth intracellular TAP (P58) and a third extracellular TAP (P60) were found. The new results suggest that the intracellular TAP were probably changed from four polypeptides (P66, P63, P60, and PS8) to three (P66, P63, and P60) during the glycosylation process. Likewise, the three extracellular TAP were changed to two (P68 and P64) as a result of further glycosylation and subsequent secretion into the extracellular medium. Inhibition of glycosylation by tunicamycin (0.5 Mg/ml) reduced the TAP secretion rate by about 39%. Extracellular TAP (P68 and P64) as well as P85«— and P58« were found to be phospho-