Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation

Abstract

Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.

Figure 1. Overview of methodology and modifications identified

Two sets of experiments were performed. The first was a qualitative assessment (non-SILAC), and the second, as illustrated here, was a SILAC experiment, in which cells from control and proteasome-inhibited cultures were mixed. We utilized two enrichment strategies for both experiments (a-b). (a) First, samples were enriched for Ub proteins. Non-Ub proteins (dashed lines) were digested and enriched for phosphopeptides. Ub proteins (solid lines) were further enriched for diGly-peptides and for phosphopeptides. (b) Proteins were digested to peptides and enriched for doubly charged peptides via strong-cation exchange (SCX). SCX fractions were then enriched for diGly-peptides prior to mass spectrometry analysis. (c-f) Illustration of four cases of how the same protein can exist in different combinations of modification states. (c) displays a phosphorylation site unique to the ubiquitylated protein isoform, while (d) and (e) display that some phosphorylation sites are found in both ubiquitylated and non-ubiquitylated isoforms. Finally, (f) displays a phosphorylation site unique to non-ubiquitylated proteins. (g) Overlap of phosphorylation sites and phosphoproteins from the Non-Ub (dashed lines) and Ub samples (solid lines).

Figure 2. Evolution and functional enrichment of modification sites

(a) GO enrichment analysis of ubiquitylated phosphoproteins vs. non-ubiquitylated phosphoproteins. GO enrichment was performed using Babelomics4. (b) For different populations of proteins we calculated the ratio of conserved phosphorylation sites over conserved amino acids from random sampling of the same number of phosphor-acceptor residues (Ser, Thr). Whiskers represent the lowest/highest data point within 1.5 times the interquartile range (*P = 0.0027, Kolmogorov-Smirnov test).

Figure 3. The effect of proteasome inhibition on protein and PTM site abundance, and properties of regulated phosphorylation sites

(a) Log₂ distributions of proteins and ubiquitylation sites abundance changes in response to proteasome inhibition (50 μm Bortezomib for 1 hour). (b) Log₂ distributions of phosphorylation and ubiquitylation sites abundance changes. (c) The distribution of sites matching a phosphodegron motif at various intervals of abundance changes after proteasome inhibition. (d) Half-life distributions for all protein identifications are compared to those proteins containing sites increasing in abundance by >75% (*P = 6.3 × 10⁻³), whiskers represent the 10^th and 90^th percentile. Protein half-life values were obtained from the literature. (e) The pairwise spatial distance (i.e. ångströms between alpha carbons) between either phosphorylation sites co-occurring with lysine ubiquitylation sites (Ub), or between the position of these same ubiquitylation sites to phosphorylation sites identified on non-ubiquitylated isoforms (Non-Ub). Distances between all pairs (white boxes) are compared to the distances between pairs for which both sites increase in abundance upon proteasome inhibition (grey boxes). (P < 0.0043). Whiskers represent the lowest/highest data point within 1.5 times the interquartile range.

Figure 4. Validation of a new phosphodegron and ubiquitin mediated regulation of phosphorylation machinery

(a-b) Yeast cells were treated with galactose to induce Swi5 expression and incubated with either cycloheximide to measure protein degradation (n = 3) (a) or Bortezomib to measure protein accumulation (b). Aliquots were taken at the indicated times and protein expression was monitored by immunoblotting with anti-HA to detect Swi5, or anti-α-tubulin for a loading control. Degradation levels measured from immunoblotting were quantified in triplicate, while Bortezomib induced accumulation was quantified from a single replicate. (b) Enrichment of ubiquitylation sites in the structure of the kinase domain. A representative structure pertaining to PDB ID: 1QMZ is shown. A total of 72 proteins showed enrichment of Ub sites within the Gly-rich loop or in Region A adjacent to the activation loop. Graph in the right represents the numeric values of enrichment over random for different positions within the kinase domain. Relevant regions within the domain are indicated. (c) Enrichment of ubiquitylation sites within the 14-3-3 phosphorylation-binding domain (PDB ID: 3MHR, 19 proteins from either human, mouse, or the yeast S. cerevisiae). The embedded table represents ubiquitylation sites identified in the phospho-binding and dimerization regions of the two 14-3-3 domain containing proteins in S. cerevisiae and the percent increase in abundance upon proteasome inhibition measured in this study.