Heat shock protein 90 modulates the unfolded protein response by stabilizing IRE1alpha

Abstract

The molecular chaperone HSP90 regulates stability and function of multiple protein kinases. The HSP90-binding drug geldanamycin interferes with this activity and promotes proteasome-dependent degradation of most HSP90 client proteins. Geldanamycin also binds to GRP94, the HSP90 paralog located in the endoplasmic reticulum (ER). Because two of three ER stress sensors are transmembrane kinases, namely IRE1alpha and PERK, we investigated whether HSP90 is necessary for the stability and function of these proteins. We found that HSP90 associates with the cytoplasmic domains of both kinases. Both geldanamycin and the HSP90-specific inhibitor, 514, led to the dissociation of HSP90 from the kinases and a concomitant turnover of newly synthesized and existing pools of these proteins, demonstrating that the continued association of HSP90 with the kinases was required to maintain their stability. Further, the previously reported ability of geldanamycin to stimulate ER stress-dependent transcription apparently depends on its interaction with GRP94, not HSP90, since geldanamycin but not 514 led to up-regulation of BiP. However, this effect is eventually superseded by HSP90-dependent destabilization of unfolded protein response signaling. These data establish a role for HSP90 in the cellular transcriptional response to ER stress and demonstrate that chaperone systems on both sides of the ER membrane serve to integrate this signal transduction cascade.

FIG. 1.

HSP90 associates with IRE1 but not with cytoplasmic domain-deleted IRE1. Cos7 cells were transfected with IRE1-FLAG or ΔIRE1-FLAG plasmids and lysed in TNESV after 24 h. Untransfected AR42J cells were treated or with GA or its derivative 514 (1 μM) of left untreated (lane 0) for 3 h, and then the cells were lysed similarly. Soluble proteins (400 μg) were immunoprecipitated with either 3 μg of anti-Flag antibody (A) or IRE1 antibody (B), and complexes were bound on protein A-Sepharose beads for 2 h at 4°C. The beads were washed four times with TNESV, bound proteins were resolved by SDS-10% PAGE and transferred to nitrocellulose membranes. Western analysis was performed with either anti-HSP90 or anti-IRE antibodies. Expression levels of transiently transfected wild-type IRE1 and ΔIRE1 are shown for comparison in panel A, and HSP90 and IRE1 steady-state levels in cell lysates are shown for comparison in panel B. HEK293 cells were transfected with IRE1-FLAG or ΔIRE1-FLAG plasmids. After 24 h, the cells were lysed and 1 mg of total proteins from each condition was subjected to GST-HSP90 binding in the presence of 400 μl of glutathione-Sepharose 4B, which had been previously saturated with GST-HSP90 (C). The binding of native IRE1 (in AR42J lysates) to either GST-HSP90 or GST alone is shown in panel D.

FIG. 2.

Geldanamycin destabilizes IRE1 but not cytoplasmic domain-deleted IRE1. Cos7 cells were transfected with full-length IRE1-FLAG or ΔIRE1-FLAG. Cos7 cells (A) or AR42J cells (expressing endogenous IRE1) (B) were incubated for 24 h with increasing concentrations of GA. (C) AR42J cells were exposed to 1 μM GA for the time periods shown. Cells were lysed, and equivalent amounts of protein were analyzed by SDS-PAGE and Western blotting with Flag antibody (A) or IRE1 antibody (B and C). In panels B and C, tubulin was blotted to demonstrate equal loading of protein.

FIG. 3.

GA decreases the half-life of newly synthesized IRE1. Transiently transfected Cos7 (A) and untransfected AR42J (B) cells were treated with GA (1 μM) 12 h after transfection or left untreated. After an additional 8 h, the medium was replaced with methionine/cysteine-free medium for 30 min, and cells were pulse-labeled with 100 μCi of [³⁵S]methionine/cysteine/ml for 60 min. At 1 h, cells were placed in complete, nonradioactive medium (time zero) and chased for the times shown. GA remained in all drug-treated samples for the duration of the experiment. Proteins were immunoprecipitated with either anti-Flag (A) or anti-IRE1 (B) antibodies. Samples were analyzed by SDS-PAGE and autoradiography. Band densities of scanned films were quantified using NIH Image software to create the graphs shown.

FIG. 4.

