The Hsp90 chaperone complex is both a facilitator and a repressor of the dsRNA-dependent kinase PKR

Abstract

PKR, a member of the eukaryotic initiation-factor 2alpha (eIF-2alpha) kinase family, mediates the host antiviral response and is implicated in tumor suppression and apoptosis. Here we show that PKR is regulated by the heat shock protein 90 (Hsp90) molecular chaperone complex. Mammalian PKR expressed in budding yeast depends on several components of the Hsp90 complex for accumulation and activity. In mammalian cells, inhibition of Hsp90 function with geldanamycin (GA) during de novo synthesis of PKR also interferes with its accumulation and activity. Hsp90 and its co-chaperone p23 bind to PKR through its N-terminal double-stranded (ds) RNA binding region as well as through its kinase domain. Both dsRNA and GA induce the rapid dissociation of Hsp90 and p23 from mature PKR, activate PKR both in vivo and in vitro and within minutes trigger the phosphorylation of the PKR substrate eIF-2alpha. A short-term exposure of cells to the Hsp90 inhibitors GA or radicicol not only derepresses PKR, but also activates the Raf-MAPK pathway. This suggests that the Hsp90 complex may more generally assist the regulatory domains of kinases and other Hsp90 substrates.

Fig. 1. PKR toxicity and accumulation are reduced in yeast strains with defective Hsp90 chaperone activity. (A) Slow growth of yeast strains expressing PKRwt is attenuated by alterations in the Hsp90 machinery. The different yeast strains containing pYES/PKRwt were grown for 8 h at 30°C under repressed (2% glucose) or partially induced (2% galactose plus 0.1% glucose) conditions. Growth was monitored by measuring OD₆₀₀ and for each strain normalized to the growth on 2% glucose. (B) Defective PKR accumulation in cdc37-34 and Δsba1 strains. Strains containing the plasmids pYES/PKRwt or PKR^K296R were cultured for 16 h on 2% galactose to induce expression of the proteins. PKR and Hsp82 were revealed by western blotting with anti-PKR and Hsp82 antibodies, respectively. Lanes 1, 2, 4, 5, 7 and 8, strains transformed with the plasmid pYES/PKRwt; lanes 3, 6 and 9, strains transformed with the plasmid pYES/PKR^K296R.

Fig. 2. GA decreases PKR levels and activity in mammalian cells. (A) Reduction of PKR protein levels upon GA treatment. HeLa cells were cultured for 16 h with or without 1 µM GA. Endogenous PKR and eIF-2α protein levels were revealed by immunoblot analysis with antibodies against PKR and eIF-2α, respectively. (B) Inhibition of PKR activity by GA in vivo. Overexpression of human PKR was induced in NIH 3T3-PKRwt in the presence or absence of 1 µM GA. Aliquots of cell lysates were examined by immunoblotting for the phosphorylation of eIF-2α with a phosphospecific anti-eIF-2α antibody, for total eIF-2α with an anti-eIF-2α antibody and for the presence of human PKR with an anti-PKR antibody. Tet, tetracycline.

Fig. 3. Hsp90 and its co-chaperone p23 form a complex with PKR in vivo. (A) Hsp90 co-precipitates with endogenous (upper panel) and overexpressed PKR (lower panel). Hsp90 immunoprecipitates from HeLa cell extracts were analyzed by western blotting with an anti-PKR antibody. Plasmid CKF/PKRwt was used to overexpress PKRwt in 293T cells. (B) PKR co-precipitates with p23. Endogenous p23 was immunoprecipitated from HeLa cell extracts with a monoclonal antibody against p23, and PKR, Hsp90 or p23 was detected by immunoblot analysis using anti-PKR, Hsp90 or p23 antibodies, respectively (upper panel). NIH 3T3-PKR^K296R cells expressing the kinase-defective mutant PKR^K296R were grown in the absence of tetracycline for 16 h and then cell lysates were subjected to immunoprecipitation experiments using an anti-p23 antibody (lower panel). C, unrelated monoclonal antibody (anti-β-galactosidase) as a negative control for immunoprecipitation; IP, immunoprecipitated proteins; input, total proteins.

