Calcium-dependent autophosphorylation of the glucose-regulated protein, Grp78.
The characteristics of phosphorylation of the 78-kDa glucose-regulated protein (Grp78), also known as the immunoglobulin heavy chain binding protein, were studied in vitro and in vivo. The purified protein from either calf liver or bovine kidney cells (MDBK) could be phosphorylated in vitro with [gamma-32P]ATP, in a reaction that is stimulated by Ca2+ and inhibited by the Ca(2+)-chelator ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). In the presence of EGTA, excess Ca2+ increased the rate of phosphorylation about 18-fold. Based on EGTA/Ca2+ titrations, the optimal Ca2+ concentration for phosphorylation was estimated to be 10-50 microM. Other divalent cations such as Mg2+, Mn2+, and Zn2+ were found to be inhibitory as was the Ca2+ antagonist lanthanum (La3+). The in vivo phosphorylation of Grp78 was studied in MDBK cells labeled with 32Pi. In the presence of inducers of Grp78 synthesis, such as ionomycin, tunicamycin, or 2-deoxyglucose, there was a large increase in the level of Grp78 in the cells but a decrease in the amount of phosphorylated protein. Two-dimensional gel analysis of Grp78 purified from bovine liver and MDBK cells identified at least four isoforms. After in vivo and in vitro phosphorylation of Grp78 all the acidic isoforms contained radioactivity but not the most basic isoform. Phosphoamino acid analysis of Grp78 showed that serine and threonine were phosphorylated in vivo and only threonine was phosphorylated in vitro.
Leustek
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Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Toledo
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H
Brot
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Weissbach
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H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D018832:Molecular Chaperones; D010766:Phosphorylation
D000596:Amino Acids; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; D002118:Calcium