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. 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Habener J.F. to PubMed Scopus Google Scholar). the that Pdx1 stability and is a Pdx1 with a ubiquitin ligase POZ protein SPOP the disordered of Pdx1 in of glucose in the β modulates Pdx1 protein stability and pancreatic and in PubMed Scopus Google Scholar, A. of a POZ protein that PubMed Scopus Google Scholar). Unlike in β cells, SPOP is and with substrates T. ubiquitin ligase adaptor SPOP in J. PubMed Scopus Google Scholar). SPOP the ubiquitin facilitates the of substrates M. T. K. POZ protein (SPOP) as an adaptor of ubiquitination by ubiquitin PubMed Scopus Google Scholar). binding to the ligase Pdx1 is and modulates Pdx1 protein stability and pancreatic and in PubMed Scopus Google and in SPOP that substrate binding with and H. E. A. A. T. M. T. L. S. of SPOP protein and to PubMed Scopus Google Scholar, M. D. D. A. D. S. L. M. A. in regulation of and PubMed Scopus Google Scholar, S. L. S. J. J. T. K. M. D. SPOP to of PubMed Scopus Google Scholar, K. J. J. L. SPOP the proteasomal of in J. Google Scholar), and the of SPOP and its has In addition to a Pdx1 intrinsically disordered that interactions within the Pdx1 not the J. Habener J.F. to PubMed Scopus Google Scholar, W.M. S. S. M. A. A. T. Docherty K. in the insulin promoter gene to PubMed Scopus Google Scholar). SPOP is of the and factor factor and and and C-terminal SPOP S. J. A. S. H. S. M. localization of SPOP to nuclear J. PubMed Scopus Google Scholar), to substrates by the of multiple J. A. J. T. motifs and in SPOP-mediated substrate PubMed Scopus Google Scholar). this SPOP substrates to present multiple binding motifs that SPOP, motifs in a substrate to a binding interaction to SPOP T. ubiquitin ligase adaptor SPOP in J. PubMed Scopus Google Scholar). has been SPOP substrates and protein S. J. A. S. H. S. M. localization of SPOP to nuclear J. PubMed Scopus Google Scholar, J. A. J. T. motifs and in SPOP-mediated substrate PubMed Scopus Google Scholar, E. D. K. T. of the SPOP the of PubMed Scopus Google Scholar). Given the and substrates by SPOP, we determine SPOP substrates by the or in their by SPOP. SPOP has been reported to substrates phase requires interaction S. J. A. S. H. S. M. localization of SPOP to nuclear J. PubMed Scopus Google Scholar, E. D. K. T. of the SPOP the of PubMed Scopus Google Scholar, T. T. T. M. of a nuclear PubMed Scopus Google Scholar). Given that only one SPOP-binding (SB) motif has been in we Pdx1 and SPOP and in in and that the not phase separate Pdx1 SPOP to the nucleoplasm. this is the of the substrate SPOP, we the interaction of with SPOP in and a second SB motif in Pdx1 that to binding but motifs that the of of SB motif in Pdx1 to the SB M. J. A. M. H. D.M. T. of of ubiquitin PubMed Scopus Google Scholar), but that the Pdx1 SB motifs a binding mode with other substrates. we find that the second SB motif is not to SPOP of nuclear speckles but that it does to Pdx1 ubiquitination results Pdx1 turnover SPOP and present the of a SPOP substrate and the of substrate SPOP its from the of SPOP localization to within the SPOP with nuclear speckles but has also been reported in other subnuclear T. T. T. M. of a nuclear PubMed Scopus Google Scholar, D. M. A. S. D. L. J. S. D. to in Scopus Google Scholar). nuclear speckles that SPOP in the of of substrate in S. J. A. S. H. S. M. localization of SPOP to nuclear J. PubMed Scopus Google Scholar). SPOP substrate to H. J. J. and of by PubMed Scopus Google Scholar). in cells, SPOP and to that from nuclear speckles and new as the sites of ubiquitination activity E. D. K. T. of the SPOP the of PubMed Scopus Google Scholar). with the of SPOP-mediated substrate ubiquitination within liquid–liquid phase separation is as a of of in S. S. T. M. J. J. L. M. phase A