Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles

Abstract

Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies.

Keywords: isodesmic self‐association; membrane‐less organelle; prostate cancer; speckle‐type POZ protein; ubiquitin ligase.

Figure 1. SPOP is composed of three distinct domains

1. Cartoon schematic of the SPOP self‐association‐incompetent mutant constructs that form complexes through only one (or neither) of the self‐association domains is shown. The previously described mutation Y353E (van Geersdaele et al, 2013) was introduced into the BACK domain so that self‐association could only occur through BTB‐mediated interactions resulting in SPOP mutBACK; previously described mutations of the conserved BTB interface (Zhuang et al, 2009) resulted in SPOP mutBTB, which self‐associates only through BACK‐mediated interactions. Constructs for expression in mammalian cells encode the full‐length protein; those for expression in bacteria comprise residues 28–359.

2. Ribbon diagram of SPOP ΔBACK [PDB ID 3HQI (Zhuang et al, 2009)] showing residues that are mutated to abolish BTB domain self‐association (L186D, L190D, L193D, I217K). The domains are colored as in (A) (MATH, green; BTB, red; and BACK, blue), with one monomer shown in gray for clarity. Introduction of these mutations does not abolish the interaction between SPOP and the CRL because they are not part of the SPOP/cullin‐3 interface (Errington et al, 2012; Zhuang et al, 2009) and because mutation of the BTB domain or deletion of the BACK domain does not prevent SPOP from promoting ubiquitination of substrates (Zhuang et al, 2009; Errington et al, 2012).

3. Ribbon diagram of SPOP ^270–374 (L273D L282D L285K) dimer crystal structure [PDB ID 4HS2 (van Geersdaele et al, 2013)] showing residue Y353, which is mutated to E to prevent BACK domain self‐association. The domains are colored as in (A) (MATH, green; BTB, red; and BACK, blue), with one monomer shown in gray for clarity.

Figure 2. SPOP localizes to nuclear speckles or other nuclear bodies

1. A–C

   Confocal microscopy images of fixed NIH 3T3 cells transiently expressing (A) full‐length HA‐SPOP, (B) GFP‐Gli3^1–455, and (C) HA‐SPOP + GFP‐Gli3^1–455 are shown. DAPI was used to stain the nucleus, SPOP localization was identified using an anti‐HA antibody, nuclear speckle localization was identified using an anti‐SC‐35 antibody, and Gli3^1–455 localization was identified via GFP fluorescence. SPOP co‐localizes with a marker for nuclear speckles or with the substrate Gli3^1–455. The areas with overlapping HA and GFP signal contain 75% of the punctate HA signal and 100% of the punctate GFP signal. For expression levels of HA‐SPOP and GFP‐Gli3^1–455 proteins, see Appendix Fig S3. Transfection efficiencies of pcDNA‐GFP‐Gli3^1–455 and pcDNA‐HA‐SPOP are 20–25% and 6–10%, respectively. When both constructs are used, ˜70–80% of transfected cells express both constructs (see Appendix Tables S1, S2 and S3).

Source data are available online for this figure.

Figure EV1. Size distribution of nuclear puncta

1. A

   Gli3^1–455‐positive bodies are not Cajal bodies, nucleoli, polycomb bodies, or PML bodies. NIH 3T3 cells were transfected with GFP‐Gli3^1–455 and stained with antibodies against coilin (a Cajal marker), B23 (a nucleolus marker), CBX8 (a polycomb marker), or PML (a PML body marker).

2. B

   NIH 3T3 cells were transfected with a construct expressing SC‐35‐GFP and GFP fluorescence was monitored in live cells. Snapshots at indicated time points show a nuclear speckle fusion event.

3. C

   NIH 3T3 cells were transfected with a construct expressing GFP‐Gli3^1–455, and GFP fluorescence was monitored in live cells. Individual nuclear bodies were photobleached, and FRAP was monitored for 1 min. Data were normalized to the maximum and minimum intensity. The mean characteristic recovery time is indicated ± SEM.

4. D

   Histograms depicting the size distribution of nuclear body areas are shown for cells transfected with HA‐SPOP alone and GFP‐Gli3^1–455 + HA‐SPOP.

5. E–G

   NIH 3T3 cells were transfected with only HA‐SPOP or GFP‐Gli3^1–455 + HA‐SPOP. Box plots of the median aspect ratio of the (E) GFP‐Gli3^1–455‐positive nuclear bodies in one cell, (F) of the intracellular median area of nuclear speckles, and (G) of the number of bodies per cell for cells transfected with HA‐SPOP alone and GFP‐Gli3^1–455 + HA‐SPOP are shown. All three were significantly different (P = 1.2 × 10⁻⁴², P = 4.6 × 10⁻¹⁹, and P = 8.7 × 10⁻⁶, respectively, according to the Wilcoxon rank‐sum test), consistent with the different nature of distinct nuclear bodies. The medians are indicated as black horizontal lines within the boxes, and boxes enclose values between the first and third quartile. Interquartile range (IQR) is calculated by subtracting the first quartile from the third quartile. All values that lay more than 1.5× IQR lower than the first quartile or 1.5× higher than the third quartile are outliers that are plotted as squares. The smallest and highest values that are not outliers are connected with the dashed line.

