Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria

Abstract

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.

Fig. 1.

Overlap of prokaryotic phosphorylation sites. The overlap between phosphorylation sites of E. coli (A), B. subtilis (B), L. lactis (C), and H. salinarum (D) is very low.

Fig. 2.

Characterization of mitochondrial mouse phosphoproteome. A, serine/threonine/tyrosine distribution. B, molecular functions that are enriched in the mitochondrial phosphoset compared with the entire mouse proteome. C, relative position-specific amino acid frequency around phosphorylated sites.

Fig. 3.

Proportion of mouse proteins that are orthologous to prokaryotic proteins. Phosphorylated (our data set) and non-phosphorylated (MitoCarta data set) mitochondrial mouse proteins are more highly conserved in prokaryotes than phosphorylated (PHOSIDA data set) and non-phosphorylated (Swiss-Prot Database) proteins, which are located in other compartments of the cell. These observations are in concordance with the endosymbiotic scenario as illustrated in the lower panel. As a measure for conservation, we used the average proportion of orthologs in 62 prokaryotes.