NOTCH1-specific phosphorylation of S1970 by Casein Kinase 1 is required for NOTCH1 transcriptional competence and signaling activity in vivo

The Notch and Wnt/β-catenin signaling pathways are essential regulators for cell-fate decisions, cellular patterning, and tissue homeostasis. Multiple studies point to their orchestrated role during development, but the molecular mechanism of the protein-protein crosstalk is largely unknown. Here, after screening effects of Wnt/β-catenin component loss on NOTCH1 protein, we identify Casein Kinase 1α (CK1α) as a positive regulator of NOTCH1 activity in vitro and in vivo . We demonstrate that CK1α associates with NOTCH1 and that its kinase activity is required to sustain Notch-driven transcription. Using UltraID proximity-assay, we revealed that CK1α is required for the NOTCH1 interactivity with transporter proteins, and MAML1 both prior and after ligand-induced activation. Combining structural modelling, NMR, and mass spectrometry, we identified Serine 1970 (S1970) as a previously unreported residue within the Notch1 Intracellular Domain (N1ICD) essential for its signaling competence. Our modeling predicts that the phosphorylation of S1970 facilitates an intra-domain conformational switch with R1937 and R1962 residues altering the assembly of the N1ICD-MAML1-RBPJk transcriptional complex. Finally, we demonstrate the biological significance of N1ICD S1970 in vivo using Xenopus laevis axis-duplication rescue assay. Our results establish CK1α as a key positive mediator of the Notch receptor transcriptional activity.