3606 Nucleic Acids Research, 2009, Vol. 37, No. 11 Figure 2. Cdc13 is directly phosphorylated by Cdk1 in vitro. (A) Schematic diagram of Cdc13 illustrates its domain structure and potential S/T phosphorylation sites. The telomerase recruitment domain (RD) and DNA-binding domain (DBD) are indicated. (B) Cdk1-mediated phosphoryla- tion of the telomerase recruitment domain of Cdc13 in vitro. Cdc28-HA and the kinase-dead Cdc28K40L-HA were puriﬁed by immunoprecipitation with an HA antibody. Immunoprecipitated kinases were analyzed by SDS–PAGE and western blotted with the HA antibody (shown at the bottom). GST-Cdc13(1–252), GST-Cdc13(252–491), GST-Cdc13(601–781) and GST alone were puriﬁed. IP-kinase assay was performed as described (30) with [g-32P] ATP and phosphorylated proteins were detected by autoradiography (shown on top). The kinase reactions were also Coomassie stained (shown below) to conﬁrm that lanes were equally loaded. (C) T308 is phosphorylated by Cdk1 in vitro. GST-Cdc13(252–491)-T308A, S336A and S460A were subjected to the IP-kinase assay as described in (B). (D) S336 is phosphorylated by Cdk1 in vitro. GST-Cdc13(252–491)-T308A, GST- Cdc13(252–491)-T308S336A and GST-Cdc13(252–491)-T308S460A were subjected to the IP-kinase assay as described in (B). (E) T308 is critical for the Mec1-mediated T306 phosphorylation in vitro. The IP-kinase assay was performed as described in (B) with equal amount of GST-Cdc13 (276–332) wild-type, S306A and T308A using immunoprecipitated Cdc28-HA or Mec1-HA. Samples were collected at 10-min intervals for 60 min. Telomere analysis of cdc13 mutants To identify potential cellular functions of Cdk1-mediated Cdc13 phosphorylation, we ﬁrst analyzed whether muta- tions in these phosphorylation sites would aﬀect the growth and replication of telomeres. In order to examine the phenotypes, point mutations were introduced into the untagged chromosomal copy of CDC13. As shown in Figure 5, single mutation at residue T308 to A caused telomere shortening. A slight growth defect was also observed in T308A cells, which was further reﬂected in its cell cycle proﬁle (Figure 4). These ﬁndings indicated that phosphorylation on this residue is important for tel- omere maintenance. Moreover, single mutation S336A or double mutation T308S336A maintained similar telomere lengths to those of wild-type and T308A, respectively (Figure 5). These data indicated that the Cdk1-mediated regulation on S336 does not contribute to the telomere maintenance. While single mutation at residue 308 from T to A caused telomere shortening and a cell cycle delay, replacement of T308 with aspartic acid (D) or glutamic acid (E) partially recovered these phenotypes (Figures 5 and S1, and data not shown). These results further con- ﬁrmed that the negative charge on this residue is impor- tant for telomere maintenance. We next examined whether predisposed telomere damage would exacerbate the Cdk1-mediated defect on Cdc13. Cdc13 and Ku are both essential for telomere integrity. The Ku complex is telomere associated and helps to protect telomeres from degradation and from inappropriate recombination (50,51). Deletion of Ku causes telomere shortening. We measured the eﬀect of T308A and S336A mutations on telomere length mainte- nance in the Ku mutant background. Mutations of T308A and S336A in the Ku mutant cells did not lead to further shortening or senescence phenotype (Figure 5 and data not shown). These results indicated that Cdk1-mediated Cdc13 phosphorylation no longer plays a role in modulat- ing telomere homeostasis in the absence of Ku. We then asked if Cdk1-mediated Cdc13 phosphoryla- tion plays a synergistic eﬀect with Tel1- or Mec1-mediated regulation on telomere lengths. Mutations of CDC13 were created in the tel1 and mec1 cells. Deletion of MEC1 leaded to mild telomere shortening, while deletion of TEL1 caused severe telomere shortening. A further reduction on telomere lengths was observed in mec1 cdc13-T308A and mec1 cdc13-T308S336A cells, but not in tel1 cdc13-T308A and tel1 cdc13-T308S336A cells (Figure 5). These results suggested that in the absence of