Identification of Critical Phosphorylation Sites Enhancing Kinase Activity With a Bimodal Fusion Framework

Phosphorylation is an indispensable regulatory mechanism in cells, with specific sites on kinases that can significantly enhance their activity. Although several such critical phosphorylation sites (phos-sites) have been experimentally identified, many more remain to be explored. To date, no computational method exists to systematically identify these critical phos-sites on kinases. In this study, we introduce PhoSiteformer, a transformer-inspired foundational model designed to generate embeddings of phos-sites using phosphorylation mass spectrometry data. Recognizing the complementary insights offered by protein sequence data and phosphorylation mass spectrometry data, we developed a classification model, CSPred, which employs a bimodal fusion strategy. CSPred combines embeddings from PhoSiteformer with those from the protein language model ProtT5. Our approach successfully identified 77 critical phos-sites on 58 human kinases. Two of these sites, T517 on PKG1 and T735 on PRKD3, have been experimentally verified. This study presents the first systematic and computational approach to identify critical phos-sites that enhance kinase activity. [Display omitted] •Developed PhoSiteformer to capture internal relationships between phos-sites.•Introduced CSPred, a bimodal framework combining PhoSiteformer and ProtT5 embeddings.•Systematically identified 77 newly critical phos-sites on 58 human kinases. Phosphorylation is essential for cellular regulation, yet identifying critical phosphorylation sites (phos-sites) on kinases remains challenging. This study introduces PhoSiteformer, a model leveraging phosphorylation mass spectrometry data to learn internal relationships between phos-sites, and CSPred, a bimodal framework combining PhoSiteformer and ProtT5 embeddings to systematically identify critical sites. The approach uncovered 77 previously unreported critical phos-sites across 58 human kinases. This work advances understanding of kinase activity and regulation, providing a computational framework to uncover essential phosphorylation events.