Presentation Title

Stress-induced activation of cytosolic p38 MAP kinase via release from the Hsp90 chaperone

Faculty Mentor

Jay Brewster

Start Date

17-11-2018 8:30 AM

End Date

17-11-2018 10:30 AM

Location

CREVELING 92

Session

POSTER 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

The endoplasmic reticulum (ER) is a cellular organelle for lipid biosynthesis, calcium storage, and the folding of proteins. Disruptions that change ER homeostasis lead to accumulation of unfolded proteins (UPs). Cells can turn on signaling pathway-the unfolded protein response (UPR) to respond to stress. PKR-like ER kinase (PERK) is a signaling activator of UPR, and it localized to MAM domains. PERK phosphorylates elF2a, which then stops the general cellular translation and turn on pro-apoptotic gene translation. One of the downstream pathways of ER stress is the activation of p38, which plays a critical role in regulating cell differentiation, proliferation, metabolism, and apoptosis. HSP90 is a highly conserved cytosolic chaperone that binds to many proteins including inactive p38. Hsp90 was found to suppress the autophosphorylation of p38, and that Hsp90 regulates p38 pathway. Prior work in our lab has shown during moderate ER stress caused by tunicamycin (Tm), PERK signaling activates apoptosis via p38. ER stress can induce a dissociation of Hsp90 from p38, and PERK inhibition can reduce dissociation. In this study, the interaction between p38 and Hsp90 was examined. Hamster kidney fibroblasts (BHK21 cells) were transfected with FLAG-DNp38.17-AAG, an inhibitor of Hsp90, was applied to cells for 10 minutes, 20 minutes and 30 minutes. Cells are gently lysis with 0.25% CHAPs. Invitrogen dynabeads protein G were probed with Hsp90 antibody or p38 antibody. Co-immunoprecipitation was performed, and standard immunoblot technique was used to analyze cell lysates. Both beads-Hsp90 Abs and beads-p38 Abs co-IP experiments support that 17-AAG inhibits Hsp90, and p38 is released when cells are exposed to 17-AAG. However, the time-dependent manner still remains unclear.

Summary of research results to be presented

Co-immunoprecipitation experiments of Hsp90 and p38 indicate that 17-AAG inhibits Hsp90, and p38 is released from Hsp90 when cells are exposed to 17-AAG. The time-dependent manner of Hsp90 and p38 association still remains unclear.

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Nov 17th, 8:30 AM Nov 17th, 10:30 AM

Stress-induced activation of cytosolic p38 MAP kinase via release from the Hsp90 chaperone

CREVELING 92

The endoplasmic reticulum (ER) is a cellular organelle for lipid biosynthesis, calcium storage, and the folding of proteins. Disruptions that change ER homeostasis lead to accumulation of unfolded proteins (UPs). Cells can turn on signaling pathway-the unfolded protein response (UPR) to respond to stress. PKR-like ER kinase (PERK) is a signaling activator of UPR, and it localized to MAM domains. PERK phosphorylates elF2a, which then stops the general cellular translation and turn on pro-apoptotic gene translation. One of the downstream pathways of ER stress is the activation of p38, which plays a critical role in regulating cell differentiation, proliferation, metabolism, and apoptosis. HSP90 is a highly conserved cytosolic chaperone that binds to many proteins including inactive p38. Hsp90 was found to suppress the autophosphorylation of p38, and that Hsp90 regulates p38 pathway. Prior work in our lab has shown during moderate ER stress caused by tunicamycin (Tm), PERK signaling activates apoptosis via p38. ER stress can induce a dissociation of Hsp90 from p38, and PERK inhibition can reduce dissociation. In this study, the interaction between p38 and Hsp90 was examined. Hamster kidney fibroblasts (BHK21 cells) were transfected with FLAG-DNp38.17-AAG, an inhibitor of Hsp90, was applied to cells for 10 minutes, 20 minutes and 30 minutes. Cells are gently lysis with 0.25% CHAPs. Invitrogen dynabeads protein G were probed with Hsp90 antibody or p38 antibody. Co-immunoprecipitation was performed, and standard immunoblot technique was used to analyze cell lysates. Both beads-Hsp90 Abs and beads-p38 Abs co-IP experiments support that 17-AAG inhibits Hsp90, and p38 is released when cells are exposed to 17-AAG. However, the time-dependent manner still remains unclear.