CDK1 dependent phosphorylation of hTERT contributes to cancer progression

Mami Yasukawa,Yasuhide Furuta,Shuichi Kaneko,Hideya Kawaji,Mikako Shirouzu,Shisako Shoji,Yoshihide Hayashizaki,Shinji Yamada,Yoko Matsuda,Takaya Abe,Yoshinari Ando,Tadashi Kondo,Mika K Kaneko,Kenkichi Masutomi,Masaki Suimye Morioka,Yukinari Kato,Mitsuhiro Machitani,Taro Yamashita,Kumiko Shiozawa

doi:10.1038/s41467-020-15289-7

Abstract

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.

Highlights

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation
Since it has been challenging to detect endogenous hTERT11,15, we extensively validated available human telomerase reverse transcriptase (hTERT)-specific antibodies against hTERT, including the mouse monoclonal antibody, the mouse mAb, the sheep polyclonal Abs abx120550, and the rabbit mAb ab3202
We previously reported that hTERT protein localizes to mitotic spindles[14] and hTERT protein expression is enriched in mitosis[12] (Fig. 1a)

Summary

Introduction

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner. Experiments involving live-cell imaging techniques combined with CRISPR-Cas[9] genome editing demonstrated that recruitment of hTERT and hTERC to telomeres occurs through dynamic interactions between telomerases and the chromosome end during S-phase[10] These observations indicate that recruitment of telomerase holoenzyme to the telomere is regulated in cell cycle-dependent manner, only a small subset of hTERT forms interactions with telomeres and Cajal bodies even in S-phase[10] and the regulation and function of the majority of hTERT outside S-phase is poorly understood. We report that hTERT is phosphorylated in a cell cycledependent manner and that this phosphorylation is essential for the RdRP activity and tumor formation via regulation of target gene expression independent of hTERT-mediated elongation of telomeres

Methods

Results

Conclusion