Evolutionary Constraints of Phosphorylation in Eukaryotes,Prokaryotes, and Mitochondria

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.Reversible protein phosphorylation on serines, threonines, and tyrosines plays a crucial role in regulating processes in all living organisms ranging from prokaryotes to eukaryotes (1). Traditionally, phosphorylation has been detected in single, purified proteins using in vitro assays. Recent advances in mass spectrometry (MS)-based proteomics now allow the identification of in vivo phosphorylation sites with high accuracy (2–7). On-line databases such as PhosphoSite (8), Phospho.ELM (9), and PHOSIDA1 (10) have collected and organized thousands of identified phosphosites. These databases as well as dedicated analysis environments such as NetworKIN (11, 12) offer and use contextual information including structural features, potential kinases, and conservation. They constitute resources that should allow the derivation of general patterns for phosphorylation events. Specifically, the recent availability of data for archaeal, prokaryotic, and diverse eukaryotic phosphoproteomes in these databases should enable investigation of the evolutionary history of this post-translational modification.Prokaryotes have two separate classes of phosphorylation events. Apart from the canonical histidine/aspartate phosphorylation, which has been studied for decades, serine/threonine/tyrosine phosphorylation is also present and has recently become amenable to analysis by MS (13). Bacterial phosphoproteins are involved in protein synthesis, carbohydrate metabolism, and the phosphoenolpyruvate-dependent phosphotransferase system. Recent phosphoproteomics studies of Bacillus subtilis, Escherichia coli, and Lactococcus lactis described around 100 phosphorylation sites on serine, threonine, and tyrosine in each of these species (13–15). Bacterial phosphorylation sites can change in response to environmental conditions (16).Interestingly, even archaea have serine/threonine and tyrosine phosphorylation. A recent study of Halobacterium salinarum described 75 serine/threonine/tyrosine phosphorylation sites on 62 proteins involved in a wide range of cellular processes including a variety of metabolic pathways (17).Although only a few hundred phosphorylation events have been found in prokaryotic species, similar experimental conditions and effort have yielded the detection of thousands of phosphorylation events in eukaryotes ranging from yeast to human (7, 18–21). Before the advent of large scale phosphoproteomics, serine/threonine/tyrosine phosphorylation has been estimated to affect one-third of all proteins (22). Recent large scale phosphoproteomics studies now suggest that more than half of all eukaryotic proteins are phosphorylated (23).A key event in evolution was the endosymbiosis of prokaryotes that enabled the development of a much more complex type of life, the eukaryotic cell. Analyses of mitochondrial genes suggest that the α-proteobacterium Rickettsia prowazekii is the endosymbiotic precursor leading to modern mitochondria (24). Almost all of the mitochondrial genes have migrated to the nuclear genome during subsequent evolution, and it is predicted that 10–15% of eukaryotic nuclear genes of organisms encode mitochondrial proteins (25).Thus, mitochondria with their unique evolutionary position between prokaryotes and eukaryotes form an interesting link for the evolutionary analysis of phosphorylation. Several studies investigated the mitochondrial phosphoproteome in different organisms using gel electrophoresis or specific enrichment methods coupled with mass spectrometry (26–28). Those studies established potential mitochondrial phosphoproteins. Three large scale studies based on affinity enrichment of phosphopeptides and mass spectrometry obtained direct experimental evidence of phosphorylation sites in mitochondria. Lee et al. (29) used a combination of different peptide enrichment strategies and found 80 phosphorylation sites of 48 different proteins from mouse liver. Very recently, a study by Deng et al. (30) characterized the murine cardiac mitochondrial mouse phosphoproteome, covering 236 phosphosites on 181 proteins. Investigating yeast, Reinders et al. (31) assigned 84 phosphorylation sites in 62 proteins.To enable comparative analysis of phosphoproteomes between all domains of life and mitochondria, here we experimentally determined a high accuracy mitochondrial mouse phosphoproteome based on technology conditions similar to those applied to the identification of prokaryotic and eukaryotic phosphoproteomes. We then performed a detailed evolutionary study of the conservation of the identified phosphoproteins and phosphorylation sites in prokaryotes and in eukaryotes. This allowed an initial comparison of the phosphoproteomes of prokaryotes, mitochondria, and eukaryotes.