GRP94 hyperglycosylation and phosphorylation in Sf21 cells

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca2+ regulator. GRP94 has been reported to be a substrate for protein kinase CK2, in vitro, although its phosphorylation in intact cells remains unreported. in SP21 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 P-32(i) led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the CRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated. even though in vitro phosphorylation of GRP93 by CK2 led to phosphorylation of all glycosylated forms. The P-32(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. (C) 2000 Elsevier Science B.V. All rights reserved.