new phase in PubMed Scopus Google Scholar, C.P. phase in and Scopus Google Scholar). interactions and within protein the phase separation S. S. L. M. D.S. M.K. in the of PubMed Scopus Google Scholar). of SPOP and the its substrates, present SB also SPOP–substrate phase Given that only one SB motif known in we to Pdx1 and SPOP undergo phase separation in cells. and SPOP in cells, and the cellular localization of the proteins by In to other SPOP substrates E. D. K. T. of the SPOP the of PubMed Scopus Google Scholar), we find that does not to in to nuclear by a nuclear protein protein the M. D. T. and nuclear Scopus Google and SPOP from nuclear speckles the diffuse nucleoplasm Pdx1 not undergo to nuclear SPOP relocalization to the of In levels by SPOP is in that SPOP to nuclear speckles that a substrate SPOP is and by the substrate that SPOP relocalization is interaction with we the protein localization a of its we that a Pdx1 that is of with SPOP is also of SPOP of nuclear speckles this is the of SPOP from nuclear speckles interaction with a substrate in the of in a nuclear Our results that the of one Pdx1 SPOP not to phase that SB motifs and we to identify SB motifs in Pdx1 that to a different of SPOP Given a motif in the Pdx1 A. J. in interaction with pancreatic transcription factor PubMed Scopus Google Scholar, M. of the the phosphorylation-dependent binding PubMed Scopus Google Scholar), we this interaction by identify specific Pdx1 that may with SPOP, we to the of as Pdx1-C, and in to of to of intrinsically disordered PubMed Scopus Google Scholar, of to and disordered PubMed Scopus Google Scholar). of with and and A and addition of SPOP to in specific to in and to in region to the of Pdx1 that by and to with SPOP, and we the of and the second as binding to that the to interaction with SPOP of interactions that the D. M. of the C-terminal from the transcription factor Pdx1 an PubMed Scopus Google Scholar). the in both of in highly an to motifs we that SB SB motif 1 and SB motif the of in with other SPOP substrates in not SB motifs. of and that the resulting to the A and and and the that one SB motif with of and to in the to the and with the a SB of with the of the in the M. of the the phosphorylation-dependent binding PubMed Scopus Google Scholar). we that the the SPOP that the binding may be by with SPOP that stability of the binding SPOP within Pdx1-C, we to the its interaction with SPOP. to and the resulting not a of the the Pdx1 protein and also to the of and may be in SB motif the substrate and of a that one of the in is with a to the the other the is with and of the from in the in the in In the of protein or in with SPOP, the in sites to of the motif as or in substrates highly to the of the M. J. A. M. H. D.M. T. of of ubiquitin PubMed Scopus Google Scholar). Most of or SPOP. other substrate also and may the to as in Pdx1 and of to the of an SPOP substrate with from the SB motif M. of the the phosphorylation-dependent binding PubMed Scopus Google the in the of SPOP, and this binding mode is by a of and to its and its only of the within the binding and and other upstream of the SB motif of to reported the Pdx1 in the and is with M. J. A. M. H. D.M. T. of of ubiquitin PubMed Scopus Google Scholar, M. of the the phosphorylation-dependent binding PubMed Scopus Google Scholar, M. of and SPOP PubMed Scopus Google Scholar, S. K. J. L. M. S. L. by as a in PubMed Scopus Google Scholar). also from the SB motif a of a also a the and of the binding motif in the is other SPOP substrates, a in 1 the in a by SPOP and within the binding and and in results in of SPOP substrate proteins E. D. K. T. of the SPOP the of PubMed Scopus Google Scholar, M. of and SPOP PubMed Scopus Google Scholar, and of SPOP, a in and PubMed Scopus Google Scholar). 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