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Journal of Biochemistry Advance Access originally published online on March 23, 2007
Journal of Biochemistry 2007 141(5):641-652; doi:10.1093/jb/mvm071
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© 2007 The Japanese Biochemical Society.

Human Telomerase Exists in Two Distinct Active Complexes In Vivo

Hideki Mizuno1,2, Shilagardi Khurts1, Takahiko Seki3, Yasuhide Hirota3, Shuichi Kaneko2 and Seishi Murakami1,*

1Department of Molecular Oncology, Cancer Research Institute, Kanazawa University; and 2Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-0934, Japan; and 3New Product Research Laboratories III R&D Division, Daiichi Pharmaceutical Company Ltd., Tokyo, Japan

*To whom correspondence should be addressed. Tel: 81-76-265-2713, Fax: 81-76-234-4532, E-mail: semuraka{at}kenroku.kanazawa-u.ac.jp

Received November 30, 2006; Accepted February 13, 2007


  Abstract

Telomerase, a stable complex of telomerase reverse transcriptase (TERT) and template RNA (TERC), is responsible for telomere maintenance. During purification trials of recombinant human telomerase of the two components reconstituted in insect cells, we identified two complexes of human telomerase of molecular masses 680 and 380 kDa, both of which retain telomerase activity in vitro. We show here that the former complex does not include Hsp90 (heat shock protein 90) and its telomerase activity is resistant to Hsp90 inhibitors, whereas the latter contains Hsp90 and its telomerase activity is sensitive to Hsp90 inhibitors. N-terminal of FLAG-hTERT in the former is exposed, as this complex was efficiently purified with anti-FLAG M2 affinity resin. We also identified two different telomerase complexes in HeLa cells, in addition to ectopically expressed hTERT. Most of endogenous hTERT and FLAG-hTERT was detected around 680 kDa. These two complexes in HeLa cells have the same properties as their respective reconstituted telomerases. The unstable property of the telomerase complex with Hsp90, especially in the presence of Hsp90 inhibitors, was due to proteasome-mediated degradation of hTERT, since proteasome inhibitors prevented hTERT degradation in vivo. To our knowledge, this is the first demonstration of two distinct active complexes of human telomerase ectopically expressed in insect and mammalian cells.

Key Words: GA, Hsp90, hTERC, hTERT

Abbreviations: GA, geldanamycin; Hsp90, heat shock protein 90; hTERC, human telomerase RNA component; hTERT, human telomerase reverse transcriptase subunit; TRAP, telomere repeat amplification protocol


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