Systematic functional prioritization of protein posttranslational modifications

Abstract

Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.

Figure 1. Evolutionary properties of eukaryotic post-translational modification sites

A) We analyzed the conservation of 10 phosphoproteomes against that of H.sapiens using protein alignments of 1-to-1 orthologs. For each species, we compared the conservation of the phosphoacceptor residues (i.e. sequence conservation) with the conservation of phosphorylation site based on the MS experimental evidence. A random expectation or null model for each case was determined based on the random shuffling of phosphosite positions within each human protein. B) Ratio of observed conservation over random expectation. C) Ratio of conservation over random expectation ratio for different PTMs. D) Predicted coverage and conservation in H.sapiens for 12 different previously reported phosphoproteomics experiments for S.cerevisiae. E) To access the impact of data quality on the conservation values we made use of different criteria to define subsets of S.cerevisiae phosphosites. For each subset we calculated the observed conservation in H.sapiens as well as the expected value based on random sampling.

Figure 2. Association of protein phosphorylation with lysine post-translational modifications

A) A Venn diagram representing the overlap of the different lysine modified proteins with the human phosphoproteome. While 33% of the human proteins are phosphorylated, 71% of the acetylated proteins, 69% of the ubiquitylated proteins and 89% of the sumoylated proteins are also phosphorylated. B) The fraction of the phosphoacceptor residues was plotted as function of the distance to modified lysine residues. Observed values were compared with expected values based on random sampling. C) Human phosphorylation sites that are near an acetylated lysine residue (<15 amino-acid distance) were more likely conserved in S. cerevisiae than average sites or an equivalent random sample. See also Figure S1 and Table S3.

Figure 3. Regulation of interface residues by PTMs

A) The conservation of all S.cerevisiae phosphosites in H.sapiens was compared with the conservation of phosphosites within PFAM domain interface residues (from the 3DID database) and at putative interface residues (from interaction models). B) We compared the conservation over random expectation of different PTMs at PFAM interface residues (from the 3DID database). The ratios of conservation over random expectation were compared using a Mann-Whitney rank test. (*) p-value <0.05. C) The Skp1:Met30 interaction model. The S162 position of the S. cerevisiae Skp1 was found to be phosphorylated in S. cerevisiae, H. sapiens, M. musculus and C. elegans. D) A protein complementation assay of cytosine deaminase activity reports on the Skp1:Met30 interaction. The phosphomimetic mutation reversed the growth pattern on selective media reporting on interaction strength by growing on +5-FC plates and without any observable growth on –Ura plates suggesting that the in vivo affinity of the Skp1:Met30 complex is reduced by phosphorylation. F) Skp1 S162D mutation shows a significant decrease in bound Met30 when compared to the S162A mutant by co-immunoprecipitation. E) Skp1 wild-type and mutants (S162A and S162D) were plated in the presence or absence of SAM. Overexpression of the S162D mutant resulted in poor growth, a phenotype that was relieved in the presence of SAM. See also Figure S2.

Figure 4. Conservation of interface phosphorylation can be achieved by regulation of different positions

A) We compared the conservation of all S. cerevisiae interface phosphorylation sites in H. sapiens (Interface residues) with the conservation of the phosphorylation of S. cerevisiae interfaces in H. sapiens without regard to the actual phosphosite position (Phosphorylated interfaces). B) A metric of phosphosite similarity (Methods) was used to compare phosphosite pairs found at the same interfaces in the two different species (S. cerevisiae and H. sapiens) with random phosphosite pairs and pairs known to be regulated by the same kinases. Open circles represent the top and bottom 5th percentiles. C) The model of the S. cerevisiae Cdc42p:Rdi1p interface was annotated with currently known phosphorylation data. The Rdi1p Y20 and S40 positions refer to the S. cerevisiae protein sequence positions. The S40 position is currently known to be phosphorylated in S. cerevisiae and C. albicans but not in human. Conversely the Y20 position is known to be phosphorylated in human but not in fungi.

Figure 5. Phosphorylation enrichment analysis identifies regulatory ‘hot-spots’

For each domain family under analysis we selected a representative structure from the PDB database (Table S4). Phosphorylation and acetylation data was mapped to the representative sequence/structure using sequence alignments and random sampling was used to calculate the enrichment over random. This value was plotted along the domain sequence position of the representative structure as a moving average and a cut-off of p-value<0.005 was used to identify significant enrichment (dotted line). See also Figure S3.

Figure 6. Phosphorylation hot-spots within the HSP70 domain family

A) A cut-off of p-value<0.005 (dotted line) was used to identify 2 regions that are significantly enriched for phosphosites in the HSP70 family. B) Phosphorylation hot-spot mutants do not complement SSA1 functions as measured by growth in liquid media. C) 10-fold dilution series of Ssa1 mutants on -URA plates and incubated at 30°C, 33°C and 37°C for 2 days. The protein abundance of the different mutants was compared to WT Ssa1 by western. D) The association of the indicated SSA1 mutants with polysomes was examined by immunoblot analysis. Ribosomal profiles (top) were determined by OD254nm and confirmed by immunoblot analysis of the ribosomal proteins Rpl3p. E) Recovery of luciferase activity is expressed as a percentage of activity before heat treatment and is an average of 2 experiments. F) Percentage of cells with multiple and single ubc9-2-GFP aggregates after heat shocked at 37°C for 30 minutes. Errors bars quantify the standard deviations of, at least, 4 technical replicates. See also Figure S4.