Fig. 4. dsRNA triggers the dissociation of PKR from Hsp90–p23. (A) Disruption of the Hsp90–PKR complex by dsRNA. HeLa cells were cultured with or without 100 µg/ml dsRNA for 15 min prior to lysis and immunoprecipitation with an anti-Hsp90 antibody. The immunoprecipitates were analyzed for the presence of Hsp90 and PKR by immunoblotting. (B) Dissociation of the PKR–p23 complex by dsRNA is dose dependent. After 16 h induction of PKR^K296R, cells were treated for 15 min with increasing dsRNA concentrations as indicated. Cell extracts were immunoprecipitated with an anti-p23 antibody and PKR was detected by western blotting. (C) Time course analysis of the dissociation of the p23–PKR complex by dsRNA. PKR^K296R was induced by removal of tetracycline. Cells were incubated with 100 µg/ml dsRNA for different periods of time as indicated, and processed as in (B).

Fig. 5. GA induces the dissociation of Hsp90–p23 from PKR. (A) Hsp90 dissociation from PKR in the presence of GA. HeLa cells were incubated with or without 1 µM GA for 15 min prior to lysis and immunoprecipitation with an anti-Hsp90 antibody. (B) Short- and long-term effects of GA on the p23–PKR complex. GA (1 µM) was added to the medium either during PKR^K296R synthesis (for 16 h) or after completion of its synthesis (for 15 min). Immunoprecipitations were performed with an anti-p23 antibody, and PKR and Hsp90 were visualized by immunoblot analysis. C, control antibody.

Fig. 6. The N- and C-terminal domains of PKR have differential requirements for the Hsp90–p23 complex. (A) Mapping of the chaperone binding region of PKR. 293T cells were transfected with plasmids CKF, CKF/PKR^K296R, CKF/Nter or CKF/Cter. Prior to cell harvesting (after 36 h), dsRNA (100 µg/ml) was added for 15 min where indicated. Cell extracts were immunoprecipitated with an anti-Flag (M2) antibody. Immunoprecipitates were examined for the presence of the different Flag-tagged variants and for p23 using anti-PKR and p23 antibodies, respectively. (B) Differential binding of the N- and C-terminal domains and full-length PKR to Hsp90. Extracts fom 293T cells transfected with plasmids CKF/PKR^K296R, CKF/Nter or CKF/Cter were immunoprecipitated in a buffer without potassium (see Materials and methods) in the presence or absence of 1 mM ATP with an anti-Hsp90 antibody.

Fig. 7. Hsp90 inhibitors activate PKR in vivo and in vitro. (A) GA and radicicol trigger eIF-2α phosphorylation. HeLa cells were incubated with 1 µM GA, 100 µg/ml dsRNA or 5 µM radicicol for different periods of time as indicated. The phosphorylation status of eIF-2α was examined by immunoblotting with a phosphospecific anti-eIF-2α antibody and total eIF-2α with a monoclonal antibody against eIF-2α. (B) GA induces PKR dimerization. 293T cells were transfected with the plasmids CKF/PKR^K296R and CK/Nter encoding the Flag-tagged full-length kinase and the untagged N-terminal region, respectively. Prior to cell harvesting, dsRNA (100 µg/ml) or 1 µM GA was added for different periods of time as indicated. Cell lysates were immunoprecipitated with an anti-Flag antibody (Flag IP) and association with the PKR N-terminus was observed by immunoblot anaysis with an antibody against the N-terminus (N-18). (C) GA promotes PKR autophosphorylation. Extracts from 293T cells, 24 h after transfection with CKF/PKR, were immunoprecipitated with an anti-Flag antibody. Autophosphorylation of Flag-PKR was revealed using a phosphospecific anti-PKR antibody. Cells were treated with 1 µM GA or dsRNA (100 µg/ml) for 15 min prior to cell harvesting. The efficiency of immunoprecipitation was monitored with an anti-Flag antibody. (D) GA activates PKR in vitro. Flag-PKR expressed for 24 h in 293T cells was immunoprecipitated and subjected to an in vitro kinase assay in the absence or presence of GA (5 µM) or dsRNA (50 ng/ml). Coomassie, levels of Flag-PKR were visualized by Coomassie Blue staining; [³²P] (kinase assay), labeled phosphate incorporation was detected by autoradiography.

Fig. 8. GA activates the Raf–MAPK pathway. HeLa cells were incubated with 10 ng/ml EGF and 5 µM GA for different periods of time as indicated. The phosphorylation status of ERK was examined by immunoblotting with a phosphospecific anti-ERK antibody and total ERK with an anti-ERK antibody.

Fig. 9. Model for Hsp90 regulation of PKR. GA blocks the maturation of PKR and activates the mature enzyme. See Discussion for details. dsRNA is represented as a double-stranded helix, and dsRNA binding motifs as small open boxes.