Protein kinases represent one of the largest and most druggable protein families. Despite considerable progress in their understanding, approximately one-third of human kinases remain poorly characterized, known as the "dark" kinome. Doublecortin-like kinase 3 (DCLK3), a member of this elusive group, has emerged for its involvement in neuroprotection in Huntington s disease and other neurodegenerative disorders. Still, its cellular substrates and regulatory functions remain unknown, hindering progress in therapeutic intervention. Unlike its paralog, DCLK1, whose regulation involves a C-terminal segment docking into the ATP-binding pocket, DCLK3 lacks such a tail, suggesting divergent regulatory mechanisms. Thro... More
Protein kinases represent one of the largest and most druggable protein families. Despite considerable progress in their understanding, approximately one-third of human kinases remain poorly characterized, known as the "dark" kinome. Doublecortin-like kinase 3 (DCLK3), a member of this elusive group, has emerged for its involvement in neuroprotection in Huntington s disease and other neurodegenerative disorders. Still, its cellular substrates and regulatory functions remain unknown, hindering progress in therapeutic intervention. Unlike its paralog, DCLK1, whose regulation involves a C-terminal segment docking into the ATP-binding pocket, DCLK3 lacks such a tail, suggesting divergent regulatory mechanisms. Through computational and experimental analyses, we discovered that DCLK3 autophosphorylates its truncated tail, tethering it to the catalytic domain in a manner distinct from DCLK1. Using a deep learning model trained on peptide-library datasets, we predicted Tau, a microtubule-associated protein, as a putative DCLK3 substrate, which we validated using in vitro assays and mass spectrometry. Additionally, DCLK3 exhibits a relatively fast turnover with a cellular half-life of approximately 15 h that can be rescued by MG132-mediated proteasomal inhibition, which results in DCLK3 polyubiquitination and cellular accumulation. Collectively, these results provide the first structural and functional insights into DCLK3, revealing a unique autoregulatory mechanism and a potential therapeutic target for neurodegenerative disorders.