Source data are available online for this figure.

Figure 3. SPOP nuclear bodies have liquid‐like character

1. A, B

   NIH 3T3 cells were (A) co‐transfected with constructs expressing full‐length SPOP and GFP‐Gli3^1–455 or (B) transfected with a construct expressing SC‐35‐GFP, and GFP fluorescence was monitored in live cells. (A) Snapshots were taken from individual time points as noted in the figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. (B) Snapshots at the indicated time points show a nuclear speckle fusion event. Additional images are shown in Fig EV1.

2. C

   Individual nuclear speckles were photobleached and FRAP was monitored for 90 s. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM.

3. D, E

   Nuclear bodies are close to spherical. (D) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA‐SPOP (151 cells) (D), or GFP‐Gli3^1–455 and HA‐SPOP (155 cells) (E) were calculated as described previously (Brangwynne et al, 2011). Representative individual images of cells are shown as insets in each panel.

Figure 4. Self‐association‐defective SPOP mutants do not localize to nuclear speckles

1. SEC chromatograms of given loading concentrations of SPOP ^28–359 are shown. The concentrations were normalized to the monomer molecular weight, that is, identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP ^28–359 contain identical numbers of protomers.

2. SEC chromatograms for SPOP constructs defective in self‐association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 μM), and the elution volume of globular molecular weight standards is noted above the graph.

3. SEC chromatograms of SPOP ^28–359 (200 μM), SPOP mutBACK (718 μM), and mixtures of the two (ratios given in the figure, SPOP at 200 μM) are shown. The elution volume of globular molecular weight standards is noted above the graph.

4. Constructs for expressing full‐length HA‐SPOP or HA‐SPOP mutants capable of oligomerization through only one (or neither) of the self‐association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus, and SPOP localization was identified using an anti‐HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined, and representative cells are shown. For additional images, see Appendix Fig S4.

Source data are available online for this figure.

Figure EV2. SPOP ΔBACK does not elute in a concentration‐dependent manner and SPOP WT forms aggregates in vitro

1. SEC chromatograms of given loading concentrations of SPOP ΔBACK are shown. The concentrations were normalized to the monomer molecular weight, that is, identical concentrations of dimeric SPOP ΔBACK and oligomeric SPOP ^28–359 contain identical numbers of protomers.

2. Protein aggregation was assayed by centrifuging protein samples, resuspending soluble pelleted material in buffer three times, and then resuspending the final insoluble pellet in sample loading dye. The ultracentrifugation conditions are expected to pellet some of the larger SPOP ^28–359 oligomeric species. These species are readily soluble in fresh buffer and do not represent aggregated material. In contrast, the majority of SPOP FL forms insoluble aggregates that do not dissociate even under extensive dilution, but can be resolubilized in denaturing gel sample buffer. These results show at least very slow off‐rates of SPOP from the aggregates, not only high stability of the aggregates, and are therefore strongly indicative of poor reversibility of aggregation.

3. SPOP ^28–359 oligomers have no apparent size limit. The apparent molecular weights (calculated from globular standards) of the major eluting species for each injection in Fig 4A and panel (A) are plotted against their elution concentrations and fit to a line. The number of monomers per oligomer was calculated by dividing the apparent molecular weight by the monomer mass and assumes regular packing of the monomer in the oligomer. The average and standard deviation of two independent experiments are shown for SPOP ^28–359. Inset shows the same data in a semi‐logarithmic plot to highlight the lower concentrations of SPOP ^28–359 assayed.

Figure 5. SPOP forms higher‐order oligomeric species in cells

1. Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP variants bound substrate with an affinity similar to that of SPOP ^28–359 (4–8 μM, see Table 2). Experimental data are shown as circles; the solid lines are fits to equation (2) (Roehrl et al, 2004).

2. In vitro cross‐linking assays were performed for SPOP ^28–359 and each mutant at 30 μM protein with the amide‐specific BS3 cross‐linker. Cross‐linking for SPOP ΔBACK and MATH domain are shown to demonstrate that cross‐linking conditions do not lead to non‐specific cross‐linking of protein species.

3. Cross‐linking reactions were performed on whole‐cell lysates from cells expressing wild‐type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB–BACK. SPOP ₁, SPOP ₂, and SPOP _n identify SPOP monomers, dimers, and larger species, respectively. For loading levels, see Appendix Fig S5.

