eIF2α integrates proteotoxic signals both from ER and cytoplasm: Hsp70-Bag3 module regulates HRI-dependent phosphorylation of eIF2α

The major heat shock protein Hsp70 has been implicated in many stages of cancer development. These effects are mediated by a scaffold protein Bag3 that binds to Hsp70 and links it to components of multiple cancer-related signaling pathways. Accordingly, the Hsp70-Bag3 complex has been targeted by small molecules, which showed strong anti-cancer effects. Here, our initial question was how JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3, causes cell death. Breast epithelial cells MCF10A transformed with a single oncogene Her2 showed higher sensitivity to JG-98 then parental MCF10A cells. RNA expression analysis showed that this enhanced sensitivity correlated with higher induction of the UPR genes. Indeed, depletion of the pro-apoptotic UPR responsive transcription factor CHOP significantly protected cells from JG-98. Surprisingly, only the eIF2α-associated branch of the UPR was activated by JG-98, suggesting that the response was not related to the ER proteotoxicity. Indeed, it was dependent on activation of a distinct cytoplasmic eIF2α kinase HRI. HRI-dependent phosphorylation of eIF2α was also activated by the cytoplasmic proteotoxicity via Hsp70-Bag3 complex, which directly associates with HRI. Dissociation of Hsp70-Bag3 complex led to Bag3-dependent degradation of HRI via autophagy. Therefore, eIF2α integrates proteotoxicity signals from both ER and cytoplasm, and the cytoplasmic response mediates cytotoxicity of the Hsp70-Bag3 inhibitors.