Misregulation of telomerase is associated with aging and cancer.49
In budding yeast, telomerase is strictly controlled, occurring only in the late S/G2 phase and acting preferentially on short telomeres. Cdc13 is a multifunctional telomere binding protein that acts as a type of molecular switch with its binding partners determining if it forms a protective cap (Stn1, Ten1) or promotes telomerase elongation (Est1) or C-strand resynthesis (Pol α). Given its multiple binding partners and functions, Cdc13 is a good candidate for regulation by phosphorylation.
We determined in vivo
phosphorylation sites on both WT and the telomerase defective Cdc13-E252K protein in asynchronous, G1 arrested (telomerase inactive), and G2/M arrested (telomerase active) cells using a new label-free nanoLC-MS/ MS approach to quantitate relative levels of phosphorylation at individual residues. This approach provides an alternative to methods that use internal peptide standards to provide the absolute abundance of a given modification.50
Our approach is useful when, as with the project described here, the sites of phosphorylation are not known, since our label-free nanoLC-MS/MS method allows the simultaneous identification and relative abundance determination of phosphorylation sites. The relative abundance of a given modification was determined by comparing across samples (i.e., between WT and mutant Cdc13 or WT from G1 versus WT from G2/M phase cells) but never between different peptides. Estimating changes
in relative abundances is valid, since the differences in ionization efficiency resulting from different PTMs on the same peptide sequence should be consistent across different runs. Indeed, our quantitative data were highly reproducible with small differences in relative abundances of modified residues across multiple technical and biological replicates ( and ).
Because our analysis was able to document modified isoforms that were present over a wide range of concentrations (), this approach should be generally useful for analysis of other hypermodified proteins. While identifications based on shotgun tandem MS and blind database searching only would have missed many lower abundance modifications, our method was able to identify these sites based on their relative retention time and MS isotopic distribution. If we had used only a traditional tandem MS search algorithm, we likely would have identified a large number of additional false sites of modification on the same peptide sequences because modified isoforms share a significant number of fragment ion peaks in tandem MS. Since our method simultaneously uses MS, MS2, and retention time to identify sites and degrees of phosphorylation, the false positive rate was low and even allowed discovery of sites in the absence of tandem MS. It should be noted here that all modified isoforms identified without available tandem MS were later targeted for MS2 in subsequent nanoLC-MS/MS runs to confirm their assignments.
We identified 21 phosphorylation sites on the 924-amino acid Cdc13 (summarized in , ). Of these 21 sites, 17 had not been detected previously in vivo
(one of the 17 sites, S225, was seen on protein phosphorylated in vitro36
). All of the phosphorylation sites are located at residues predicted to be in flexible regions of Cdc13 that are more likely to be solvent accessible.28
In contrast, phosphorylation sites detected only in the Cdc13 DBD (S467, S472, T473) were not on exposed surfaces and probably are not relevant to intact protein. However, S170, a residue that was only phosphorylated in OB1-RD, is on a disordered loop,28
indicating it is on the protein surface in the context of OB1-RD but not in the context of fulllength Cdc13. Therefore, the S170-containing surface may be responsible for a long-range interaction within the Cdc13 protein.
The phosphorylated residues were not spread uniformly throughout the protein. To our surprise, regions that make critical biological contacts, such as the core of the DBD and OB1, and the highly conserved N-terminal half of RD that interacts with Est1, were devoid of detectable phosphorylation (, ). Perhaps the phosphorylation sites surrounding these interaction interfaces do not regulate these functions as an on-off switch, as do the phosphorylation sites on the CDKs.51,52
Rather, we suggest they are present to modulate and coordinate these interactions, or mediate long-range interactions between Cdc13 domains.
