Applying a Conservation-Based Approach for Predicting Novel Phosphorylation Sites in Eukaryotes and Evaluating Their Functional Relevance

Protein phosphorylation, a key post-translational modification, is central to cellular signaling and disease pathogenesis. The development of high-throughput proteomics pipelines has led to the discovery of large numbers of phosphorylated protein motifs and sites (phosphosites) across many eukaryotic species. However, the majority of phosphosites are reported from human samples, with most species having a few experimentally confirmed or computationally predicted phosphosites. Furthermore, only a small fraction of the characterized human phosphoproteome has an annotated functional role. A common way of predicting functional phosphosites is through conservation-based sequence analysis, but large-scale evolutionary studies are scarce. In this study, we explore the conservation of 20,751 confident human phosphosites across 100 eukaryotic species and investigate the evolution of associated protein domains and kinases. We categorize protein functions based on phosphosite conservation patterns and demonstrate the importance of conservation analysis in identifying organisms suitable as biological models for studying conserved signaling pathways relevant to human biology and disease. Finally, we use human protein sequences as a reference for propagating over 1,000,000 potential phosphosites to other eukaryotes. Our results can improve proteome annotations of several species and help direct research aimed at exploring the evolution and functional relevance of phosphorylation.