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

ST6Gal-I (alpha2,6-sialyltransferase) is expressed as two isoforms, STTyr and STCys, which exhibit differences in catalytic activity, trafficking through the secretory pathway, and proteolytic processing and secretion. We have found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and Thr residues. Immunoprecipitation of 35S- and 32P-labeled proteins expressed in COS-1 cells suggests that the STTyr isoform is phosphorylated to a greater extent than the STCys isoform. Analysis of domain deletion mutants revealed that STTyr is phosphorylated on stem and catalytic domain amino acids, whereas STCys is phosphorylated on catalytic domain amino acids. An endoplasmic reticulum retained/retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type STTyr isoform. This suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Golgi. Treatment of cells with the ionophore monensin does not significantly block phosphorylation of the STTyr isoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence of kinase activities in the cis-medial Golgi and the substantial phosphorylation of the luminal sequences of a glycosyltransferase.