Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries

Abstract

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Figure 1:. Large scale phosphoproteomics of Tel1-dependent phosphorylation.

(A) Overview of DNA lesions activating PIKKs, with Mec1 and Tel1 canonically becoming activated by different forms of DNA damage. (B) Schematic of large-scale phosphoproteomic pipeline. Large SILAC yeast cultures are required to obtain sufficient starting material for tryptic digestion and phosphopeptide enrichment. Extensive HILIC prefractionation reduces sample complexity and allows for in-depth LC-MS/MS analysis, followed by database searching and Bowtie replicate filtering. (C) Bowtie filtering of forward and reverse replicates performed for each experimental condition, in which experimental conditions are inverted between SILAC channels. The replicates are then plotted against one another to determine which phosphopeptides feature appropriately inverting quantitative ratios, discarding any that do not invert. (D) The quantitative ratios averaged between forward and reverse replicates for 17,486 phosphopeptides that passed Bowtie filtering. Among these are phosphopeptides on Yku80 and components of the MRX complex, most of which are found to be Tel1-dependent.

Figure 2:. Tel1-dependent phosphorylation events are enriched for the S/T-Q motif on proteins involved in the DNA damage response and telomere maintenance.

(A) The averaged quantitative ratio for 17,486 phosphopeptides that passed Bowtie filtering with S/T-Q sites highlighted in green. Tel1-dependent phosphorylation events are over 3X enriched for the canonical preferred PIKK S/T-Q motif. (B) Comparison of the number of Tel1-dependent phosphorylation events detected in the current study versus our previous investigation of the Tel1 signaling network. (C) The top 10 GO terms (minus terms containing “process”) enriched among proteins featuring Tel1-dependent S/T-Q sites. (D) Telomere-related GO term enrichment among proteins featuring Tel1-dependent S/T-Q sites.

Figure 3:. A novel D/E-S/T motif is more enriched than the S/T-Q motif among Tel1-dependent phosphorylation events.

(A) Heatmap illustrating enrichment of amino acid residues at loci relative to phosphorylation events found to be Tel1-dependent. Acidic residues, particularly aspartic acid, are found to be enriched in the −1 locus. Enrichment for the S/T-Q motif is also illustrated here. (B) The averaged quantitative ratio for 17,486 phosphopeptides that passed Bowtie filtering with D/E-S/T sites highlighted in pink and the sites with the combined motif (D/E-S/T-Q) highlighted in purple. (C) Prevalence of four phospho-motifs among subsets of the Tel1 signaling dataset. The most enriched motif in phosphopeptides that decreased upon deletion of Tel1 was D/E-S/T, with S/T-Q and D/E-S/T-Q motifs also showing high enrichment among this same subset of the data. (D) The top 10 GO terms (minus terms containing “process”) enriched among proteins featuring Tel1-dependent D/E-S/T sites. (E) Telomere-related GO term enrichment among proteins featuring Tel1-dependent D/E-S/T sites.

Figure 4:. The D/E-S/T motif is more enriched in Tel1-dependent phosphorylation events compared to Mec1-dependent phosphorylation events.

(A) Tel1 phosphoproteomic data from the current study plotted against Mec1 phosphoproteomic data from Sanford et al. 2021 in which both Mec1 and adaptor protein Rad9 were deleted to investigate Mec1-dependent signaling events. Increased abundance of Tel1-dependent phosphorylation events upon loss of Mec1 supports the notion that Tel1 engages in some amount of compensatory signaling when Mec1 activity is compromised. (B) Enrichment of the S/T-Q motif among Tel1- and Mec1-dependent phosphorylation events illustrates a higher enrichment for this motif among Mec1-dependent phosphorylation events. (C) Enrichment of the D/E-S/T motif among Tel1- and Mec1-dependent phosphorylation events strongly suggests that D/E-S/T motif enrichment is exclusive to Tel1-dependent signaling. (D) Structural superimposition of yeast Mec1 and Tel1 (PDB IDs 7Z87 and 5YZ0 respectively) highlighting the substrate-binding pocket and key residues involved in the recognition of peptide substrates via their positions −1 (Tel1 H²⁵²⁶, R²⁵⁴⁴, and N²⁶¹⁶; Mec1 R²¹³⁹, K²¹⁵⁶ and E²²²⁸) and +1 (Tel1 L²⁶⁴² and Y²⁷⁸⁰; Mec1 L²²⁵⁴ and Y²³⁶¹). Inserts on the right show the electrostatic potential of surfaces around the −1 binding pocket for each kinase, blue indicating positive charge and red indicating negative charge. In all figures, the peptide substrate shown in yellow is superimposed from a structure of ATM bound to p53 (PDB ID 8OXO) with the original Leu at the −1 position remodeled as Asp (asterisk). ATP loops of kinases are omitted to facilitate visualization. Carbon atoms are tan for Mec1 and green for Tel1.

Figure 5:. Tel1-dependent D/E-S/T phosphorylation events are not dependent on downstream kinases Hrr25, Dun1, or Rad53.

(A) S/T-Q sites on kinases Hrr25 and Dun1 were found to be Tel1-dependent. (B) Sequence map of Hrr25 showing where an analog-sensitivity mutation was made (Ile 82) as well as the location of the Tel1-dependent S/T-Q phosphorylation event (Ser 438). (C) Sequence map of Dun1 showing the location of the Tel1-dependent S/T-Q phosphorylation event (Ser 345). (D) Tel1 phosphoproteomic data plotted against Hrr25 phosphoproteomic data generated using acute kinase inhibition via analog sensitivity mutation, with no significant co-dependence of signaling events observed. The D/E-S/T motif was not found to be enriched among Hrr25-dependent phosphorylation events. (E) Tel1 phosphoproteomic data plotted against Dun1/Rad53 phosphoproteomic data generated using deletion of both kinases, with no significant co-dependence of signaling events observed. The D/E-S/T motif was not found to be enriched among Dun1/Rad53-dependent phosphorylation events.

Figure 6:. An expanded network of Tel1-dependent phosphorylation targeting S/T-Q and D/E-S/T motifs.

(A) Overview of Tel1-dependent phosphorylation events that were able to be localized and their motifs. *D/E-S/T and S/T-Q motif prevalence calculated excluding the combined motif, D/E-S/T-Q. (B) Tel1-dependent phosphorylation events with either the S/T-Q or D/E-S/T motif implicated in the DNA damage response, DNA-templated transcription, telomere organization, or noncoding RNA processing. Proteins denoted as having “both” motifs either contain multiple sites with both motifs represented or at least one site with the combined motif, D/E-S/T-Q. (C) Breakdown of phosphorylation events implicated in telomere organization as denoted by panel B.