Intrinsically disordered substrates dictate SPOP subnuclear localization and ubiquitination activity

Emery T Usher,Nafiseh Sabri,Roman Rohac,Amie K Boal,Tanja Mittag,Scott A Showalter

doi:10.1016/j.jbc.2021.100693

Emery T Usher, Nafiseh Sabri + Show 4 more

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https://doi.org/10.1016/j.jbc.2021.100693

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Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Most SPOP substrates present multiple SPOP-binding (SB) motifs and undergo liquid–liquid phase separation with SPOP. Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is downregulated by interaction with SPOP. Unlike other substrates, only one SB motif has previously been reported within the Pdx1 C-terminal intrinsically disordered region (Pdx1-C). Given this difference, we aimed to determine the specific mode of interaction of Pdx1 with SPOP and how it is similar or different to that of other SPOP substrates. Here, we identify a second SB motif in Pdx1-C, but still find that the resulting moderate valency is insufficient to support phase separation with SPOP in cells. Although Pdx1 does not phase separate with SPOP, Pdx1 and SPOP interaction prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. Accordingly, we find that SPOP-mediated ubiquitination activity of Pdx1 occurs in the nucleoplasm and that highly efficient Pdx1 turnover requires both SB motifs. Our results suggest that the subnuclear localization of SPOP–substrate interactions and substrate ubiquitination may be directed by the properties of the substrate itself.

Highlights

Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation
We found that a Pancreatic and duodenal homeobox 1 (Pdx1) construct that is incapable of interacting with SPOP is incapable of drawing SPOP out of nuclear speckles (Fig. 2B)
Biophysical studies of SPOP–substrate interactions have centered on substrates that contain several SB motifs that support liquid–liquid phase separation (LLPS) of SPOP– substrate in cells [22, 23]

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Introduction

Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Among the regulatory signals that control Pdx stability and function is a degradation pathway wherein Pdx associates with a ubiquitin ligase adaptor, speckle-type POZ protein (SPOP). SPOP substrates tend to present multiple binding motifs that individually have relatively weak affinities for SPOP, yet several motifs in a single substrate contribute to a substantially strengthened binding interaction to oligomeric SPOP (Fig. 1C) [14] This phenomenon has been demonstrated for SPOP substrates transcriptional activator GLI3, androgen receptor (AR), and death domain-associated protein 6 (DAXX) [21,22,23]. Given that only one SPOP-binding (SB) motif has been identified in Pdx, we tested whether Pdx and SPOP engage and function in phase-separated

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