Requirement of the p38 mitogen-activated protein kinase signalling pathway for the induction of the 78 kDa glucose-regulated protein/immunoglobulin heavy-chain binding protein by azetidine stress: activating transcription factor 6 as a target for stress-induced phosphorylation

Malfolded protein formation and perturbance of calcium homoeostasis results in the induction of the endoplasmic reticulum (ER) chaperone protein, namely the 78 kDa glucose-regulated protein (GRP78)/immunoglobulin heavy-chain binding protein. Various ER stress inducers can activate grp78, but signal transduction mechanisms are not well understood. We report in the present study that the induction of endogenous grp78 mRNA by the amino acid analogue azetidine (AzC) requires the integrity of a signal transduction pathway mediated by p38 mitogen-activated protein kinase (p38 MAPK). In contrast, induction of grp78 by thapsigargin that depletes the ER calcium storage can occur even when the p38 MAPK pathway is blocked. Treatment of cells with AzC results in the sustained activation of p38 MAPK. We identified an ER transmembrane activating transcription factor 6 (ATF6) as a target of p38 MAPK phosphorylation in AzC-treated cells. ATF6 undergoes proteolytic cleavage on AzC treatment, releasing a nuclear form that is an activator of the grp78 promoter. We show here that constitutively active mitogen-activated protein kinase kinase 6, a selective p38 MAPK activator, enhances the ability of the nuclear form of ATF6 to transactivate the grp78 promoter. Our results provide direct evidence that different ER stress inducers use diverse pathways to activate grp78 and that in addition to activation by proteolytic cleavage, ATF6 undergoes specific ER stress-induced phosphorylation. We propose that phosphorylation of ATF6 is a novel mechanism for augmenting its potential as a transcription activator.