Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments - PubMed (original) (raw)

. 2018 Oct 4;72(1):19-36.e8.

doi: 10.1016/j.molcel.2018.08.027. Epub 2018 Sep 20.

Joel H Otero 1, Daniel C Scott 1, Elzbieta Szulc 2, Erik W Martin 1, Nafiseh Sabri 1, Daniele Granata 3, Melissa R Marzahn 1, Kresten Lindorff-Larsen 3, Xavier Salvatella 4, Brenda A Schulman 5, Tanja Mittag 6

Affiliations

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments

Jill J Bouchard et al. Mol Cell. 2018.

Abstract

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.

Keywords: Cul3; DAXX; NMR; androgen receptor; biomolecular codensates; multivalency; nuclear bodies; polymerization; prostate cancer; ubiquitination.

Copyright © 2018 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1.

Figure 1.. SPOP and DAXX co-localize in liquid organelles, and colocalization is disrupted by SPOP cancer mutations.

(A) Sequence and cartoon schematics for SPOP (left) and DAXX (right) constructs used in this study. Sequence cartoons at the top represent domain architecture. SPOP contains a substrate binding MATH domain, and two dimerization domains, BTB and BACK. DAXX contains a DAXX helical bundle (DHB) domain, a helical region, and a C-terminal disordered domain (Escobar-Cabrera et al., 2010). Predicted SPOP-binding motifs (based on the consensus sequence motif, nonpolar-polar-S-S/T-S/T (Zhuang et al., 2009)) are shown in orange, with stronger binding sites shaded darker. Lys residues available for ubiquitination are shown as K. (Below) Cartoon schematics represent self-association and substrate binding behavior, with mutated interfaces in SPOP shown curved instead of straight to indicate the inability to self-associate or bind substrate. WT SPOP forms higher-order oligomers of different sizes (Marzahn et al., 2016); we show hexamers as an example. Cancer mutations W131G and F133V in the MATH domain (green) reduce substrate binding. Mutation of the dimerization interface of the BACK domain (blue, mutation Y353E (van Geersdaele et al., 2013)) results in SPOP mutBACK dimers (Marzahn et al., 2016); the addition of mutations of the BTB interface (red, L186D, L190D, L193D, I217K, (Zhuang et al., 2009)) results in SPOP mutBTB/BACK monomers (Marzahn et al., 2016). SPOP constructs for expression in mammalian cells encode the full-length protein; those for expression in bacteria 28–359. DAXX mammalian expression constructs encode full-length protein unless labeled cDAXX which comprises residues 495–740, as the bacterial expression constructs. H-cDAXX harbors a His6-tag. In cDAXX-0sb, the major SB motifs are mutated. (For details see Fig. 3 and Table S1.) (B)SPOP and DAXX localize to SPOP/DAXX bodies. HeLa cells were transfected with the indicated GFP-DAXX and/or SPOP-mCherry and analyzed by confocal microscopy. mCherry (red) and GFP fluorescence (green) were observed for SPOP and DAXX, while SC-35 and PML (both magenta) were used as markers for nuclear speckles and PML bodies, respectively, and detected by IF. DAPI (blue) marks the nucleus. (See also Fig. S1A-D.) (C) PML bodies behave like liquid droplets. HeLa cells were transfected with GFP-DAXX and GFP monitored in live cells. Snapshots at the indicated time points show a PML body fusion event in the area boxed on the left. (See also Video S1.) (D)SPOP and DAXX form nuclear bodies with liquid properties. HeLa cells were transfected with GFP-DAXX and SPOP-mCherry and analyzed as in (C). (See also Video S2.) (E)SPOP cancer mutants fail to localize to SPOP/DAXX bodies. HeLa cells were transfected with GFP-DAXX and either WT V5-SPOP or mutants F133V or W131G, and analyzed as in (B). (See also Fig. S1G.)

Figure 2.

Figure 2.. SPOP and DAXX undergo phase separation in vitro and in cells, which depends on SPOP oligomerization.

