Abstract
Maintenance of telomere is regulated by active telomerase complex, including telomerase holoenzyme and its associated proteins. The activity of telomerase is precisely controlled in cells, and its dysregulation is one of the hallmarks of cancer. The telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) plays a central role for telomerase activity. In this study, we indentified that Polo-like kinase 1 (Plk1) is a novel telomerase-associated protein. Plk1 can interact with hTERT independently of its kinase activity. More importantly, we found that Plk1 is associated with active telomerase complex. In addition, we demonstrated that knockdown of Plk1 caused the reduction of telomerase activity, whereas overexpression of Plk1 increased telomerase activity. Further analysis showed that overexpression of Plk1 led to a significant increase of hTERT protein by prolonging its half-life but did not affect the level of hTERT mRNA. Furthermore, we found that Plk1 enhanced the chromatin loading of hTERT and inhibited its ubiquitination. This implied that Plk1 affected hTERT stability by inhibiting its ubiquitin-mediated degradation. Collectively, these observations suggested that Plk1 is a positive modulator of telomerase by enhancing the stability of hTERT.
Highlights
HTERT plays a central role for telomerase activity and telomeric chromatin maintenance
The co-immunoprecipitation data showed that ectopically expressed Cdk2 and Polo-like kinase 1 (Plk1) can interact with FLAG-human telomerase reverse transcriptase (hTERT) in 293T cells (Fig. 1A)
The results indicated that both in-house and Abcam antibodies can recognize endogenous hTERT protein in hTERT-positive cells (293T, HeLa, and H1299 cells) and full-length FLAG-hTERT expressed in U2OS but not antigen epitope-truncated hTERT (Fig. 1B)
Summary
Cell Culture and Synchronization—HEK293T, HeLa, and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and H1299 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 g/ml streptomycin, and 100 units/ml penicillin at 37 °C with 5% CO2. The cell extracts were subjected to 8% SDS-PAGE, transferred onto PVDF membrane (Millipore) followed by blocking with Tris-buffered saline/Tween 20 (TBST) containing 5% skim milk for 1 h at room temperature, and probed with the indicated primary antibody at 4 °C overnight. The immunoprecipitates were dissolved in SDS loading buffer, separated by SDS-PAGE, and immunoblotted with the indicated antibodies. The bound proteins were dissolved in SDS loading buffer, separated by SDS-PAGE, and immunoblotted with Plk antibody. 2 g of protein of cell lysates were mixed with 50 M dNTPs and 80 ng/l TS primer (5Ј-AATCCGTCGAGCAGAGTT-3Ј) in TRAP buffer (20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM EGTA, 0.1 mg/ml BSA) and incubated for 30 min at 30 °C and for 10 min at 94 °C. Primers for -actin were: forward, 5Ј-GTGAAGGTGACAGCAGTCGGTT-3Ј; reverse, 5Ј-GAAGTGGGGTGGCTTTTAGGA-3Ј
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