Abstract
Identifying effective therapies targeting multi-protein complexes that lack catalytic sites or cofactor pockets remains a long-standing challenge. The proto-oncogene, ubiquitin E3 ligase SCFSkp2, is one such target. SCFSkp2 promotes the proteasomal degradation of the cyclin-dependent kinase inhibitor p27, which controls cell cycle progression. Targeted knockout of Rb1/Trp53 causes metastatic prostate cancer in mice; additional knockout of Skp2 completely blocks tumorigenesis. We compared gene-edited mice that carried two different single amino acid changes in the SCFSkp2 complex, structurally predicted to inhibit the degradation of p27. Mutation of the SCFSkp2 accessory protein Cks1 (Cks1N45R) completely blocked Rb1/Trp53-driven prostate tumorigenesis, phenocopying Skp2 knockout, whereas a mutation directly stabilizing p27 (p27T187A) did not. This was consistent with structural models that predicted the binding of both p27 and p27T187A to the SCFSkp2/Cks1/Cdk2/CyclinA/p27 complex, and their subsequent ubiquitination and degradation, albeit at different rates. Two binding modes, which differ in their dependence on phosphorylated T187, are predicted by the model. Studies confirmed the role of p27 in mediating tumorigenesis in Rb1/Trp53 mutant tumors and revealed a mutually destabilizing Skp2 and p27 feedback loop. The integration of gene editing, drug-surrogate mutations, and mouse tumor models offers a blueprint for studying SCFSkp2 and other multi-subunit biomedical targets.