We have shown that Cdc13 is phosphorylated by Cdk1 in vivo
during late S to G2 phase of the cell cycle, a time in the cell cycle that coincides with the previously reported recruitment of the telomerase complex to telomeres (Schramke et al., 2004
; Taggart et al., 2002
). Our data also provide the first evidence that phosphorylation of Cdc13 is important in regulating the recruitment efficiency of the telomerase complex to telomeres by competing with the Stn1-Ten1 complexes during telomere elongation. Uncoupling of this cell cycle-dependent event by inactivation of Cdc13 phosphorylation results in less telomerase recruitment to telomeres during cell cycle progression and compromised telomere elongation.
To put these findings in context, we note that Cdk1 phosphorylation of Cdc13 at T308 will, in turn, need to be coordinated with other levels of Cdc13 regulation. In addition to Cdk1, the Tel1 and Mec1 kinases (homologues of mammalian ATM and ATR) have previously been shown to play important roles in telomere length maintenance. Tel1 and Mec1 control the DNA damage response in budding yeast through phosphorylation of proteins involved in checkpoint control (such as Rad53) and DNA replication (such as RPA) (Brush et al., 1996
; Sanchez et al., 1996
). In budding yeast, in contrast to Mec1, Tel1 plays a minor role in the cellular response to DNA damage, but a major role in telomere length maintenance. Loss of Tel1 results in telomere shortening (Lustig and Petes, 1986
). While tel1 mec1
double mutants have a senescence phenotype (Ritchie et al., 1999
), they have normal telomerase enzymatic activity (Chan et al., 2001
). Consistent with this observation, Tel1 and Mec1 are required for normal levels of association of Est1 and Est2 with telomeres, suggesting a potential role in promoting the recruitment of telomerase complex to telomeres (Goudsouzian et al., 2006
). Recent studies also showed that Tel1 kinase activity is crucial for preferential recruitment of telomerase to a shortened telomere (Chang et al., 2007
; Hector et al., 2007
; Hirano and Sugimoto, 2007
; Sabourin et al., 2007
). Furthermore, Tel1 and Mec1 can phosphorylate Cdc13 at multiple sites in vitro
. Single mutation of two of these in vitro
phosphorylation sites (Serine 249 and Serine 255) to alanine resulted in telomere shortening and double mutation of these two potential phosphoylation sites resulted in progressive telomere shortening and senescence in vivo
(Tseng et al., 2006
). While combination of Cdc13 T308A with S249A and S255A mutations did not result in obvious synergistic telomere shortening (Figure S11
), it is still conceivable that phosphorylation of Cdc13 by Cdk1 and by Tel1 and/or Mec1 may act synergistically to determine which telomere is elongated through preferential recruitment of a functional telomerase complex in late S to G2 phase. Further identification of potential Tel1/Mec1 dependent phosphorylation sites in Cdc13 and confirmation of these Tel1/Mec1 phosphorylation events in vivo
using phospho-specific antibodies will be important to probe their potential roles in telomerase action on telomeres.
Based on the results reported here and on previous data, we propose the following model: Cell cycle progression into late S phase results in telomere end processing and elongation of the G-strand overhang. Cdk1 phosphorylates Cdc13 at T308. As depicted in , the threonine 308 phosphorylated Cdc13 on the G-strand overhang results in preferential recruitment of the telomerase complex through its interaction with Est1, because the T308 phosphorylation biases the binding of Cdc13 toward Est1. Conversely, lack of T308 phosphorylation results in reduced interaction of Cdc13 and Est1, hence increasing the association of Cdc13 with the Stn1/Ten1 complex. Thus, this Cdk1 dependent phosphorylation of Cdc13 provides a mechanism that coordinates cell cycle progression with the recruitment of telomerase relative to the Stn1-Ten1 complexes to telomeres in late S to G2. Deletion of the Stn1 C terminus results in loss of Cdc13 binding, hence alleviating negative telomere length regulation by the Stn1-Ten1 complex and resulting in over-elongation of telomeres. The results using the stn1-ΔC199 mutant also argue against the cdc13-T308A mutation affecting the processing of single-strand G-rich overhangs. If cdc13-T308A caused an end processing phenotype, we should have seen a telomere length difference between CDC13 and cdc13-T308A yeast strains in the stn1-ΔC199 background. In addition to Cdk1 dependent phosphorylation, we suggest that the preferential recruitment of Tel1 to short telomeres and subsequent phosphorylation of Cdc13 (at serine 249 and serine 255 or other unidentified phosphorylation sites) by Tel1/Mec1 on the short telomere may preferentially synergize with the cdk1 phosphorylation to promote the recruitment of telomererase complex (likely through preferential binding to Est1) to that short telomere in a cell cycle-dependent manner ().
A schematic model for potential function of Cdc13 threonine 308 phosphorylation
Previous data have shown that loss of Stn1 results in significant telomere elongation, while Stn1 overexpression results in telomere shortening (Chandra et al., 2001
; Dahlseid et al., 2003
; Grossi et al., 2004
). It was therefore proposed that the interaction of the Stn1-Ten1 complex with Cdc13 could compete with the recruitment of telomerase complex by Cdc13, thereby inhibiting telomerase dependent telomere addition (Chandra et al., 2001
; Pennock et al., 2001
). Consistent with this hypothesis, we found that the telomere shortening induced by loss of Cdk1 dependent phosphorylation of Cdc13 is fully alleviated in stn1-ΔC199
yeast, in which the interaction of Stn1 and Cdc13 is disrupted. Hence, since Cdc13 can no longer interact with Stn1, phosphorylation of Cdc13 threonine 308 is no longer necessary to promote the competing interaction with Est1. Using ChIP analysis, we also detected recruitment of Stn1 to telomeres during late S to G2 phase of the cell cycle in Cdc13 or cdc13-T308A
yeast. Such cell cycle-dependent recruitment of Stn1 to telomeres was recently reported by others (Puglisi et al., 2008
). Interestingly, the magnitude of Stn1 telomere binding is independent of telomere length (Puglisi et al., 2008
). Our data also suggest that the interaction of both Cdc13-3HA and Cdc13-T308A-3HA with overexpressed 13myc-Est1 is stronger than with 13myc-Stn1, so that even with the Cdc13 threonine 308 to alanine mutation, Est1 can still recruit the telomerase complex in the presence of Stn1-Ten1 competition, albeit less efficiently. This can explain why we only see a moderate telomere shortening in cdc13-T308A
yeast, but not an ever shorter telomere (EST) phenotype or senescence.
It will also be of interest to find out whether similar regulatory mechanisms modulate telomerase action in human cells. Such regulatory mechanisms may provide new targets for potential cancer therapy and for anti-aging research.