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

Turetti, F.; Maia, L. A.; Hajsmanova, H.; Slawski, J.; Dehaen, W.; Dokoupil, M.; Collu, G.; Gybel, T.; Paruch, K.; Andersson, E. R.; Bryja, V.; Perlikova, P.; Tripsianes, K.; Harnos, J.; Masek, J.

doi:10.64898/2026.05.05.722849

bioRxiv · 10.64898/2026.05.05.722849

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

Turetti, F.·Maia, L. A.·Hajsmanova, H.·Slawski, J.·Dehaen, W.·Dokoupil, M.·Collu, G.·Gybel, T.·Paruch, K.·Andersson, E. R.·Bryja, V.·Perlikova, P.·Tripsianes, K.·Harnos, J.·Masek, J.

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Abstract

The Notch and Wnt/{beta}-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/{beta}-catenin component loss on NOTCH1 protein, we identify Casein Kinase 1 (CK1) as a positive regulator of NOTCH1 activity in vitro and in vivo. We demonstrate that CK1 associates with NOTCH1 and that its kinase activity is required to sustain Notch-driven transcription. Using UltraID proximity-assay, we revealed that CK1 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 CK1 as a key positive mediator of the Notch receptor transcriptional activity.

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Turetti, F., Maia, L. A., Hajsmanova, H., Slawski, J., Dehaen, W., Dokoupil, M., Collu, G., Gybel, T., Paruch, K., Andersson, E. R., Bryja, V., Perlikova, P., Tripsianes, K., Harnos, J., Masek, J.. 2026-05-07. NOTCH1-specific phosphorylation of S1970 by Casein Kinase 1 is required for NOTCH1 transcriptional competence and signaling activity in vivo. https://doi.org/10.64898/2026.05.05.722849

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