GRP94 hyperglycosylation and phosphorylation in Sf21 cells

• Published in: Biochimica et biophysica acta 2000-04, Vol.1496 (2), p.296-310
• Main Author: Cala, Steven E
• Format: Article
• Language: English
• Subjects: Animals; Baculoviridae - genetics; Binding Sites; Casein Kinase II; Cell Line; CK2; Dogs; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Gene Expression; Glycosylation; GRP94; HSP70 Heat-Shock Proteins - chemistry; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; Insecta; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Chaperones - chemistry; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Phosphorus Radioisotopes; Phosphorylation; Protein Biosynthesis; Protein Isoforms - genetics; Protein-Serine-Threonine Kinases - metabolism; Sarcoplasmic Reticulum - metabolism; Time Factors; Transfection
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/S0167-4889(00)00028-8

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca 2+ regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with 32P i led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The 32P i incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.