The Role of Higher-Order SPOP Oligomers for Localization to Cellular “Bodies” and Ubiquitination Activity

Abstract

Light microscopically detectable, non-membrane bound cellular “bodies” are large protein assemblies with liquid-like properties, but the biophysical basis of their formation is unclear. Weak, multivalent protein interactions can result in higher-order complexes and can enable the formation of cellular bodies. The inherent size heterogeneity of higher-order complexes renders them difficult to characterize biophysically. As a result, their size distributions remain largely unquantified, limiting molecular insight into their biological functions. We report a novel mechanism governing cellular body formation of the Speckle-type POZ protein (SPOP), which was recently identified as tumor suppressor, is a ubiquitin ligase substrate adaptor that localizes to nuclear puncta. We demonstrate that its cellular localization is dependent upon the ability of SPOP to form higher-order homo-oligomers through indefinite self-association, mediated by two distinct oligomerization domains. Furthermore, in vitro ubiquitination of substrates is enhanced through higher-order self-association of SPOP, suggesting that SPOP puncta are hotspots of substrate ubiquitination. One of SPOP's domains dimerizes with nanomolar affinity yielding stable SPOP dimers as “building blocks” for indefinite self-association, while the other domain dimerizes with micromolar affinity, rendering SPOP oligomers highly dynamic. Together, this results in isodesmic self-association, in which each addition of a dimer occurs with the same affinity, independent of the oligomer size. From this model, we describe the size distribution of SPOP oligomers, providing for the first time a quantitative analysis of protein assemblies participating in the formation of cellular bodies. Mutations within both oligomerization domains have been observed in a variety of cancers, supporting our conclusion that SPOP self-association is important for its biological function. We propose that dynamic, higher-order protein self-association is a general mechanism underlying the formation of cellular bodies, which may serve as switches to fine-tune signaling cascades.