Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13

Abstract

Budding yeast telomerase is mainly activated by Tel1/Mec1 (yeast ATM/ATR) on Cdc13 from late S to G2 phase of the cell cycle. Here, we demonstrated that the telomerase-recruitment domain of Cdc13 is also phosphorylated by Cdk1 at the same cell cycle stage as the Tel1/Mec1-dependent regulation. Phosphor-specific gel analysis demonstrated that Cdk1 phosphorylates residues 308 and 336 of Cdc13. The residue T308 of Cdc13 is critical for efficient Mec1-mediated S306 phosphorylation in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13 S/TP motifs phosphorylated by Cdk1 caused cell cycle delay and telomere shortening and these phenotypes could be partially restored by the replacement with a negative charge residue. In the absence of Ku or Tel1, Cdk1-mediated phosphorylation of Cdc13 showed no effect on telomere length maintenance. Moreover, this Cdk1-mediated phosphorylation was required to promote the regular turnover of Cdc13. Together these results demonstrate that Cdk1 phosphorylates the telomerase recruitment domain of Cdc13, thereby preserves optimal function and expression level of Cdc13 for precise telomere replication and cell cycle progression.

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

“…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.…”

“…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).…”

“…It recruits telomerase to its site of action through an electrostatic interaction between Cdc13 and Est1 (refs 20,21). Some recent studies have suggested that Cdk1, Tel1 and Mec1 may phosphorylate the telomerase recruitment domain of Cdc13 to promote this Cdc13-Est1 interaction at the late S phase [22][23][24][25][26][27] .…”

“…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 .…”

CDK1 differentially regulates G-overhang generation at leading- and lagging-strand telomeres in telomerase-negative cells in G2phase

“…27 In budding yeast, which contains only one CDK, cdc28, Cdc28 is required for the generation of G-overhangs by controlling end resection [28][29][30] as well as by regulating the recruitment of telomerase to telomeres. 31,32 The role of CDK1 in regulating telomere maintenance in human cells is largely elusive.…”