The Hsp90 Chaperone Regulates p38 Activation During ER Stress

Abstract

Diseases such as diabetes, Alzheimer’s disease, and Parkinson’s disease can be characterized by the presence of endoplasmic reticulum stress. The endoplasmic reticulum is an organelle that plays a major role in protein synthesis, but an accumulation of misfolded proteins within the ER lumen causes the organelle to release a stress signal through the unfolded protein response (UPR). Despite being present in many disorders, there are alternative signaling routes within the UPR branches that are not fully understood. Perk dimerization initiates one of three main signaling cascades in the UPR by phosphorylating eIF2α and causing a cell‐wide pause in mRNA translation; however, a study has shown that outside of the established pathway, activation of Perk results in p38 activation, which induces apoptosis during prolonged ER stress. The mechanism responsible for p38 activation is still unknown. One route for elucidating the regulation of p38 activation during ER stress is the Hsp90 chaperone. The Hsp90 chaperone complex sequesters p38 in the cytoplasm and prevents its auto‐activation under normal conditions, so we hypothesized that Perk activation may mediate post‐translational modification of the Hsp90 complex in a manner that would release p38, allowing it to activate. In this study, Tunicamycin‐induced ER stress and Hsp90 inhibition were applied to BHK21 fibroblasts, a cell line in which ER stress has been well characterized, and dissociation of the p38‐Hsp90 complex was measured through immunoblot and co‐immunoprecipitation analysis. Initial immunoblot results from 50nM and 200nM of tunicamycin reconfirm that moderate doses of tunicamycin induced p38 activation in a dose dependent manner. Co‐immunoprecipitation of Hsp90 reconfirmed that tunicamycin‐induced ER stress causes p38 to dissociate from Hsp90. Previous experiments with co‐immunoprecipitation of p38 by Jingyi Chen revealed that treatment with 50nM of tunicamycin resulted in 0.3 decrease in fold induction after 15 minutes. Unexpectedly, we found that inhibition of Hsp90 causes it to exhibit increased binding of p38 with an peak average fold increase of 7 after treatment with 5uM of Hsp90‐I (17‐AAG) for 30 minutes, which suggests Hsp90 as a potential regulator for p38. Because p38 plays an important role in the induction of many intracellular signaling pathways, learning ways to regulate its actions within the context of ER stress may be beneficial in mediating symptoms of disease.