Both GA and 514, a GA derivative that has a 2-log-greater affinity for HSP90 than for GRP94, inhibit the UPR after prolonged exposure, but only GA induces the UPR after short exposure. (A) AR42J cells (which express high levels of IRE1) were exposed to either GA (1 μM) or 514 (1 or 10 μM) for 12 h. IRE1 and tubulin proteins were detected in whole-cell lysates by Western blotting. (B) AR42J cells were pretreated with either the GA derivative 514 or GA for 12 h before the stress-inducer TM was added (for an additional 4 h). BiP protein was detected by [³⁵S]methionine pulse-labeling for 30 min, and tubulin protein was detected by Western blotting. (C) AR42J cells were exposed to TM, GA, or 514 for 4 h and processed for BiP and tubulin protein detection as was done for panel A. (D) Cos7 cells were transfected with pCGN-ATF6, an ATF6 expression plasmid containing a HA epitope tag. Twenty hours after transfection, cells were either left untreated (lanes 1 and 7), treated with TM for 4 h (lane 2), pretreated with GA for 12 h, followed by TM for an additional 4 h (lane 3), pretreated with 514 for 12 h, followed by TM for 4 h (lane 4), treated with only GA for 12 h (lane 5), or treated with only 514 for 12 h (lane 6). In order to better detect the ATF6 cleavage product induced by ER stress, we treated all cells with the proteasome inhibitor PS-341 (0.5 μM) during exposure to the ER stress agent. In lanes 1, 5, 6, and 7, PS-341 was added for the final 4 h of incubation prior to lysis. Following cell lysis, equivalent amounts of soluble proteins were separated by SDS-PAGE and analyzed for stress-induced ATF6 cleavage by Western blotting using an anti-HA antibody.

FIG. 5.

ER stressors, including GA, dissociate IRE1 from BiP but do not dissociate HSP90 from IRE1. (A) AR42J cells were treated with GA (2 μM) for 1 or 2 h, TM (2.5 μg/ml) for 2 h, or DTT (10 mM) for 30 min. After lysis, 400 μg of soluble proteins were immunoprecipitated with 2 μg of IRE1 antibody, and the complexes were bound on protein A-Sepharose beads. Bound proteins were resolved by SDS-7.5% PAGE and transferred to nitrocellulose membranes. Western analysis was performed with anti-IRE1 and anti-BiP antibodies. (B) In a similar experiment, HSP90 coprecipitation following anti-IRE1 immunoprecipitation was examined (by an HSP90 Western blot) in cells treated with TM or DTT. The specificity of HSP90 coprecipitation with IRE1 is shown by lack of an HSP90 signal in anti-FLAG immunoprecipitates.

FIG. 6.

The ER transmembrane kinase PERK associates with HSP90 but is functionally less sensitive to GA than is IRE1. (A) AR42J cells were treated with GA (1 μM) for the times indicated, cells were lysed, and proteins were resolved by SDS- 10% PAGE and transferred to nitrocellulose membranes. Western analysis of PERK steady-state levels was performed with an anti-PERK antibody. Tubulin was blotted to demonstrate equal loading of protein. (B) AR42J cells were left untreated or treated with GA (1 μM for 4 h) and then lysed, and 400 μg of soluble proteins were immunoprecipitated with 2 μg of anti-PERK antibody. Antibody complexes were bound on protein A-Sepharose beads, washed, eluted, and resolved by SDS-10% PAGE. After transfer to a nitrocellulose membrane, Western analysis of coprecipitated HSP90 was performed. The immunoprecipitates were also blotted for PERK to demonstrate its equivalent pulldown in the presence and absence of GA. (C) AR42J cells were treated with GA (1 μM) for the indicated times. The cells were then pulse-labeled with 100 μCi of [³⁵S]methionine/cysteine/ml for 30 min and lysed. Equal amounts of total soluble proteins were analyzed by SDS-PAGE and autoradiography. (D) AR42J cells were treated with thapsigargin (Th; 0.5 μM) for 0.5 h (filled diamond), GA (1 μM) for 0.5, 1, 2, or 24 h (filled circles) or with a combination of GA and Th (filled square and filled triangle; GA was added for the indicated time, followed by Th for 30 min). Cells were then pulse-labeled and processed as for panel C. Equal amounts of total cell proteins were analyzed by SDS-PAGE and autoradiography.