Related Experiment Videos
GRP94 hyperglycosylation and phosphorylation in Sf21 cells
1Program in Molecular and Cellular Cardiology, Wayne State University School of Medicine, 421 East Canfield Avenue, Rm 1107, Detroit, MI, USA. s.cala@wayne.edu
Biochimica Et Biophysica Acta
|April 20, 2000
Summary
Glucose-regulated protein 94 (GRP94) is phosphorylated by protein kinase CK2 in intact cells, specifically on CK2-sensitive sites. This phosphorylation occurs constitutively and is linked to GRP94
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein.
- GRP94 acts as a protein chaperone and calcium (Ca2+) regulator.
- Previous studies indicated GRP94 as a substrate for protein kinase CK2 in vitro, but its phosphorylation in intact cells was unknown.
Purpose of the Study:
- To investigate the phosphorylation of GRP94 in intact cells.
- To determine the role of protein kinase CK2 in GRP94 phosphorylation.
- To elucidate the cellular localization and modification of GRP94.
Main Methods:
- Overexpression of canine GRP94 in Sf21 insect cells.
- Treatment with tunicamycin to analyze protein modifications.
- Metabolic labeling with 32P(i) to detect phosphorylation.
- Phosphopeptide mapping to identify phosphorylation sites.
Main Results:
- Overexpressed GRP94 appeared as multiple molecular-mass isoforms in Sf21 cells.
- Tunicamycin treatment reduced GRP94 isoforms to a single form, indicating post-translational modifications.
- Constitutive phosphorylation of GRP94 was observed, occurring specifically on CK2-sensitive sites.
- Only the lowest mobility GRP94 isoform was phosphorylated in intact cells, contrasting with in vitro findings.
Conclusions:
- Protein kinase CK2 plays a role in the cell biology of GRP94.
- Phosphorylation of GRP94 by CK2 occurs in intact cells, specifically on CK2-sensitive sites.
- These findings suggest a role for CK2 in regulating ER/SR resident proteins.