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.
Highlights
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
The basis of our evolutionary studies is high accuracy phosphoproteomes that have been published during the last 3 years and that are deposited in PHOSIDA [10]
Cellular proteomes are digested to peptides, and phosphopeptides are enriched using a TiO2 metal affinity matrix in the presence of 2,5-dihydrobenzoic acid [41] and measured by tandem mass spectrometry on a linear ion trap Orbitrap mass spectrometer using multistage activation [42]
Summary
Cell Culture and Primary Cell Isolation—3T3-L1, brown preadipocytes, C2C12, and Hepa 1-6 cell lines were subcultured and differentiated in DMEM supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics in 5% CO2 at 37 °C. Phosphosites fulfilling the following two criteria are defined as unambiguously identified sites: 1) their localization probability for the assignment is at least 0.75, and 2) the PTM score difference from the second possible localization assignment is 5 or higher These cutoffs proved to yield highly accurate results in previous studies [18]. We created a bootstrap distribution for a given species from 10,000 sets of serines, threonines, and tyrosines that were randomly selected from phosphorylated proteins that had an ortholog. Those residues were included in the analysis that showed the same predicted structural constraints as phosphorylated residues (high accessibility and localization in loops).
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