Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

Young-Seuk Bae,Mi Kyung Kim,Yu Sam Kim,In Kwon Chung,Mi Ran Kang,Hyung Wook Nam

doi:10.1074/jbc.m710065200

Abstract

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

Highlights

The telomerase catalytic subunit gene into normal somatic cells prevents telomere erosion and extends their proliferative life span [9]
casein kinase 2 (CK2)-mediated phosphorylation is required for the efficient telomere binding of telomeric repeat binding factor 1 (TRF1)
Our findings suggest a novel role of CK2 that functions as a positive regulator for determining the levels of TRF1 at telomeres

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screening—Yeast two-hybrid screening was performed as described previously [29]. In vitro kinase assays were performed with [␥-32P]ATP (Amersham Biosciences) and GST fusion proteins as described before [31]. Electrophoretic Mobility Shift Assays—Nuclear extracts were prepared from MCF7 cells expressing the FLAG-TRF1, and EMSA was performed as described [32]. Labeled DNA probes (0.5 ng) were incubated with nuclear extracts (6 ␮g of protein) and poly(dI-dC) (0.5 ␮g) as the nonspecific competitor (Amersham Biosciences) for 20 min at 25 °C and were fractionated on an 8% nondenaturing polyacrylamide gel. Lysates were immunoprecipitated with anti-FLAG antibody and supplemented with protein A-Sepharose beads. 72 h after transfection, nuclear extracts were immunoblotted using anti-TRF1 (Sigma), anti-TRF2 (Upstate), anti-CK2␣, and anti-CK2␤ (Santa Cruz Biotechnology) antibodies. Nano-LC and ESI MS/MS Analysis—In gel protein digestion was performed as described previously [34]. The column outlet was directly coupled to the high voltage ESI source, which was interfaced to the QSTAR Pulsar quadrupole time-of-flight mass spectrometer (Applied Biosystems)

RESULTS

C Mock RNAi CK2β RNAi

DISCUSSION