MOTIVATION: Phosphorylation, a post-translational modification regulated by protein kinase enzymes, plays an essential role in almost all cellular processes. Understanding how each of the nearly 500 human protein kinases selectively phosphorylates their substrates is a foundational challenge in bioinformatics and cell signaling. Although deep learning models have been a popular means to predict kinase-substrate relationships, existing models often lack interpretability and are trained on datasets skewed toward a subset of well-studied kinases. RESULTS: Here we leverage recent peptide library datasets generated to determine substrate specificity profiles of 300 serine/threonine kinases to develop an explainable Transformer model for kinase-peptide interaction prediction. The model, trained solely on primary sequences, achieved state-of-the-art performance. Its unique multitask learning paradigm built within the model enables predictions on virtually any kinase-peptide pair, including predictions on 139 kinases not used in peptide library screens. Furthermore, we employed explainable machine learning methods to elucidate the model's inner workings. Through analysis of learned embeddings at different training stages, we demonstrate that the model employs a unique strategy of substrate prediction considering both substrate motif patterns and kinase evolutionary features. SHapley Additive exPlanation (SHAP) analysis reveals key specificity determining residues in the peptide sequence. Finally, we provide a web interface for predicting kinase-substrate associations for user-defined sequences and a resource for visualizing the learned kinase-substrate associations. AVAILABILITY AND IMPLEMENTATION: All code and data are available at https://github.com/esbgkannan/Phosformer-ST. Web server is available at https://phosformer.netlify.app.

Protein language models, trained on millions of biologically observed sequences, generate feature-rich numerical representations of protein sequences. These representations, called sequence embeddings, can infer structure-functional properties, despite protein language models being trained on primary sequence alone. While sequence embeddings have been applied toward tasks such as structure and function prediction, applications toward alignment-free sequence classification have been hindered by the lack of studies to derive, quantify and evaluate relationships between protein sequence embeddings. Here, we develop workflows and visualization methods for the classification of protein families using sequence embedding derived from protein language models. A benchmark of manifold visualization methods reveals that Neighbor Joining (NJ) embedding trees are highly effective in capturing global structure while achieving similar performance in capturing local structure compared with popular dimensionality reduction techniques such as t-SNE and UMAP. The statistical significance of hierarchical clusters on a tree is evaluated by resampling embeddings using a variational autoencoder (VAE). We demonstrate the application of our methods in the classification of two well-studied enzyme superfamilies, phosphatases and protein kinases. Our embedding-based classifications remain consistent with and extend upon previously published sequence alignment-based classifications. We also propose a new hierarchical classification for the S-Adenosyl-L-Methionine (SAM) enzyme superfamily which has been difficult to classify using traditional alignment-based approaches. Beyond applications in sequence classification, our results further suggest NJ trees are a promising general method for visualizing high-dimensional data sets.

MOTIVATION: The human genome encodes over 500 distinct protein kinases which regulate nearly all cellular processes by the specific phosphorylation of protein substrates. While advances in mass spectrometry and proteomics studies have identified thousands of phosphorylation sites across species, information on the specific kinases that phosphorylate these sites is currently lacking for the vast majority of phosphosites. Recently, there has been a major focus on the development of computational models for predicting kinase-substrate associations. However, most current models only allow predictions on a subset of well-studied kinases. Furthermore, the utilization of hand-curated features and imbalances in training and testing datasets pose unique challenges in the development of accurate predictive models for kinase-specific phosphorylation prediction. Motivated by the recent development of universal protein language models which automatically generate context-aware features from primary sequence information, we sought to develop a unified framework for kinase-specific phosphosite prediction, allowing for greater investigative utility and enabling substrate predictions at the whole kinome level. RESULTS: We present a deep learning model for kinase-specific phosphosite prediction, termed Phosformer, which predicts the probability of phosphorylation given an arbitrary pair of unaligned kinase and substrate peptide sequences. We demonstrate that Phosformer implicitly learns evolutionary and functional features during training, removing the need for feature curation and engineering. Further analyses reveal that Phosformer also learns substrate specificity motifs and is able to distinguish between functionally distinct kinase families. Benchmarks indicate that Phosformer exhibits significant improvements compared to the state-of-the-art models, while also presenting a more generalized, unified, and interpretable predictive framework. AVAILABILITY AND IMPLEMENTATION: Code and data are available at https://github.com/esbgkannan/phosformer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.