Activation of p38 mitogen-activated protein kinase and mitochondrial Ca2+-mediated oxidative stress are essential for the enhanced expression ofgrp78 induced by the protein phosphatase inhibitors okadaic acid and calyculin A

Abstract

We have reported that treatment with okadaic acid, a potent protein phosphatase inhibitor, has the ability to enhance the synthesis of the 78-kDa glucose-regulated protein (GRP78). This article reports our investigation of another protein phosphatase inhibitor, calyculin A, demonstrating the signaling pathways elicited by the protein phosphatase inhibitors that lead to the induction of grp78. Our data showed that the induction process is abolished 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 blotting 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 substantiated by using transient transfection assays with a plasmid, pGRP78-Luc, which contains a 0.72-kbp stretch of the grp78 promoter. By exploiting the same transfection assay, we demonstrated that the up-regulation of the grp78 promoter by the protein phosphatase inhibitors is suppressed in the presence of the cytoplasmic calcium chelator bis(aminophenoxy)ethane N,N'-tetraacetic acid, the mitochondria calcium uniporter inhibitor ruthenium red as well as the antioxidants N-acetyl cysteine and pyrrolidinedithiocarbamate. Taken together, our results lead us to conclude that treatment with the protein phosphatase inhibitors would activate the signaling pathways involving p38(MAPK) and mitochondrial calcium-mediated oxidative stress and that these pathways must act in concert in order to confer the induction of grp78 by okadaic acid and calyculin A.

p38 mitogen‐activated protein kinase dephosphorylation is regulated by protein phosphatase 2A in human platelets activated by collagen

“…PP2A is known to inhibit p38 activity in vitro [17]. Similar experiments support such a role for PP2A in the rat tumor cell line 9L RBT and in both human skin fibroblasts and NIH 3T3 cells [31,32], where the use of OA similarly led to an increase of p38 activity. Together with these reports, our data suggest that there may be a widespread role for PP2A in terminating signaling through p38 MAPK.…”

Macrophage Apoptosis by Anthrax Lethal Factor Through p38 MAP Kinase Inhibition

“…BS somatic cells exhibit increases in chromosome aberrations, sister chromatid exchanges (SCEs), homologous chromatid interchanges, and micronuclei that are a consequence of chromosome breakage (1,15,16). Although the cytogenetic analysis of somatic cells from human BLM heterozygotes remains to be completed (17), spermatozoa from two of three obligate heterozygotes have been shown to display excess numbers of chromosome breaks and rearrangements (18).…”

“…Treatment of J774A.1 macrophage-like cells with the protein phosphatase inhibitor calyculin A was found to sensitize them to LT-induced apoptosis (12). Treatment of different cells with such phosphatase inhibitors activates numerous protein kinases, including MAPKs (15) and inhibitor of NF-B (IB) kinase (IKK) (16). Hence, calyculin A could activate J774A.1 macrophages.…”