Hsp90 inhibitors decrease the association between rpS3 and Hsp90. (A) HEK293T cells were treated with 1.5 μM GA for 4 h. Total cell extracts were prepared and immunoprecipitated with anti-Hsp90 antibody, and the precipitate was monitored for the presence of Hsp90 or rpS3 by immunoblotting with anti-Hsp90 or anti-rpS3. (B and C) HEK293T cells were transfected with GFP-tagged (B) or FLAG-tagged (C) rpS3. After 24 h, cells were incubated for 4 h in the absence or presence of GA (1.5 μM). The cells were then harvested and lysed. Total cell lysates were subjected to immunoprecipitation with anti-Hsp90 polyclonal antibody. The immunoprecipitated proteins were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Coimmunoprecipitates were blotted using anti-GFP (B) or anti-FLAG (C) antibodies. (D) Subcellular localization of rpS3 and Hsp90 in 293T cells. 293T cells were treated with GA for 4 h, fixed with paraformaldehyde, and immunostained with both polyclonal anti-rpS3 (red) and monoclonal anti-Hsp90 (green) antibodies. Cells were detected by confocal microscopy. (E) HEK293T cells were incubated in the presence or absence of GA for 4 h. Total cell lysates were fractionated to isolate the nucleoli, as described in Materials and Methods. Fractionated proteins were analyzed by immunoblotting with anti-rpS3 or anti-Hsp90. The fraction marker used tubulin (cytoplasm) and CK2α (nucleolus) antibodies.

Reduction of rpS3 was prevented by proteasome inhibitor treatment. (A) HEK293T cells were transiently transfected with GST-rpS3. After 24 h, cells were treated with GA (1.5 μM) after pretreatment with 20 μM of proteasome inhibitor MG-132 for 2 h. Cell lysates were prepared and resolved by 10% SDS-PAGE. Phospho-ERK was used to verify the effect of GA treatment. (B) HEK293T cells were treated with ALLN proteasome inhibitor, followed by treatment with radicicol. Cells were harvested and immunoblotted. Raf-1 was used to verify the effect of ALLN.

Hsp90 inhibitors induce the association of rpS3 with Hsp70. (A) HEK293T cells were treated with GA for 4 h. Total cell extracts were prepared and immunoprecipitated with anti-Hsp90 antibody, and the precipitate was then monitored for the presence of Hsp90 or rpS3 by immunoblotting with anti-Hsp90 or anti-rpS3. PIRS was used as a negative control. (B) HT1080 cells stably expressing FLAG-rpS3 were treated with 1.5 μM of GA for 4 h, and total cell lysates were then immunoprecipitated with anti-Hsp90 antibody. The precipitates were separated by SDS-PAGE, transferred, and immunoblotted using the indicated antibodies. (C) HT1080 cells stably expressing FLAG-tagged rpS3 were incubated with 1.5 μM of GA for the indicated amounts of time. Cell lysates were immunoprecipitated with anti-FLAG mAb. The precipitates were resolved by SDS-PAGE and detected by immunoblot analysis using anti-Hsp70 antibody. The graph was normalized by dividing the value of the binding result by the value of the lysate and expressed as fold increase.

Hsp90 interacts with rpS3 in vitro and in vivo. (A) GFP-Hsp90 fusion protein was expressed in transfected HEK293T cells. The cell lysates were then immunoblotted. GST-rpS3 fusion protein was expressed in E. coli, purified on glutathione (GSH)-Sepharose 4B beads and stained with Coomassie Blue to demonstrate the expression of the fusion proteins (A, left panels). The HEK293T cell extracts were subsequently incubated with bead-bound GST-rpS3. After the beads were washed, proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Bound GFP-Hsp90 was detected by immunoblotting with anti-GFP antibodies (A, right panel). (B) The cell lysates were immunoprecipitated with anti-Hsp90 antibody, and the precipitates were immunoblotted with anti-Hsp90 and anti-rpS3 antibodies. PIRS stands for preimmune rabbit serum, used as a negative control. (C) Deletion constructs of GFP-rpS3. (D) GST-rpS3 derivatives were tested for interaction with GFP-Hsp90. GST-rpS3 and GFP-Hsp90 constructs were cotransfected into HEK293T cells. The expression levels of GST-rpS3 and GFP-Hsp90 were assessed by immunoblot analysis (D, left panel). The lysates were incubated with polyclonal anti-GST antibody, immunoprecipitated with anti-IgA-Sepharose, and subjected to 10% SDS-PAGE (D, right panel).

