Novel phosphorylation sites in the S. cerevisiae Cdc13 protein reveal new targets for telomere length regulation

Abstract

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. Multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics.

Figure 1

Schematic illustration of the LC-MS/MS experimental and computational approach for phosphorylation identification and quantitation.

Figure 2

(A) Schematic illustration of Cdc13 domain organization. (B) Phosphatase treatment of Cdc13. 1.5 µg of purified Cdc13 was incubated with 40 U of λ protein phosphatase (NEB, lanes 2 and 3) and 1 mM MnCl₂ (lanes 3 and 4) in 1X vendor-supplied buffer at 30 °C for 30 min. Samples were resolved on 8% SDS-PAGE gel and stained with Coomassie blue.

Figure 3

Summary of Cdc13 phosphorylation sites with respect to domain structure.

Figure 4

Cell cycle analysis of phospho-peptide levels from WT Cdc13. Results are presented as averages from at least two independent measurements. Error bars represent 1 standard deviation. Peptides that showed significant difference in abundance between G1 and G2/M phases are indicated by an asterisk.

Figure 5

Comparison of phospho-peptide quantification between WT Cdc13 and Cdc13-E252K mutant (MT). Results are presented as the Log2 value of the WT/MT ratio, and the error bar represents 1 standard deviation. Peptides that showed significant difference in abundance between WT and mutant are indicated by an asterisk.

Figure 6

Telomere length analysis of Cdc13 phosphorylation mutants. cdc13Δ cells carrying WT or mutant Cdc13 on a CEN TRP1 plasmid were analyzed for their telomere lengths as described in Methods. Mutants analyzed are (A) S314A, S314D, S324A, S324D, S314A S324A, and S314D S324D, and (B) cdc13-2 and cdc13-2 S314A mutants. (C) Western blot analysis of myc9-tagged Cdc13 mutants expressed from Cdc13 promoter on a CEN TRP1 plasmid (upper panel). The same blot was probed with anti-α tubulin as loading control (lower panel). UT: untagged.