Using ProPeL to discover the binding specificity of human DYRK1a

Abstract

Pairing kinases to their substrates is central to elucidating signal transduction pathways and understanding their functions in normal and diseased states. Kinases discriminate between potential substrates in part by linear sequence “motifs” surrounding the residue to be phosphorylated, yet motifs for the majority of the 518 human kinases remain unknown. One largely uncharacterized kinase is Dual specificity tyrosine‐phosphorylation‐regulated kinase 1a (DYRK1a), which is implicated in Down's Syndrome, Alzheimer's Disease, alternative splicing and synaptic trafficking. Using our new Proteomic Peptide Library (ProPeL) methodology, in which we express a human kinase in E. coli and analyze the resulting phosphopeptides with our pLogo and motif‐x software, we extracted a strong sequence motif for DYRK1a from 933 unique phosphorylation sites. We also detected 8 novel DYRK1a autophosphorylation sites. Currently, the only known tyrosine targets of DYRK1a are on DYRK1a itself. Strikingly, however, we observed 66 unique phosphotyrosine sites on substrates other than DYRK1a, suggesting that its ability to phosphorylate tyrosine residues may not be limited to autophosphorylation. Finally we predicted human DYRK1a substrates using the authors’ scan‐x tool. This study was funded in part by grants from the University of Connecticut Research Foundation (DS) and Genomes to Life from US DoE (GMC).