Regulation of survivin function by Hsp90 - PubMed (original) (raw)

Regulation of survivin function by Hsp90

Paola Fortugno et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin-Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin-Hsp90 complex may provide a rational approach for cancer therapy.

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Figures

Fig. 1.

Survivin–Hsp90 interaction. (A) Heat shock. HeLa cells exposed to 42°C for 1 h were harvested at the indicated time intervals and analyzed by Western blotting. (B) Cell cycle analysis. HeLa cells were harvested 8 h after heat shock and analyzed for DNA content by flow cytometry. The percentage of G2/M cells is indicated. (C)Affinity purification. HeLa cell extracts were fractionated on a survivin-Sepharose column. Eluted proteins (arrows) were identified by Western blotting. (D) Coimmunoprecipitation. Raji cell extracts were immunoprecipitated with IgG or an Ab to survivin, and pellets (P) and supernatants (S) were analyzed by Western blotting. (E) In vivo pull-down. Asynchronous or taxol-treated HeLa cell extracts were incubated with Sepharose (None) or survivin-Sepharose (Survivin), and pellets or supernatants (25% of reaction) were analyzed for coassociated Hsp90 by Western blotting. MW, molecular weight.

Fig. 2.

Structure/function of survivin–Hsp90 interaction. (A) In vitro pull-down. Survivin was incubated with Sepharose-GST, -GST-Hsp90, or the indicated -GST-Hsp90 N, M, or C domains, and protein binding was determined by Western blotting. (B) Immunoprecipitation. HeLa cells transfected with the indicated FLAG-Hsp90 domains were immunoprecipitated with a mAb to FLAG and analyzed by Western blotting. MW, molecular weight. (C) Peptide mapping. The indicated survivin peptides were used to compete the binding between Hsp90 and survivin by ELISA. (D) Folding requirement. Protein binding to immobilized survivin (Upper) or Hsp90 (Lower) was determined by ELISA. (E) In vivo turnover. HeLa cells transfected with GFP-survivin (Left) or GFP-survivin(C84A) (Right) were treated with cycloheximide, and protein turnover at the indicated time intervals was determined by Western blotting with an Ab to GFP.

Fig. 3.

Regulation of survivin stability by Hsp90. (A) Requirement for Hsp90 chaperone function. HeLa cells were treated with GA and analyzed after 24 h by Western blotting. MW, molecular weight. (B) Cell cycle analysis. Untreated (Left) or GA-treated (Right) HeLa cells were harvested after 24 h and analyzed for DNA content by flow cytometry. The percentage of cells at each cell cycle transition is indicated. Apoptotic cells were electronically excluded. (C) Proteasome inhibition. GA-treated HeLa cells with or without the proteasome inhibitor lactacystin were analyzed after 30 h by Western blotting. (D) GA-induced apoptosis. GA-treated HeLa cells were analyzed for plasma membrane integrity (red fluorescence) and caspase-3/7 activity (green fluorescence) by multiparametric flow cytometry. The percentage of cells in each quadrant is indicated. (E) Apoptosome requirement. WT or Apaf-1–/– MEF were treated with GA and analyzed for apoptosis (Upper) or G2/M arrest (Lower) by DNA content and flow cytometry. (F) Modulation of GA-induced apoptosis. YUSAC-2 cells conditionally expressing survivin after Tet withdrawal (25) were treated with GA with or without Tet and analyzed for apoptosis by DNA content and flow cytometry. Data in E and F are the means ± SD of three independent experiments.

Fig. 4.

Ab disruption of survivin–Hsp90 complex induces apoptosis and cell cycle arrest. (A) In vitro targeting. Survivin was incubated with mAbs 58 or 8E2 to survivin (24) or IgG, and binding to GST-Hsp90 or GST-N-Hsp90 (GST-Hsp90 N) was determined by Western blotting. (B) In vivo targeting. HeLa cells were loaded intracellularly with mAb 8E2 or IgG, treated with cycloheximide, and analyzed by Western blotting (Upper). MW, molecular weight. Protein amounts were quantified by densitometry (Lower). (C) Caspase activity. YUSAC-2 cells loaded with mAb 8E2 or IgG were analyzed for caspase-3/7 activity by Asp-Glu-Val-Asp expression and flow cytometry in the absence (Tet–) or presence (Tet+) of Tet. Data are mean ± SD of three independent experiments. (Inset) Proteolytic processing of caspase-9 in HeLa cells transduced with mAbs 8E2 or 58 to survivin. The positions of proform (Casp.9) or cleaved (Cleaved) caspase-9 are indicated. (D) Mitotic defects. HeLa cells loaded with mAbs 8E2 or 58 to survivin or IgG were stained with an Ab to β-tubulin and analyzed by fluorescence microscopy. (Magnification, ×400.) Multinucleated cells in mAb 8E2-transduced cultures are indicated by arrows. (E) Summary of cell division defects. Mitotic or multinucleated cells in Ab-transduced HeLa cells were scored by fluorescence microscopy. Data are the means ± SEM of four independent determinations.

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Regulation of survivin function by Hsp90 - PubMed (original) (raw)