Okadaic acid as an inducer of the 78-kDa glucose-regulated protein in 9L rat brain tumor cells.
Okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, has been widely used as a tool for unravelling the regulation of cellular metabolic processes involving protein phosphorylation/dephosphorylation. It has recently been found that OA can induce reversible hyperphosphorylation of vimentin and reorganization of intermediate filaments [Lee et al., J. Cell. Biochem. 49: 378-393, 1992]. We report here that OA specifically induced the synthesis of a 78-kDa protein, which was identified as the 78-kDa glucose-regulated protein (GRP78) by two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis and peptide mapping. The induction of GRP78 by OA was dose-dependent and reversible. For 7 h treatments, GRP78 synthesis was initially enhanced under 50 nM OA and became the highest (about 6-fold) under 200 nM OA. Meanwhile, under 200 nM OA, GRP78 synthesis was initially enhanced after 4 h and reached its maximal level (about 8-fold) after 15 h of treatment. Subsequently, upon removal of OA, the level of OA-induced GRP78 was reduced to basal level after 12 h of recovery. Induction of GRP78 synthesis by OA was abolished in cells pretreated with actinomycin D and cycloheximide, indicating that it was regulated at the transcriptional level and its induction required de novo protein synthesis. Furthermore, OA suppressed protein glycosylation, and the result lent support to the hypothesis that suppression of protein glycosylation may correlate with induction of GRP78 synthesis.
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
M C
MC
Hou
C H
CH
Shen
W C
WC
Lee
Y K
YK
Lai
D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004988:Ethers, Cyclic / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005910:Glioma / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D004988:Ethers, Cyclic; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D003609:Dactinomycin; D019319:Okadaic Acid; D000001:Calcimycin; D003513:Cycloheximide; D010749:Phosphoprotein Phosphatases