Protein proximity networks and functional evaluation of the Casein Kinase 1 γ family reveals unique roles for CK1γ3 in WNT signaling

The WNT/ β -catenin signaling pathway is evolutionarily conserved and controls normal embryonic development, adult tissue homeostasis and regeneration. Aberrant activation or suppression of WNT signaling contributes to cancer initiation and progression, developmental disorders, neurodegeneration, and bone disease. Despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and ‘dark’. Here we studied the function of the CSNK1 γ subfamily of human kinases. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1 γ 1, CSNK1 γ 2, and CSNK1 γ 3 revealed numerous established components of the β -catenin- dependent and independent WNT signaling pathway, as well as novel interactors. In gain-of- function experiments leveraging a panel of transcriptional reporters, CSNK1 γ 3 but not CSNK1 γ 1 or CSNK1 γ 2 activated β -catenin-dependent WNT signaling and the Notch pathway. Within the family, CSNK1 γ 3 expression uniquely induced LRP6 phosphorylation. Conversely, siRNA- mediated silencing of CSNK1 γ 3 alone had no impact on WNT signaling, though co-silencing of all three family members decreased WNT pathway activity. We characterized two moderately selective and potent small molecule inhibitors of the CSNK1 γ family. These inhibitors and a CSNK1 γ 3 kinase dead mutant suppressed but did not eliminate WNT-driven LRP6 phosphorylation and β -catenin stabilization. Our data suggest that while CSNK1 γ 3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway. ### Competing Interest Statement The authors have declared no competing interest.