Abstract
The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.
Highlights
We investigate the role of HSP90 in the processing of the platelet derived growth factor receptor ␣ (PDGFR␣), a protein implicated in transformation, in human tumors originating in the mesenchyme
PDGFR␣ Expression Is Not Affected by Exposure to 17-AAG in Untransformed Cells—To test whether the effects of 17-AAG are cancer cell-specific, we examined its effects on untransformed cells
The present study demonstrates that geldanamycin derivatives induce degradation of the mature PDGFR␣
Summary
Materials—17-AAG, PDGF-BB, chloroquine, monensin, leupeptin, and MG132 were purchased from Sigma. Immunoblotting—Actively growing cells were lysed into RIPA buffer containing leupeptin (1 g/ml), aprotinin (1 g/ml), PMSF (400 M), and Na3VO4 (1 mM). 500 g of protein from the supernatant was incubated overnight at 4 °C with anti-PDGFR␣ or with anti-cdc antibodies, with 30 l of a slurry of Protein G Plus-agarose beads for 90 min at 4 °C. Protein-antibody-bead complexes were centrifuged in a wash buffer containing 0.2% Triton and boiled for 5 min in 1ϫ SDS protein loading dye. After blocking with 3% goat serum in PBS, cells were incubated with anti-PDGFR␣ antibody (Santa Cruz Biotechnology, 1:50) for 2 h at room temperature, followed by a 30-min incubation with Alexa Fluor 488 anti-rabbit secondary antibody (1:1000, Molecular Probes, Eugene, OR). Cells were subsequently treated with a peroxidase labeled anti-BrdUrd antibody for 90 min, and the colorimetric reaction was developed with a tetramethylbenzidine-based substrate. Statistical Analysis—A two-tailed Student’s t test compared results of cell proliferation assays
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