CHIP Promotes Human Telomerase Reverse Transcriptase Degradation and Negatively Regulates Telomerase Activity

Ji Hoon Lee,Prabhat Khadka,Seung Han Baek,In Kwon Chung

doi:10.1074/jbc.m110.149831

Abstract

The maintenance of eukaryotic telomeres requires telomerase, which is minimally composed of a telomerase reverse transcriptase (TERT) and an associated RNA component. Telomerase activity is tightly regulated by expression of human (h) TERT at both the transcriptional and post-translational levels. The Hsp90 and p23 molecular chaperones have been shown to associate with hTERT for the assembly of active telomerase. Here, we show that CHIP (C terminus of Hsc70-interacting protein) physically associates with hTERT in the cytoplasm and regulates the cellular abundance of hTERT through a ubiquitin-mediated degradation. Overexpression of CHIP prevents nuclear translocation of hTERT and promotes hTERT degradation in the cytoplasm, thereby inhibiting telomerase activity. In contrast, knockdown of endogenous CHIP results in the stabilization of cytoplasmic hTERT. However, it does not affect the level of nuclear hTERT and has no effect on telomerase activity and telomere length. We further show that the binding of CHIP and Hsp70 to hTERT inhibits nuclear translocation of hTERT by dissociating p23. However, Hsp90 binding to hTERT was not affected by CHIP overexpression. These results suggest that CHIP can remodel the hTERT-chaperone complexes. Finally, the amount of hTERT associated with CHIP peaks in G(2)/M phases but decreases during S phase, suggesting a cell cycle-dependent regulation of hTERT. Our data suggest that CHIP represents a new pathway for modulating telomerase activity in cancer.

Highlights

Absence of a telomere maintenance pathway, dividing somatic cells show a progressive loss of telomeric DNA during successive rounds of cell division because of a DNA end replication problem (8, 9)
We found that the amount of hTERT associated with CHIP peaks in G2/M phases during which telomerase does not act on telomeres but decreases during S phase, suggesting a cell cycle-dependent regulation of hTERT by CHIP
To determine whether hTERT and CHIP associate in vivo, H1299 cells co-transfected with hTERT-HA and CHIP-His were treated with the proteasome inhibitor MG132 to block degradation of ubiquitinated hTERT and subjected to immunoprecipitation. hTERT-HA was detected in anti-His immunoprecipitates when CHIP-His was expressed (Fig. 1B)

Summary

Introduction

Absence of a telomere maintenance pathway, dividing somatic cells show a progressive loss of telomeric DNA during successive rounds of cell division because of a DNA end replication problem (8, 9). In the TRAP assay with the CHIP mutants, we observed that overexpression of H260Q resulted in a significant reduction in telomerase activity compared with the vector control (Fig. 2E). Whereas endogenous hTERT was predominantly localized to the nucleus in the control cells, transfection of wild-type CHIP or H260Q resulted in a cytoplasmic accumulation of hTERT (Fig. 2F).

Results

Conclusion