Mutations within the predicted fragment-binding region of FAM83G/SACK1G abolish its interaction with the Ser/Thr...

SACK1G (aka FAM83G, PAWS1) plays a central role in activating canonical WNT signalling through interaction with the Ser/Thr kinase CK1α. The loss of CK1α binding and WNT signalling underlies the pathogenesis of palmoplantar keratoderma (PPK) caused by several reported mutations in the SACK1G gene. We modelled the scaffold anchor of CK1 (SACK1) domain of SACK1G and used fragment-bound structures of the SACK1B (FAM83B) dimer to guide our analysis. This allowed us to computationally predict several key residues near the fragment-binding site in SACK1G that may be important for its function. We mutated these residues, introduced them into SACK1G-/- DLD-1 colorectal cancer cells and investigated their ability to bind endogenous CK1α. We uncovered two SACK1G mutations, namely Y204A and I206A, that abolish interaction with CK1α similarly to the PPK pathogenic mutant A34E. Consistent with this loss of SACK1G-CK1α interaction, the molecular glue degrader of CK1α, DEG-77, fails to co-degrade the Y204A and I206A mutants while it still co-degrades native SACK1G. Our findings demonstrate the utility of our computational methods to uncover functional residues on proteins based on fragment-binding sites.