There was a cluster of ten phosphorylation sites within a 47 amino acid region at the boundary of the RD/OB2 domains (aa 306 to 352; ). This cluster includes the two previously identified phosphorylation sites (S306 and T308) and the one identified here (S314) whose phosphorylation affects telomere length or telomerase action (). All three of these residues are located within the C-terminal half of the RD. The extension of this phosphorylation cluster into the OB2 domain suggests that OB2 may have a previously unidentified role in affecting telomerase recruitment in addition to its previously suggested roles as an effector of the affinity and sequence specificity of the DBD.53
Of particular interest, phosphorylation of two in vitro
identified Tel1/Mec1 targets, S249 and S255 within the RD,36
was not detected despite repeated attempts. The lack of detectable phosphorylation on these two residues was probably not due to false negativity or low sensitivity from the search algorithms, since we detected phosphorylation at two other Tel1/Mec1 sites, S225 and S306, as well as modifications present in ~0.01% of the sample (as in phosphorylation of 331-KLS
IR-343, ). A lack of in vivo
phosphorylation at these residues provides an explanation for seemingly contradictory results from earlier studies. S249 and S255 are both phosphorylated by Tel1 and Mec1 in vitro
, and changing both residues to alanine confers a telomerase defective phenotype.36
Nonetheless, a cdc13-Q250A Q256A
allele is not telomerase deficient.39
Our results support the interpretation that these residues are not Tel1/Mec1 substrates in vivo
. Since these two SQ motifs are extremely conserved in Cdc13 from closely related fungi,28,39
we propose that they play a structural, rather than a signaling, role. In this model, the telomerase-null phenotype of the cdc13-S249A S255A
allele is explained by a local loss of structural integrity rather than a lack of phosphorylation. These results emphasize the importance of determining sites of modification in vivo
even when genetic data are available.
To gain information about the importance of phosphorylation of given residues, we determined if modifications were correlated with cell cycle phase () and/or WT versus telomerase deficient protein (). There were no differences between mutant and WT protein in terms of sites of phosphorylation. This finding may indicate that regulated phosphorylation occurs upstream of telomerase recruitment. Alternatively, quantitative rather than qualitative differences in phosphorylation may explain functional differences, as there were six sites that showed different levels of phosphorylation between WT and mutant Cdc13 (S314, S324, S333, S336, S347, S883). In addition, there were five residues (T308, S314, S324, S341, S708) that showed cell cycle-specific differences. In both cases, these differences could be substantial. For example, phosphorylation of S314, S324 on the peptide 311-KTS
GSK-327 was 6-fold more abundant in WT protein in the G2/M versus the G1 phase () and >4,000-fold less frequent in mutant Cdc13 (). As the abundance of S314 phosphorylation was affected both by the cdc13-2
mutation and by the cell cycle, we mutated this site to alanine or aspartate. Although S314 mutations conferred effects on telomere length, consistent with phosphorylation at this residue having a negative effect on telomerase (), the effects were subtle, similar to what has been seen previously for mutation of the Cdk1 target T308.37,38
Thus, individual phosphorylation events may act redundantly to influence telomere length, and multiple mutations may be needed for large effects. Consistent with this view, we observed that the S314A S324A double mutation resulted in longer telomeres while either mutation alone had no effect (). Our analysis provides many more candidates for residues that can be mutated singly and in combination with other mutations for effects on telomerase recruitment and telomere structure.
CDK/MAPKs phosphorylate S/TP motifs and ATM/ATR kinases phosphorylate S/TQ motifs. There are seven S/TP and ten S/TQ motifs in Cdc13. Five of the seven S/TP motifs were phosphorylated in vivo
, but only two were CDK1 targets in vitro
Four of the ten S/TQ motifs can be phosphorylated by Tel1 or Mec1 in vitro
but only two (S225, S306) appeared to be phosphorylated in vivo
. Clearly, factors other than sequence specificity contribute to substrate recognition and phosphorylation in vivo
. The three S/TP motifs that are not direct CDK1 targets in vitro
but were phosphorylated in vivo
may be phosphorylated by another kinase with the same consensus motif. Indeed, the kinases responsible for most of the detected phosphorylation events (17 out of 21) could not be determined by the sequence context of the modified residue. However, a recent high-throughput study identified three kinases, Cak1, Hsl1, and Vhs1, that affect cell cycle progression and that also interact with Cdc13.54
All three are excellent candidates to explain phosphorylation of residues for which there is, as yet, no evident responsible kinase.
This study has provided a wealth of knowledge about Cdc13 phosphorylation, but there is still much to learn. Although our study identified 17 novel phosphorylation sites, the biological functions of most of these are unknown. A full understanding of their importance will likely involve analysis of multiply modified proteins to circumvent biological redundancy. By using information on the abundance of phosphorylation events as a function of the cell cycle and in vivo function, we provide a first step for a rational next stage analysis.