3610 Nucleic Acids Research, 2009, Vol. 37, No. 11 An intriguing ﬁnding of this study is that Cdk1- mediated phosphorylation is required for, in addition to optimal telomerase recruitment, the proper turnover of Cdc13. While the protein level of Cdc13 is well regulated throughout the cell cycle, which peaks at late S/G2 phase and drops at the G1 phase, we found that phosphorylation at T308 is critical for the rapid degradation of Cdc13. What kind of disturbances would be caused by under- degraded Cdc13 existing in the nucleus after the G2 phase of the cell cycle is still unclear. Further identiﬁcation of the proteolysis pathway of Cdc13 will be important to study this eﬀect by uncoupling the function of tuning the protein level of Cdc13 from the function of telomerase recruitment of Cdc13 contributed by Cdk1. Phosphoproteomics has been used to proﬁle thousands of phosphorylation in cells. Using mass spectrometry to analyze phosphorylation sites in wild-type S. cerevisiae, both S306 and T308 of Cdc13 were found to be phos- phorylated in vivo (57). In our previous study, mutations at the Tel1/Mec1 phosphorylation sites did not lead to an alteration of protein levels of Cdc13 (30). Interestingly, the T308 residue is very close to a Mec1-regulated site, S306, and T308 is critical for the full activity of Mec1 on S306, at least in our in vitro assay (Figure 2E). These results indicate that a conformational change at T308 might mod- ulate Mec1-mediated Cdc13 phosphorylation. However, the Cdk1-mediated telomere shortening observed in the T308 strain was abolished in the absence of Tel1. Altogether these data suggest that Tel1 makes a greater impact, while Mec1 and Cdk1 kinases play supporting roles, to optimize the reaction of telomerase recruitment. Previous studies showed that Cdk1 mutation could produce long single strand G-tails at telomeres (26,28), which was not observed in our T308S336A cells (data not shown). It remains possible, in addition to Cdc13, Cdk1 may target other proteins during telomere replica- tion. In that case, the telomeric eﬀect of Cdk1 on Cdc13 (26,28) should be masked in the cdc28-4 cells, that is, the cdc28-4 mutation causes a greater telomeric alteration than cdc13-T308A. Further studies to illustrate other tar- gets of Cdk1 in telomere replication will be required in order to address this possibility. SUPPLEMENTARY DATA Supplementary Data is available at NAR Online. ACKNOWLEDGEMENTS We thank Virginia Zakian and Huilin Zhou for the discus- sion and sharing of their unpublished data. We also thank Drs Steven Reed, David Morgan, Daniel Gottschling, Virginia Zakian, John Diﬄey, Orna Cohen-Fix and Katsunori Sugimoto for providing plasmids and strains. 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