Abstract
The Hsp90-associated protein p23 modulates Hsp90 activity during the final stages of the chaperone pathway to facilitate maturation of client proteins. Previous reports indicate that p23 cleavage induced by caspases during cell death triggers destabilization of client proteins. However, the specific role of truncated p23 (Delta p23) in this process and the underlying mechanisms remain to be determined. One such client protein, hTERT, is a telomerase catalytic subunit regulated by several chaperone proteins, including Hsp90 and p23. In the present study, we examined the effects of p23 cleavage on hTERT stability and telomerase activity. Our data showed that overexpression of Delta p23 resulted in a decrease in hTERT levels, and a down-regulation in telomerase activity. Serine phosphorylation of Hsp90 was significantly reduced in cells expressing high levels of Delta p23 compared with those expressing full-length p23. Mutation analyses revealed that two serine residues (Ser-231 and Ser-263) in Hsp90 are important for activation of telomerase, and down-regulation of telomerase activity by Delta p23 was associated with inhibition of cell growth and sensitization of cells to cisplatin. Our data aid in determining the mechanism underlying the regulation of telomerase activity by the chaperone complex during caspase-dependent cell death.
Highlights
Telomerase is a specialized reverse transcriptase responsible for the maintenance and preservation of telomere ends in germ cells, immortalized cells, and cancer cells (1). hTERT,3 the reverse transcriptase subunit of telomerase, possesses catalytic activity, whereas the associated RNA component, human telomerase RNA, serves as a template for the synthesis of telomeric sequences (2)
Telomerase activity is tightly regulated in cells, and hTERT is a key molecule in this process (2)
HTERT protein levels are primarily controlled by transcriptional regulation in normal human cells, recent reports suggest the additional involvement of ubiquitination and proteasome-mediated degradation due to Hsp90 malfunction (28)
Summary
Cell Lines and Constructs—HeLa and 293 cells were cultured in minimum essential medium or Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA) in 5% CO2. The resin was washed three times with co-immunoprecipitation buffer (Pierce) containing 4 mM ATP, 20 mM Na2MoO4, and 1 mM MgCl2. Hsp binding was measured by combining 2 g of native purified Hsp (Assay Designs Stressgen) with 2 g of p23 in a final volume of 200 l binding buffer (10 mM Tris-HCl, 50 mM KCl, 8 mM MgCl2, 2 mM dithiothreitol, 4 mM ATP, 10 mM Na2MoO4, 0.01% Nonidet P-40, pH adjusted to 7.5), including an ATP-regeneration system consisting of 10 mM phosphocreatine and 7 units of creatine phosphokinase, as described previously (23). Cells were incubated for 60 min at 37 °C, washed in cold phosphate-buffered saline, and lysed in M-PER reagent containing 1ϫ protease inhibitor mixture for 30 min on ice. Immunoprecipitation with anti-Hsp was performed as described above. Amplified products were separated by electrophoresis on a 10% polyacrylamide gel and stained with ethidium bromide
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