Systematic Characterization of Cancer‐Associated SPOP Mutants Reveals Novel and Reprogrammable Degradative Activities

Speckle‐type POZ protein (SPOP) functions as the substrate adaptor of the Cullin3‐RING ligase complex and is recurrently mutated in multiple cancer types. Among these, F102C and F133L are frequent prostate cancer mutations within the substrate‐binding domain, yet their biochemical consequences remain incompletely understood. Using quantitative proteomics, we show that SPOP‐F133L, unlike SPOP‐F102C, retains degradative activity toward the nuclear basket proteins NUP153 and TPR, indicating substrate‐dependent loss‐of‐function. Moreover, SPOP‐F133L induces partial down‐regulation of p53 through a Cullin‐RING ligase‐dependent, post‐translational mechanism, revealing a potential neo‐substrate relationship. Finally, we demonstrate that both SPOP‐F102C and SPOP‐F133L support targeted protein degradation in an engineered cellular system. These findings define the degradative capacities of SPOP mutants and highlight opportunities to repurpose these variants as mutant‐selective E3 ligases for therapeutic applications.