(A)SPOP and DAXX undergo phase separation in vitro. Fluorescence microscopy images of increasing concentrations of WT SPOP (green) alone, H-cDAXX (red) alone, and SPOP + H-cDAXX at a constant molar ratio of 1 SPOP : 5 DAXX. Camera settings are the same across rows. The panel boxed red is shown as separate green, red and DIC channels below. All samples in (A) and (C) contain 10% w/v ficoll 70, 500 nM ORG-SPOP and/or Rhodamine-H-cDAXX. Samples were in 25 mM Tris pH 7.6, 150 mM NaCl, and 1 mM T-CEP. (See also Fig. S2A-C.) (B) SPOP multivalency is required for SPOP/DAXX phase separation in vitro. Fluorescence microscopy images of SPOP variants (green) with reduced self-association ability, in the presence or absence of H-cDAXX (red). Camera settings are the same down columns. (C)SPOP multivalency is required for SPOP/DAXX co-localization in cells. HeLa cells were transfected with GFP-DAXX and V5-WT SPOP or mutants mutBACK or mutBTB/BACK and analyzed by confocal microscopy. GFP fluorescence was observed for DAXX, while V5-SPOP (red), and PML bodies (magenta) were detected by IF. (See also Fig. S2D.) (D) SPOP mutants are defective in DAXX ubiquitination in cells. Western Blots showing GFP-cDAXX ubiquitination in HEK293 cells that were transfected with His6-Ubiquitin, Myc-Cul3, HARbx1 and one of the SPOP variants each. The asterisk * indicates the IgG heavy chain. (See Fig. S2E for in cell ubiquitination assay with pull-down on His6-Ubiquitin.)

Figure 3.

Figure 3.. Multiple weak SPOP-binding motifs in DAXX mediate phase separation with SPOP

(A) cDAXX is intrinsically disordered and binds SPOP via several SB motifs. 15N,13C CON NMR spectrum of cDAXX at 600 MHz and 25 °C, without SPOPMATH (black) and in the presence of 2 molar equivalents of SPOPMATH (red). (For spectra annotated with all assignments, see Fig. S3A, B.) (B) Titration of SPOPMATH into cDAXX leads to identification of SB motifs. Intensity ratios of CON correlations for cDAXX upon titration with SPOPMATH (I/I0) are plotted as a function of residue number. Broadening of CON resonances of cDAXX in the presence of SPOPMATH reveals multiple SB motifs, the 5 predicted (solid orange lines), one unpredicted (dashed orange lines), and other broadened regions. (C) Sequence schematic for cDAXX constructs updated based on binding data in (B), (D) and (E). Stronger SB motifs are shown in darker shades of orange. In cDAXX-0sb, the nonpolar residue in each SB motif was replaced with a polar residue, the second residue replaced with a proline, and the rest of the motif sequence scrambled. (See Table S1 and S2 for sequences.) (D - G) cDAXX binds SPOP in an SB motif-dependent manner. Representative fluorescence anisotropy competition binding isotherms for peptides containing cDAXX binding sites (D) or mutated binding sites (E) into SPOPMATH and fluorescein-Puc91−106, and direct binding isotherms for SPOPMATH (F) and WT SPOP (G) into full-length Rhodamine-cDAXX constructs. Symbols are experimental data points; continuous or dashed lines are non-linear least-squares fits (Roehrl et al., 2004). All measurements were conducted in triplicate. (Average K _D_s are shown in Tables S2 and S3, respectively.) (H) DAXX-0sb does not phase separate with SPOP in vitro. Fluorescence microscopy images of SPOP with cDAXX or cDAXX-0sb. All samples contain 10% w/v ficoll 70, 500 nM ORG-SPOP and/or Rhodamine-cDAXX. (I) DAXX-0sb does not localize predominantly to SPOP/DAXX bodies in cells. HeLa cells were transfected with GFP-cDAXX or GFP-cDAXX-0sb and SPOP-mCherry and analyzed by confocal microscopy. cDAXX-0sb in the absence of endogenous SPOP localizes to PML bodies (Fig. S3C). (J)Quantification of partition coefficient of GFP-cDAXX and GFP-cDAXX-0sb into SPOP/cDAXX bodies in (I). Each point in the whisker plot signifies an individual cell, the mean is shown as a line. Error bars indicate the SD. (K)The cDAXX-0sb mutant is defective for ubiquitination in cells. Western Blots showing GFP-cDAXX and GFP-cDAXX-0sb ubiquitination in HEK293 cells that were transfected and analyzed as in Fig. 2D.

Figure 4.

Figure 4.. Material properties of SPOP/DAXX mesoscale assemblies.

