3604 Nucleic Acids Research, 2009, Vol. 37, No. 11 Immunoprecipitation-kinase analysis Log phase cells were lyzed and immunoprecipitated by incubation with an HA antibody and protein A beads. The immunoprecipitation (IP)-kinase assay was per- formed as described (48). Cdc28-HA or the kinase-dead Cdc28K40L-HA were puriﬁed from a protease-defected strain, BJ2168, carrying plasmid pKB174 (CDC28-HA) or pKB173 (cdc28K40L-HA). KSC1333 (MEC1-HA sml1::HIS3) (48) was kindly provided by Katsunori Sugimoto. De novo telomere addition assay De novo telomere addition assay was performed as previously described (49). UCC5706 strain was kindly provided by Daniel Gottschling. CDC13, cdc13-T308A, cdc13-T308D and cdc13-T308E were generated by one- step chromosomal insertion using XhoI-digested pRS304CDC13, pRS304cdc13-T308A, pRS304cdc13- T308D and pRS304cdc13-T308E, respectively. RESULTS Cdk1-dependent Cdc13 phosphorylation in vivo Under the examination of Tel1/Mec1-mediated phosphor- ylation, we observed a TEL1/MEC1-independent phos- phorylation of Cdc13 (Figure 1A). In order to elucidate how these phosphorylations mediate other functions of Cdc13 and to identify the kinase that contributes to TEL1/MEC1-independent phosphorylation, we ﬁrst investigated whether Cdc13 was phosphorylated in a cell cycle-dependent manner. Western blot analysis showed that Cdc13 is regulated in a cell cycle-dependent manner (Figure 1B) and hyperphosphorylated from late S (40 min in Figure 1C) to G2 (60 min in Figure 1C) stages. To determine whether Cdk1 is involved in this cell cycle- dependent phosphorylation, we examined Cdc13 phos- phorylation in cdc28-4, SIC1 overexpressed and cdc28-as1 strains. All three strains can be manipulated to equip defective Cdk1 activity (37,38,40). The migration of Cdc13 in gels was faster in these Cdk1-crippling back- grounds (Figure 1D), suggesting that Cdc13 might be phosphorylated in a CDK1-dependent manner in vivo. The telomerase recruitment domain of Cdc13 is phosphorylated by Cdk1 in vitro One apparent hypothesis for the CDK1-dependent Cdc13 phosphorylation is that Cdc13 may be directly phosp- horylated by Cdk1. The CDK family of kinases has a preference for phosphorylation of serine in SP motifs or threonine in TP motifs (25). A total of seven such motifs were identiﬁed in Cdc13 (Figure 2A). Two of these sites in Cdc13 were found in the telomerase recruitment domain (T308 and S336) (Figure 2A). For the ﬁrst step in deter- mining whether Cdc13 is a direct substrate of Cdk1, we tested whether Cdk1 could phosphorylate Cdc13 in vitro. Cdk1 immunoprecipitates were incubated under in vitro kinase conditions in the presence of g-[32P]-ATP and recombinant GST-Cdc13(1–252), GST-Cdc13(252–491) or GST-Cdc13(601–781) covering all seven Cdk1 consensus target sites. The reactions were subjected to SDS–PAGE and autoradiography (Figure 2B). The phos- phorylated GST-Cdc13(252–491) subunit was observed, while neither GST alone nor other GST-Cdc13 proteins showed detectable phosphorylation by Cdk1 (Figure 2B). To conﬁrm that Cdk1 kinase activity was indeed necessary for Cdc13 phosphorylation, assays were conducted using extracts isolated from a mutant strain expressing an HA- tagged Cdk1-kd protein with the amino acid lysine 40 mutated in its catalytic domain (41) (Figure 2B). Based on assays using equivalent amounts of protein, inactiva- tion of Cdk1 kinase activity completely eliminated Cdc13 phosphorylation. These results demonstrated that Cdk1 kinase phosphorylates Cdc13 directly. There are three S/TP sites (residue 308, 336 and 460) within the 225–491 fragment of Cdc13. To determine the exact phosphorylation sites, we puriﬁed mutant GST- Cdc13 containing single or double alanine (A) mutations at these sites. These substrates were then used in the IP- kinase assay. Compared to wild-type Cdc13, phosphory- lation of the T308A Cdc13 subunit was drastically reduced (Figure 2C). These results imply that T308 is the major phosphorylation site of Cdc13. Double mutations at T308S336A, but not at T308S460A, caused a further reduction of phosphorylation, comparing to T308A alone (Figure 2D). Therefore, our data suggested that the phos- phorylation sites of Cdc13 by Cdk1 are located at amino acids 308 and 336. Since the Cdk1-mediated regulation on residue T308 is very close to a Mec1-mediated regulation on S306 of Cdc13 (30), we asked whether Cdk1 would modulate the action of Mec1 on Cdc13, or vice versa. As shown in Figure 2E, Cdk1 was able to phosphorylate Cdc13-S306A and Mec1 was also able to phosphorylate Cdc13-T308A. However, an attenuated Mec1-mediated phosphorylation was observed in Cdc13-T308A. On the other hand, the mutation on S306 made no major impact on the Cdk1-mediated modiﬁcation on T308. These ﬁndings indi- cated that the Cdk1-mediated phosphorylation might be critical to promoting the action of Mec1 on Cdc13. The telomerase recruitment domain of Cdc13 is phosphorylated by Cdk1 in vivo To verify whether residues 308 and 336 are indeed Cdk1-mediated phosphorylation sites in vivo, we created Cdc13 mutant strains at these sites. Compared to the wild-type strain, the cdc13-T308A, cdc13-S336A and cdc13-T308S336A mutants retained residual levels of phosphorylated Cdc13 isoforms (Figure 3A), which should be contributed from Tel1/Mec1-mediated phos- phorylation. However, hyperphosphorylated forms of Cdc13 in cdc13-T308A and cdc13-T308S336A cells were reduced at the G2/M stage (Figure 3A), suggesting that T308 is phosphorylated in vivo. Due to the high molecular weight of Cdc13, the resolution of regular SDS–PAGE was not high enough to discern multiple phosphorylated forms of Cdc13. Therefore, a phosphor-speciﬁc gel using acrylamide-pendant Phos-tagTM (Phos-tag Consortium) (45) was applied to augment the diﬀerence of phosphor- ylation. Phosphorylation on T308 and S336 became more