SPOP Mutations Disrupt Phase Separation to Impair Its Activity

SPOP is a tumor suppressor protein and substrate adaptor of the cullin3-RING ubiquitin ligase (CUL3) that is frequently mutated in a variety of tumor types. Tumor-associated SPOP mutations disrupt substrate binding and ubiquitination, leading to increased expression of oncogenic substrates, but the mechanisms by which SPOP assembles with its substrates and gets recruited to nuclear bodies remain poorly understood. Bouchard, Otero, and colleagues investigated the mechanisms underlying colocalization of SPOP and its substrates, including the death domain–associated protein (DAXX) and the androgen receptor (AR). When SPOP and DAXX were coexpressed, they both relocalized into nuclear bodies (termed SPOP/DAXX bodies) distinct from the PML bodies where DAXX usually resides and the nuclear speckles where SPOP generally localizes. These SPOP/DAXX bodies were characterized as liquid membraneless organelles, and the relocalization to these membraneless bodies was disrupted by prostate cancer–associated SPOP mutations. The phase separation was mediated by multiple weak interactions between DAXX and SPOP, which are facilitated by multiple SPOP-binding motifs in DAXX and oligomerization of SPOP. These features were required for SPOP/DAXX phase separation, as was shown by mutational analysis. Mutations in SPOP or DAXX disrupted phase separation, preventing the colocalization of SPOP and DAXX, and reducing ubiquitination of target proteins. The SPOP/DAXX bodies were active ubiquitination hubs, recruiting CUL3 to facilitate ubiquitination of DAXX. Similarly, other SPOP substrates harbored weak SB motifs, suggesting that SPOP may recruit them via phase separation. Consistent with this hypothesis, AR formed liquid droplet-like assemblies with SPOP, indicating a phase separation similar to DAXX. Taken together, these findings reveal that cancer-associated SPOP mutations disrupt the liquid–liquid phase separation that normally concentrates the components required for substrate ubiquitination, resulting in loss of function.

Bouchard JJ, Otero JH, Scott DC, Szulc E, Martin EW, Sabri N, et al. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments. Mol Cell 2018;72:19–36.e8.

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