Nucleic Acids Research, 2009, Vol. 37, No. 11 3605 Figure 1. Cdk1-dependent phosphorylation of Cdc13 in vivo. (A) MEC1/TEL1-independent phosphorylation of Cdc13. Cdc13 was chromosomally tagged with Myc9. Lysates from wild-type and mec1 tel1 strains were immunoprecipitated by a Myc antibody and incubated in PPase buﬀer, PPase buﬀer with PPase or PPase buﬀer containing PPase and PPase inhibitors (PI). Reactions were subjected to a 5.7% SDS–PAGE and transferred for western blot analysis using a Myc antibody. Non-, hypo- and hyper-phosphorylated Cdc13 are marked with none, single and double asterisks, respectively. (B) G2/M phase-speciﬁc phosphorylation of Cdc13. Lysates from a factor- or nocodazole-treated cells were extracted by TCA precip- itation and Cdc13 phosphorylation was analyzed by western blot analysis. (C) Cell cycle-dependent phosphorylation of Cdc13. Overnight culture was grown to early log phase in YEPD, and arrested in G1 with a factor. When over 90% of the cells, as determined by microscopy, exhibited cell cycle arrest, cells were released into cell cycle. FACS analysis (showed at the right) of this sample indicated that cells had a 1N content of DNA. After release from a factor, cells were collected at 20-min intervals for 180 min and Cdc13 was analyzed by western blot analysis. (D) Cdk1-dependent phosphorylation of Cdc13. Cdc13 phosphorylation was analyzed in cdc28-4, SIC1 overexpressed and cdc28-as1 strains, which all can be manipulated to provide defective Cdk1 activities (37,38,40). cdc28-4 (a temperature sensitive mutant) was synchronized by nocodazole at 238C and shifted to 378C for 3 h; pGAL-Sic1 N transformants were treated with dextrose (D) or galactose (G) for 3 h, and W303 (wild-type) and cdc28-as1 were treated with or without 1-NMPP1 for 3 h. Lysates were extracted and Cdc13 phosphorylation was analyzed by western blot analysis. distinguishable in the Phos-tag gel (Figure 3B). Under the help of Phos-tag, increases of migration were detected in both T308A and S336 A (Figure 3B). These data indicated that both T308 and S336 are phosphorylated in vivo. Since previous studies demonstrated that Tel1 makes the major contribution to the activation of Cdc13 to recruit telomerase (31–33), to understand if the phosphor- ylation of T308 and S336 were Tel1-dependent, these mutants were introduced into the tel1 cells. As shown in Figure 3C, even in the absence of TEL1, reduction of the phosphorylation level was further observed in T308A mutation, comparing to the wild-type Cdc13. These ﬁnd- ings argued that Cdk1-mediated phosphorylation does not rely on the action of Tel1-mediated phosphorylation on Cdc13. We next examined whether T308 is authentically the Cdk1-phosphorylated site in vivo, T308A mutation was constructed in the cdc28-as1 strains. Compared to the normal cdc28-as1 strain, T308A mutation did not further convert the electrophoretic behavior of Cdc13 to a single sharper band with greater mobility (Figure 3D). Similar results were also observed in the Sic1-overexpressed strain (Figure 3D). Therefore, we concluded that Cdc13 is phos- phorylated by Cdk1 in vivo at T308. Cdk1 phosphorylates Cdc13 at the late S and G2 phases for proper cell cycle progression Tel1 is highly enriched at short telomeres from S through G2 phase (31–33). To determine whether Cdk1 targets Cdc13 at a similar timing to the action of Tel1, the status of Cdc13 phosphorylation was followed throughout the cell cycle. As observed in Figure 4, Cdc13 hyperpho- sphorylation was peaked at the late S and G2 phases, which is identical to the timing of the action of Tel1 as previously reported (31–33). This peak was diminished in the cdc13-T308A strain, further indicating that Cdk1 plays a critical role on the hyperphosphorylated peak from late S to G2 phase observed in the wild-type cells (Figure 4A). Following release from the arrest, a slight cell cycle delay was observed in the cdc13-T308A strain (Figure 4B, 40 min). In the absence of TEL1, a hyperphosphorylated Cdc13 peak at the late S and G2 phases was still observed (Figure 4A). This abnormal cell cycle progression was not due to activation of the checkpoint (Figure 4C). These results suggested that Cdk1-mediated phosphorylation of Cdc13 shares the same cell cycle stage with the Tel1/ Mec1-mediated telomerase recruitment. However, this phosphorylation is Tel1-independent and required for accurate cell cycle progression.