Protein phosphorylation is a key mechanism to regulate protein functions. However, the contribution of this protein modification to species divergence is still largely unknown. Here, we studied the evolution of mammalian phosphoregulation by comparing the human and mouse phosphoproteomes. We found that 84% of the positions that are phosphorylated in one species or the other are conserved at the residue level. Twenty percent of these conserved sites are phosphorylated in both species. This proportion is 2.5 times more than expected by chance alone, suggesting that purifying selection is preserving phosphoregulation. However, we show that the majority of the sites that are conserved at the residue level are differentially phosphorylated between species. These sites likely result from false-negative identifications due to incomplete experimental coverage, false-positive identifications and non-functional sites. In addition, our results suggest that at least 5% of them are likely to be true differentially phosphorylated sites and may thus contribute to the divergence in phosphorylation networks between mouse and humans and this, despite residue conservation between orthologous proteins. We also showed that evolutionary turnover of phosphosites at adjacent positions (in a distance range of up to 40 amino acids) in human or mouse leads to an over estimation of the divergence in phosphoregulation between these two species. These sites tend to be phosphorylated by the same kinases, supporting the hypothesis that they are functionally redundant. Our results support the hypothesis that the evolutionary turnover of phosphorylation sites contributes to the divergence in phosphorylation profiles while preserving phosphoregulation. Overall, our study provides advanced analyses of mammalian phosphoproteomes and a framework for the study of their contribution to phenotypic evolution.
Understanding how differences in cellular regulation lead to phenotypic differences between species remains an open challenge in evolutionary genetics. The extensive phosphorylation data currently available allows to compare the human and mouse phosphoproteomes and to measure changes in their phosphoregulation. We found a general conservation of phosphorylation sites between these two species. However, a fraction of sites are conserved at the sequence level (the same amino acid is present in both species) but differ in their phosphorylation status. These sites represent candidate sites that have the potential to explain differences between human and mouse signalling networks that do not depend on the divergence of orthologous residues. Furthermore, we identified several sites where to a phosphorylation site in one species corresponds a non-phosphorylatable residue in the other one. These cases represent clear differences in protein regulation. Recent studies suggest that phosphorylation sites can shift position during evolution, leading to configurations in which pairs of divergent phosphorylation sites are functionally redundant. We identified more than 100 putative such cases, suggesting that divergence in amino acid does not necessarily imply functional divergence when comparing phosphoproteomes. Overall, our study provides new key concepts and data for the study of how regulatory differences may be linked to phenotypic ones at the network level.