RpS3 was degraded by Hsp90 inhibitors. (A) HT1080 cells stably expressing the wild-type rpS3 tagged with FLAG were treated with different concentrations of GA for 16 h. To measure the FLAG-rpS3 levels, total cell lysates were blotted using anti-FLAG and anti-actin antibodies. (B) To measure the levels of endogenous rpS3 protein, HEK293T cells were incubated with different concentrations of GA for 20 h. Total cell lysates were blotted using anti-rpS3 and anti-actin antibodies. (C) To measure the endogenous rpS3 protein levels, HEK293T cells were treated with GA (1.5 μM) in a time-dependent manner. Each lane of SDS-PAGE contains 80 μg of total lysates. Error bars represent SDs from the mean of at least three independent experiments.

RpS3 degradation is mediated through the ubiquitin-dependent proteasome pathway. (A) HEK293T cells were transiently transfected with FLAG-rpS3. After 24 h, cells were treated with 20 μM of MG-132 for 4 h. Cells were harvested, lysed, and immunoprecipitated with anti-FLAG monoclonal antibody (mAb). Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (B) HEK293T cells were cotransfected with FLAG-rpS3 and HA-tagged ubiquitin and were harvested after 24-h incubation. The lysates were immunoprecipitated with anti-FLAG mAb. Ubiquitin-conjugated rpS3 was detected by immunoblotting. (C) HEK293T cells were transfected with GFP-rpS3. After 24 h, cells were treated with 20 μM of radicicol for 16 h. Recombinant proteins were immunoprecipitated with anti-GFP antibody and were then resolved by PAGE (left panel). The levels of ubiquitinated rpS3 were determined by Western blot analysis (right panel). The two left lanes of the gels are for GFP controls, and the two right lanes of the gels are for GFP-S3. (D) HEK293T cells were transfected with pEGFPc1-rpS3 to express GFP-rpS3. After 24 h, transfected cells were incubated with 0, 2.5, 5.0, and 7.5 μM of radicicol for 16 h. GFP-rpS3 was immunoprecipitated with anti-GFP mAb, and polyubiquitinated rpS3 was detected using anti-ubiquitin mAb. (E) HEK293T cells were transfected with GFP-tagged rpS3. After 24 h, transfected cells were pretreated with 20 μM of MG-132 (lanes 4 and 5) for 2 h and were then treated with 1.5 μM of GA (lanes 3 and 4) for 6 h. The GFP-rpS3 protein immunoprecipitated with anti-GFP antibodies was separately detected using anti-GFP and anti-ubiquitin monoclonal antibodies.

Hsp90 inhibition affects the state of ribosomes. (A) HEK293T cells were treated with 1.5 μM of GA for 4 h. Total cell extracts were prepared and immunoprecipitated with anti-Hsp90 antibody, and the precipitate was then monitored for the presence of Hsp90 or rpS6 by immunoblotting with anti-Hsp90 or anti-rpS6 (top panel). HEK293T cells were transfected with FLAG-tagged rpS11. After 24 h, cells were treated with 1.5 μM GA for 4 h. Total cell extracts were prepared and immunoprecipitated with anti-Hsp90 or control antibodies (bottom panel). (B) The 40S ribosomal subunit is influenced by GA. HT1080 cells were treated with GA for 4 h. Cell extracts from control or GA-treated HT1080 cells were centrifuged at low speed. Ribosomes were precipitated by ultracentrifugation at 150,000 × g for 2 h through a sucrose cushion. Ribosomal (pellet) and nonribosomal (upper and lower) fractions were subjected to immunoblot analysis with anti-rpS3, anti-rpS6, anti-Hsp90, or anti-JNK antibodies. (C) Stably expressed FLAG-rpS3 showed a similar result as with endogenous rpS3. Cell extracts from FLAG-rpS3-expressing HT1080 cells were ultracentrifuged at 150,000 × g for 2 h through a sucrose cushion. Ribosomal (pellet) and nonribosomal (upper and lower) fractions were subjected to immunoblot analysis with anti-FLAG, anti-Hsp90, or anti-JNK antibodies. (D) Hsp90 interacts with free rpS3 and protects from degradation. The upper fraction of (C) was used for immunoprecipitation with the anti-FLAG antibody, followed by immunoblotting with anti-FLAG or anti-Hsp90 antibodies. (E) HEK293T cells were left untreated or treated with 1.5 μM of GA for 12 h and subjected to [³⁵S]-metabolic labeling for 10, 20, and 30 min. Equal amounts of proteins were resolved by 10% SDS-PAGE and analyzed by autoradiography.