Sialyltransferase isoforms are phosphorylated in the cis-medial Golgi on serine and threonine residues in their luminal sequences

ST6Gal-I (alpha 2,6-sialyltransferase) is expressed as two isoforms, STTyr and STCys, which exhibit differences in catalytic activity, trafficking thr ough the secretory pathway, and proteolytic processing and secretion. We ha ve found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and T hr residues. Immunoprecipitation of S-35- and P-32-labeled proteins express ed in COS-1 cells suggests that the STTyr isoform is phosphorylated to a gr eater extent than the STTyr isoform, Analysis of domain deletion mutants re vealed that STTyr is phosphorylated on stem and catalytic domain amino acid s, whereas STTyr is phosphorylated on catalytic domain amino acids, An endo plasmic reticulum retained/retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type STTyr isoform, T his suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Gels, Treatment of cells with the ionophore monensin does not significa ntly block phosphorylation of the STTyr isoform, suggesting that phosphoryl ation is occurring in the cis-medial Golgi prior to the monensin block. Thi s study demonstrates the presence of kinase activities in the cia-medial Ge ls and the substantial phosphorylation of the luminal sequences of a glycos yltransferase.