Telomerase Inhibitor PinX1 Provides a Link between TRF1 and Telomerase to Prevent Telomere Elongation

Tae Ho Lee,Xiao Zhen Zhou,Kun Ping Lu,Rong Shi,Pengyu Huang,Christina Y. Soohoo

doi:10.1074/jbc.m110.180174

Tae Ho Lee, Xiao Zhen Zhou + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.180174

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Abstract

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Highlights

GST-TRF1 fragments encompassing residues 1– 439, 1–336, and 1–220, but not 1–130, bound to PinX1 that was synthesized by in vitro transcription and translation (Fig. 1C). These results indicate that the PinX1 binding domain is located in the Telomere restriction fragment (TRF) homology (TRFH) domain of TRF1, as shown for several other TRF1-binding proteins [71, 80, 100]
All GST-PinX1 mutant proteins containing residues 291–310 with or without any N-terminal or C-terminal flanking sequences were able to bind to TRF1, whereas mutant proteins lacking this domain could not (Fig. 1, D and E). These results indicate that the 20-amino acid fragment (C20) from residue 291 to 310 of PinX1 is both necessary and sufficient to mediate the interaction with TRF1
PinX1 was originally identified as a TRF1-interacting protein, nothing is known about the biological function of this interaction

Summary

Introduction

The localization of these mutant proteins were determined by co-transfecting HT1080 cells overnight with vectors expressing RFP-tagged TRF1 and GFP-tagged wild-type full-length PinX1 or its TID with or without mutations followed by subjecting them to fluorescence microscopy (D). We examined the role of the TRF1-PinX1 interaction for PinX1 or TID to localize to telomeres and to induce telomere shortening independent of their ability to inhibit telomerase activity in cells.

Results

Conclusion