Cooperativity within proximal phosphorylation sites is revealed from large-scale proteomics data

Abstract

Background: Phosphorylation is the most prevalent post-translational modification on eukaryotic proteins. Multisite phosphorylation enables a specific combination of phosphosites to determine the speed, specificity and duration of biological response. Until recent years, the lack of high quality data limited the possibility for analyzing the properties of phosphorylation at the proteome scale and in the context of a wide range of conditions. Thanks to advances of mass spectrometry technologies, thousands of phosphosites from in-vivo experiments were identified and archived in the public domain. Such resource is appropriate to derive an unbiased view on the phosphosites properties in eukaryotes and on their functional relevance.

Results: We present statistically rigorous tests on the spatial and functional properties of a collection of approximately 70,000 reported phosphosites. We show that the distribution of phosphosites positioning along the protein tends to occur as dense clusters of Serine/Threonines (pS/pT) and between Serine/Threonines and Tyrosines, but generally not as much between Tyrosines (pY) only. This phenomenon is more ubiquitous than anticipated and is pertinent for most eukaryotic proteins: for proteins with > or = 2 phosphosites, 54% of all pS/pT sites are within 4 amino acids of another site. We found a strong tendency for clustered pS/pT to be activated by the same kinase. Large-scale analyses of phosphopeptides are thus consistent with a cooperative function within the cluster.

Conclusions: We present evidence supporting the notion that clusters of pS/pT but generally not pY should be considered as the elementary building blocks in phosphorylation regulation. Indeed, closely positioned sites tend to be activated by the same kinase, a signal that overrides the tendency of a protein to be activated by a single or only few kinases. Within these clusters, coordination and positional dependency is evident. We postulate that cellular regulation takes advantage of such design. Specifically, phosphosite clusters may increase the robustness of the effectiveness of phosphorylation-dependent response.

Reviewers: Reviewed by Joel Bader, Frank Eisenhaber, Emmanuel Levy (nominated by Sarah Teichmann). For the full reviews, please go to the Reviewers' comments section.

Figure 1

Statistics of phosphosites origin and types. (A) Analysis of the different types of phosphosites complied from SysPTM, Phospho.ELM and PHOSIDA. (B) The distribution of phosphosites according to their organisms. Organisms that have less than 1% of the total phosphosites are not shown. It accounts together for less than 1%. See Table 1 for further information.

Figure 2

Distances of nearest phosphosites. (A) Analysis of ~51,000 non- redundant S/T phosphosites from unique proteins (B) Analysis of ~3160 non-redundant Y phosphosites. For each distance, the frequency is shown relative to the frequency of randomly selected from the relevant amino acids (see Methods). (C) Analysis of S/T phosphosites as in A, the distance to the nearest Y phosphosite is reported. The tail distribution of phosphosites including a distance >30 amino acids is provided in Additional file 5.

Figure 3

Distances of nearest phosphosites partitioned by model organisms and non redundant sequences. Analysis of ~51,000 phosphosites was performed as in Figure 2. The data were separated according to major organisms including human, mouse, Drosophila, Arabidodpsis and yeast. In all organisms, 32-37% of the pS/pT sites are within a distance smaller than 3. The data from UniRf90 show the reduction of UniProtKB phosphoproteins to a non-redundant set in which no two proteins share more than 90% sequence identity. Results from the non-redundant set (UniRef90) are identical to the complete set.

Figure 4

A representative set of pS/pT clustered-rich proteins. Short segments (75 amino acids each) that are exceptionally rich in clustered phosphosites are shown. These proteins have >5 proximal phosphosites clusters and >5 independent evidence from the literature. We marked clusters by a stringent definition where the distance between two consecutive pS/pT sites is at most n+3 (n denotes the position of pS/pT). The frames around the phosphosites denote the following: black, only one pair of pS/pT; orange, extended cluster according to the maximal distance of n+3 between neighboring pS/pT sites; blue, a mixed cluster of pS/T and pY. Phosphosites that are inferred from the identification of phosphosites in a close homologue are marked in a black font. For a complete list of clustered-rich proteins see Additional file 1

Figure 5

Patterns in phosphorylation of adjacent phosphosites. For each pair of phosphosites (from the entire sources for phosphoproteins), the peptides that contain both of them are searched. It is then asked if from these peptides, there are peptides that contain both sites in their phosphorylated state (marked as 'both', B), only the first site is phosphorylated (marked as 'left', L) or only the second site is phosphorylated (marked as 'right', R). Each pair of sites is assigned a pattern according to the types of peptides we have seen. For example, the rightmost bar contains pairs for which we have only seen peptides in which both sites are phosphorylated (marked only with B). Note that the amount of pairs not seen in any constellation is only ~5%, indicating a high coverage of the set of experimental results that were applied for this analysis.

Figure 6

Structural and biochemical features of pS/pT sites. (A) The tendency of pS/pT sites to be inside/outside a domain. The proportions of being inside or outside a Pfam domain are measured for: (i) all amino acids, (ii) all S/T phosphosites, (iii) only S/T phosphosites with a near neighbor, (iv) all Y phosphosites and (v) only Y phosphosites with a near neighbor. (B) Distribution of secondary structure elements. The proportions of being coiled, in α-Helix or β-sheet for: (i) S/T positions that are not phosphosites (~12,000 random positions) (ii) all S/T phosphosites (~18,300 sites) where these are divided to: (iii) only S/T phosphosites with a near neighbor (~8400 sites) (iv) only S/T phosphosites without a near neighbor (~9900 sites). (C) Distribution of ordered and disordered elements. The proportions of being in disordered regions: (i) S/T positions that are not phosphosites (~36,700 random positions) (ii) all S/T phosphosites (~36,000 sites) where these are divided to: (iii) only S/T phosphosites with a near neighbor (~16,700 sites) (iv) only S/T phosphosites without a near neighbor (~19,200 sites).