(A) SPOP and H-cDAXX form filamentous assemblies as well as liquid droplets. Fluorescence microscopy images of SPOP/H-cDAXX as a function of protein concentration. All samples contain 10% w/v ficoll 70, 500 nM ORG-SPOP and/or Rhodamine-H-cDAXX. Images in red boxes are shown zoomed in with DIC overlaid at the right. (B) Quantification of protein concentration in mesoscale assemblies in the first row of (A, blue box). Error bars represent the SD from three replicate images. (For standard curves and additional conditions see Fig. S4A-C.) (C) Filamentous assemblies are not irreversible aggregates. (top) Time course of fluorescence microscopy/DIC images of a 15 μM SPOP : 50 μM H-cDAXX sample, which develops its typical droplet appearance over time. (middle) Addition of extra H-cDAXX to a filamentous sample incubated for 2 hr. The assemblies change from the filamentous to the droplet-like morphology. (bottom) Fusion events between SPOP/H-cDAXX droplets (red boxes). (D) Schematic of the proposed nature of assemblies at different SPOP/H-cDAXX molar ratios. SPOP alone forms oligomers (left). Oligomers are stabilized in the presence of low molar ratios of H-cDAXX, leading to large filamentous assemblies (middle left). At higher molar ratios of H-cDAXX, intermolecular interactions are favored, SPOP oligomers are smaller, and H-cDAXX contributes to liquid behavior (middle right). H-cDAXX alone forms droplets (right). (See also Fig. S4 D-G.)

Figure 5.

Figure 5.. SPOP cancer mutants disrupt phase separation and DAXX degradation.

(A) SPOP cancer mutants are defective at phase separation in vitro. Fluorescence microscopy/DIC images of WT SPOP or cancer mutants as a function of H-cDAXX concentration. All samples contain 10% w/v ficoll 70, 500 nM ORG-SPOP construct and/or Rhodamine-cDAXX. Camera settings were optimized in samples containing ~1:1 molar ratios for each row. (B) SPOP cancer mutants are defective at co-localization with DAXX in HeLa cells. SC-35 (magenta) was used as marker for nuclear speckles. Cells with SPOP-DAXX co-localization or lack thereof are indicated. (For co-staining with PML, see Fig. S5B.) (C) SPOP cancer mutants co-localize with DAXX when expressed at high levels. Whisker plot showing the signal intensity of V5-SPOP or V5-F133V (red points) and GFP-DAXX (green points) from (B) in which the V5-SPOP construct and GFP-DAXX co-localize or fail to co-localize. Each point represents a single cell. Horizontal lines indicate the mean; error bars indicate SD.

Figure 6.

Figure 6.. SPOP/DAXX bodies are active ubiquitination compartments.

(A) SPOP recruits Cul3 to SPOP/DAXX bodies. HeLa cells were transfected with the indicated constructs. Cul3-Myc (magenta) was detected by IF. (B) SPOP recruits Cul3 and Rbx1 to SPOP/DAXX bodies. Cul3-Myc (blue) and Rbx1-HA (magenta) were detected by IF. (C) Cul3 partitions into SPOP/DAXX assemblies in vitro. Fluorescence microscopy images of N8~Cul3/Rbx1 (blue channel), SPOP (green) and H-cDAXX (red). The blue-only channel images were pseudo-colored to black/white for clarity. All samples contain 500 nM of each Alexa647N8~Cul3/Rbx1, ORG-SPOP, and Rhodamine-H-cDAXX. Samples were in 25 mM HEPES pH 7.5, and 150 mM NaCl (top row) and 25 mM Tris pH 7.6, 150 mM NaCl, and 1 mM T-CEP (bottom row). (D) Conjugated ubiquitin in SPOP/DAXX bodies depends on SPOP-Cul3 interaction. HeLa cells were transfected with the indicated constructs. Cul3-Myc (blue) and conjugated ubiquitin (magenta, with FK2 antibody) were detected by IF. (E) Disruption of SPOP/Cul3 interaction results in increased GFP-cDAXX levels and decreased conjugated ubiquitin levels. Quantification of signals from GFP (green bars), conjugated ubiquitin (magenta bars), and conjugated ubiquitin normalized by GFP (open bars) for n=20 cells per condition in (D). (F) Schematic representation of in vitro ubiquitination assay. Transfer of ubiquitin is monitored by SDS-PAGE and the incorporation of fluorescent *UB into assemblies microscopically. (G) Ubiquitinated H-cDAXX accumulates in SPOP/H-cDAXX assemblies in vitro. Fluorescence microscopy/DIC images showing the time-course of in vitro ubiquitination assays described in (F) at the indicated SPOP (green) / H-cDAXX (red) molar ratios plus 1.25 μM N8~Cul3/Rbx1, 20 nM ARIH1 as indicated on the left, and 1.5 μM UbcH7~*UB (blue). All reactions contain either ficoll 70 or sucrose as indicated, and 500 nM ORG-SPOP and Rhodamine-H-cDAXX; *UB denotes stoichiometrically labeled Alexa647-Ubiquitin. (See Fig. S6C for images of control reaction conditions.) (H) Ubiquitination can occur in the presence or absence of SPOP/DAXX assemblies. Representative fluorescent scan of non-denaturing gels showing time course of in vitro ubiquitination reactions described in (F). Blue UBCH7~*UB band diminishes and blue H-cDAXX~*UB band appears over the course of reactions containing WT ARIH1 + ficoll70 or sucrose, but not in reactions containing no ARIH1 or the catalytically inactive mutant, ARIH1C375S. (I) Quantification of in vitro ubiquitination assay from blue fluorescence intensity of assemblies in fluorescence microscopy images in (G and Fig. S6C)—left; and gel band intensity of product HcDaxx~*UB in (H and Fig. S6C)—right. Data points represent average of triplicate experiments. Error bars indicate SD.

