TY - JOUR
T1 - Intrinsically disordered substrates dictate SPOP subnuclear localization and ubiquitination activity
AU - Usher, Emery T.
AU - Sabri, Nafiseh
AU - Rohac, Roman
AU - Boal, Amie K.
AU - Mittag, Tanja
AU - Showalter, Scott A.
N1 - Funding Information:
Funding and additional information—This work was funded by NIH Grant R01DK121509 and US National Science Foundation Grant MCB-1515974 to S. A. S., NIH NRSA predoctoral fellowship F31DK124047 to E. T. U. (formerly G. A. U.), NIH Grant R01GM112846 (to T. M.), and the St Jude Children’s Research Hospital Research Collaborative on Membraneless Organelles in Health and Disease (to T. M.), and NIH Grant R35GM119707 (to A. K. B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
Acknowledgments—We thank Monique Bastidas, Melissa Rolls, and Gavin Palowitch for help with data collection, Matt Cuneo for technical assistance, and Jill Bouchard for helpful discussion. General Medical Sciences and Cancer Institutes Structural Biology Facility at the Advanced Photon Source (GM/CA@APS) has been funded in whole or in part with federal funds from the National Cancer Institute (ACD-12002) and the National Institute of General Medical Sciences (AGM-120060). We also acknowledge the resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract No. DE-AC02-06CH11357. The EIGER detector at General Medical Sciences and Cancer Institutes Structural Biology Facility - X-ray science division (GM/CA-XSD) was funded by NIH Grant S10 OD012289. The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231.
Publisher Copyright:
© 2021 THE AUTHORS.
PY - 2021/1/1
Y1 - 2021/1/1
N2 - 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.
AB - 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.
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U2 - 10.1016/j.jbc.2021.100693
DO - 10.1016/j.jbc.2021.100693
M3 - Article
C2 - 33894201
AN - SCOPUS:85105953208
VL - 296
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
M1 - 100693
ER -