Activation of p38 mitogen-activated protein kinase and mitochondrial Ca2+-mediated oxidative stress are essential for the enhanced expression of grp78 induced by the protein phosphatase inhibitors okadaic acid and calyculin A

We have reported that treatment with okadaic acid, a potent protein phospha tase inhibitor, has the ability to enhance the synthesis of the 78-kDa gluc ose-regulated protein (GRP78). This article reports our investigation of an other protein phosphatase inhibitor, calyculin A, demonstrating the signali ng pathways elicited by the protein phosphatase inhibitors that lead to the induction of grp78. Our data showed that the induction process is abolishe d by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Phosphorylation-activation of p38(MAPK) in the treated cells was indicated by its own phosphorylation, as shown by double Western blotti ng analyses and directly confirmed by the in vitro kinase assay using MAPK- activated protein kinase-2, a well-known downstream effector of p38(MAPK), as a substrate. The involvement of p38(MAPK) in this process is further sub stantiated by using transient transfection assays with a plasmid, pGRP78-Lu c, which contains a 0.72-kbp stretch of the grp78 promoter. By exploiting t he same transfection assay, we demonstrated that the up-regulation of the g rp78 promoter by the protein phosphatase inhibitors is suppressed in the pr esence of the cytoplasmic calcium chelator bis(aminophenoxy)ethane N,N'-tet raacetic acid, the mitochondria calcium uniporter inhibitor ruthenium red a s well as the antioxidants N-acetyl cysteine and pyrrolidinedithiocarbamate . Taken together, our results lead us to conclude that treatment with the p rotein phosphatase inhibitors would activate the signaling pathways involvi ng p38(MAPK) and mitochondrial calcium-mediated oxidative stress and that t hese pathways must act in concert in order to confer the induction of grp78 by okadaic acid and calyculin A. (C) 2000 Wiley-Liss, inc.