Figure 7.

Figure 7.. SPOP phase separates with androgen receptor and may phase separate with other substrates in an evolutionarily conserved fashion.

(A) Sequence schematic for AR and the N-terminal fragment used for in vitro experiments (nAR). AR contains an N-terminal disordered domain, DNA binding domain (DBD), and a ligand binding domain (LBD) (Centenera et al., 2008). (B) SPOP phase separates in vitro with nAR. Fluorescence microscopy images of SPOP (green) and nAR (red). All samples contain 10% w/v ficoll 70, and 500 nM ORG-SPOP and/or Rhodamine-nAR. Samples were in 20 mM NaPO4 pH 7.4, 50 mM NaCl, 2 mM T-CEP, and 1 mM EDTA. (C) SPOP co-localizes with the AR in cells. HeLa cells were transfected with the indicated constructs and analyzed by confocal microscopy. (D) Covariation analysis of SPOP shows evolutionary coupling across the BTB and BACK interfaces. Co-evolutionary couplings in SPOP from covariation of ~2600 SPOP homologues (Table S7) sharing all three structural domains. The co-evolutionary couplings (top 600) are reported in the upper triangle of the matrix as black dots with size proportional to the relative coupling strength, overlapping both intra- and intermolecular contacts. The couplings are compared to contacts between pairs of residues with a distance of up to 5 Å between sidechain heavy atoms, based on a structural model of SPOP28−359 (built using two available crystal structures (PDB ID 3HQI (Zhuang et al., 2009) and 4HS2 (van Geersdaele et al., 2013)), and no further assumptions); intra- and intermolecular contacts are shown in blue and red, respectively. (E) BTB and BACK interface residues coevolve with residues across the interface, not with domain core residues. Evolutionary domains, obtained by the analysis of the patterns of couplings (Granata et al., 2017) are reported on the SPOP monomer structure model for the subdivision in 2 groups of coevolving residues (Q=2) (top), and on the oligomer structure model for 5 groups of coevolving residues (Q=5) (bottom). Other meaningful subdivisions are reported in Fig. S7B-D. (F) Schematic of proposed mechanism. SPOP phase separates with multivalent substrates, and is able to target and ubiquitinate substrates localized to membrane-less organelles. SPOP cancer mutants are defective at phase separation and therefore co-localization and ubiquitination.

Comment in

References

    1. An J, Wang C, Deng Y, Yu L, and Huang H (2014). Destruction of full-length androgen receptor by wild-type SPOP, but not prostate-cancer-associated mutants. Cell Rep 6, 657–669. - PMC - PubMed
    1. Anthis NJ, and Clore GM (2013). Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 22, 851–858. - PMC - PubMed
    1. Banani SF, Lee HO, Hyman AA, and Rosen MK (2017). Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18, 285–298. - PMC - PubMed
    1. Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, and Vasos PR (2005). Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Edit 44, 3089–3092. - PubMed
    1. Bermel W, Bertini I, Felli IC, Kummerle R, and Pierattelli R (2006a). Novel 13C direct detection experiments, including extension to the third dimension, to perform the complete assignment of proteins. J Magn Reson 178, 56–64. - PubMed

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