The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation

Abstract

The DNA damage-responsive protein kinases ATM and ATR phosphorylate SQ/TQ motifs that lie in clusters in most of their in vivo targets. Budding yeast Cdc13p contains two clusters of SQ/TQ motifs, suggesting that it might be a target of Mec1p/Tel1p (yeast ATR/ATM). Here we demonstrated that the telomerase recruitment domain of Cdc13p is phosphorylated by Mec1p and Tel1p. Gel analysis showed that Cdc13p contains a Mec1/Tel1-dependent post-translational modification. Using an immunoprecipitate (IP)-kinase assay, we showed that Mec1p phosphorylates Cdc13p on serine 225, 249, 255 and 306, and Tel1p phosphorylates Cdc13p on serine 225, 249 and 255 in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13p SQ motifs phosphorylated by Mec1p and Tel1p caused multiple telomere and growth defects. In addition, normal telomere length and growth could be restored by expressing a Cdc13–Est1p hybrid protein. These results demonstrate the telomerase recruitment domain of Cdc13p as an important new telomere-specific target of Mec1p/Tel1p.

PP2A and Aurora differentially modify Cdc13 to promote telomerase release from telomeres at G2/M phase

“…We previously found that Tel1 and Mec1 phosphorylate Cdc13 at S249 and S255 in vitro 22 . Several laboratories also discovered genetic and physical evidence of the importance of Tel1 in telomerase recruitment [23][24][25] .…”

“…1a) [22][23][24][25][26][27] . To examine the possibility that recruitment of telomerase by Cdc13 may be repressed by phosphatases, we determined the phosphorylation level of Cdc13 throughout the cell cycle for wild type (WT) and various phosphatase deletion strains.…”

“…Our previous study showed that Cdc13 is phosphorylated at S225, S249, S255 and S306 by Tel1 and Mec1, and that S249 and S255 are two important residues to promote telomerase recruitment 22 . To test whether Pph22 targets these two phosphorylation sites, we raised a Cdc13 S249/S255 phosphospecific polyclonal antibody and the antibody exhibited higher binding affinity to phospho-S249/S255 and phospho-S255 than phospho-S249 (Supplementary Figs 2a, b and d).…”

“…Using mass spectrometry (MS) to analyse phosphorylation sites of overexpressed Cdc13-Myc 9 in S. cerevisiae, we identified new phosphorylation sites S314, S324 and S333 on Cdc13 (Fig. 3a) that have not been well studied previously 22,26,27,42 ( Supplementary Fig. 10).…”

“…Cdc13 binds Est1 to recruit telomerase [35][36][37] , and recent reports have suggested that the phosphorylations on the telomerase recruitment domain of Cdc13 may promote telomerase recruitment 22,26,27 . However, this proposed signalling has been controversial 38,39 .…”