{"doi":"10.1016/j.jbc.2021.100693","title":"Intrinsically disordered substrates dictate SPOP subnuclear localization and ubiquitination activity","abstract":"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. 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. Regulation of protein stability is a critical determinant of cellular health and function. In pancreatic β cells, the transcription factor pancreatic and duodenal homeobox 1 (Pdx1; also known as glucose-sensitive factor (1Marshak S. Totary H. Cerasi E. Melloul D. Purification of the beta-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15057-15062Crossref PubMed Scopus (151) Google Scholar), insulin promoter factor 1 (2Ohlsson H. Karlsson K. Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene.EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (745) Google Scholar), insulin upstream factor 1 (3Boam D.S. Docherty K. A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer.Biochem. J. 1989; 264: 233-239Crossref PubMed Scopus (65) Google Scholar), and islet/duodenum homeobox 1 (4Miller C.P. McGehee Jr., R.E. Habener J.F. IDX-1: A new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (370) Google Scholar)) modulates insulin production in response to blood-glucose levels (5MacFarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. ","journal":"Journal of Biological Chemistry","year":2021,"id":170794,"datarank":0.49983067652628066,"base_score":3.332204510175204,"endowment":3.332204510175204,"self_citation_contribution":0.49983067652628066,"citation_network_contribution":0.0,"self_endowment_contribution":0.49983067652628066,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":27,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9594,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":518663,"name":"Nafiseh Sabri","orcid":null,"position":1,"is_corresponding":false},{"id":703860,"name":"Roman Rohac","orcid":"0000-0002-5954-0998","position":2,"is_corresponding":false},{"id":642752,"name":"Amie K. Boal","orcid":"0000-0002-1234-8472","position":3,"is_corresponding":false},{"id":257878,"name":"Tanja Mittag","orcid":"0000-0002-1827-3811","position":4,"is_corresponding":false},{"id":498779,"name":"Scott A. Showalter","orcid":"0000-0001-5179-032X","position":5,"is_corresponding":false},{"id":498778,"name":"Emery T. Usher","orcid":"0000-0002-8303-9992","position":0,"is_corresponding":true}],"reference_count":62,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:46:24.040760Z","pmid":"33894201","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}