Dysregulation of Phase Separation in Cancer

Abstract

Liquid-liquid phase separation leads to demixing of proteins from solution and results in a dense, protein-rich phase, which co-exists with a light phase depleted of protein. Recent findings support a model in which phase separation is the biophysical driving force for the formation of membrane-less organelles in the cell, such as stress granules, nucleoli and nuclear speckles. Current open questions are: (i) How is phase separation propensity encoded in the protein sequence, (ii) are dense liquid droplets used as reaction compartments in the cell, and (iii) is physiological phase separation disrupted in disease states? To address them, we study the interaction of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate adaptor of a ubiquitin ligase, with its substrates. SPOP localizes to different liquid membrane-less organelles in the cell nucleus, where it encounters its substrates, but it is never found diffuse in the cell. However, its recruitment mechanism to these organelles is not understood. Here, we show for the first time that SPOP undergoes liquid-liquid phase separation with substrate proteins, and that this mechanism underlies its recruitment to membrane-less organelles. Multivalency of SPOP and substrate for each other drive their ability to phase separate. Moreover, we present strong evidence that the SPOP/substrate assemblies are active ubiquitination compartments in vitro and in cells. SPOP cancer mutations reduce the propensity for phase separation and tune the material properties of mesoscale assemblies. In the cell, cancer mutants fail to localize to the proper organelles and to recruit substrate. We propose that SPOP has evolved a propensity for phase separation in order to target substrates localized in membrane-less compartments. Our results provide mechanistic insights into the contributions of structured and disordered domains to phase separate, enzymatic activity inside liquid organelles, and disruption of phase separation by cancer mutations.