Figure 6. The SPOP oligomerization domains dimerize with different affinities

1. A–C

   The BTB domain dimerizes with nanomolar affinity. (A) SV‐AUC data for 0.2 nM AF488‐SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c(s) analysis in SEDFIT (Schuck, 2000), and the results of this fit are shown (solid lines, rainbow color scheme). (B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown. (C) The isotherm of weight‐average s‐values versus concentration (black circles) was fit to a dimer self‐association model and reveals a K_D of 1.11 nM [95% confidence interval 0.88, 1.40 nM] (solid line).

2. D

   The BACK domain dimerizes with micromolar affinity. Experimental weight‐average molar mass (M_w) from CG‐MALS for SPOP mutBTB (black circles) was fitted to a self‐association model (see text for details) for BACK–BACK dimerization (black line) yielding a K_D of 36 ± 3 μM (average and standard deviation from three independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer–trimer, tetramer, and pentamer equilibrium models are depicted as black solid line and as dashed and gray lines, respectively. Residuals for all models are depicted in the lower panel.

Figure 7. Indefinite SPOP self‐association fits to an isodesmic model

1. Cartoon schematic depicting the proposed isodesmic self‐association model. K_{D 1} and K_{D 2} represent the domain‐mediated dimerization affinities for the BTB domain and BACK domains, respectively. K_{D 2} is identical for all association steps independent of oligomer size. At large oligomer sizes, K_{D 2} may increase due to entropic penalties, resulting in K_{D 2}*. The N‐ and C‐termini contain neither defined domains nor low‐complexity sequences but may add additional self‐association behavior as evidenced by aggregation; these interactions were not dissected due to the poor reversibility of aggregation (Fig EV2B).

2. Experimental weight‐average molar mass (M_w) from CG‐MALS for SPOP ^28–359 (orange circles) was fitted to an isodesmic self‐association model in which SPOP dimers are the self‐associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers [(SPOP ₂)₁₁]. The fits from three independent experiments yielded a K_{D 2} of 2.4 ± 0.4 μM. Lines for fits of the data to self‐association models that assume formation of individual oligomeric species instead of isodesmic self‐association are shown for reference (gray lines).

3. Experimental weight‐average molar mass (M_w) from CG‐MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self‐association domains are interaction‐competent. This figure is comprised of data shown in panel (B), Fig 6D and Appendix Fig S6A for direct comparison.

4. Ribbon diagram illustrating structural model of an octamer SPOP assembly. In a 27 μM SPOP solution, three percent of the oligomeric assemblies would be equal in size or larger. The domains are colored as in panel (A) and Fig 1 (MATH, green; BTB, red; and BACK, blue), with alternating monomers shown in gray for clarity. The model was built using two available crystal structures [PDB ID 3HQI (Zhuang et al, 2009) and 4HS2 (van Geersdaele et al, 2013)], and no further assumptions.

Figure EV3. A family of BTB–BACK domain‐containing proteins in rodents may self‐associate isodesmically

1. A

   The BACK domain dimer is stabilized via a hydrogen bond between N326 and Y353 and a salt bridge between E334 and R354. The two BACK domain monomers are shown in different shades of blue.

2. B

   The long form of the BACK domain, as exemplified by KLHL3 (shown in yellow, PDB ID 4HXI), occludes the dimerization interface of the short SPOP BACK domain.

3. C, D

   The human genome contains only two genes encoding a protein with a BTB and short version of the BACK domain, SPOP and SPOPL, the latter of which does not dimerize because of a sterically hindering insertion. The family of BTB–BACK domain‐containing proteins is expanded in rodents. (C) Alignment of mouse protein sequences containing a BTB and a short BACK domain with human SPOP. The N326/Y353 and E334/R354 interactions are not conserved but could potentially be replaced by hydrophobic/aromatic and negatively charged/histidine interactions, respectively. (D) Alignment of rat protein sequences containing a BTB and a short BACK domain with human SPOP. In the TDPZ variant shown on the bottom, residues E334 and R354 are conserved, potentially enabling a salt bridge. This protein may undergo isodesmic self‐association into higher‐order oligomers.

Figure 8. SPOP forms dynamic higher‐order oligomers that can be analyzed quantitatively

1. Native MS spectrum of 30 μM SPOP ^28–359 confirms that SPOP self‐associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies.

2. Graphical representation of the SPOP concentration of each oligomeric species within a 27 μM SPOP ^28–359 sample. The cartoons' sizes are scaled based upon the fraction present within the total sample.

3. Concentration dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG‐MALS data.

Figure EV4. Detailed MS analysis of SPOP oligomers

1. Schematic representation of putative SPOP assemblies. Under each assembly state, the number of monomers and the theoretical mass are shown.

2. Mass spectrum of denatured SPOP. To evaluate the purity of the sample, SPOP (50 pmol) was loaded onto a monolithic column (Rozen et al, 2013) and eluted in a linear gradient of 20–50% ACN over 30 min. The protein eluted as a single peak at 17.6–19.8 min, at approximately 33% ACN. The spectrum shows a major population of 37,656 ± 5 Da (orange circles) and a smaller population of 37,730 ± 13 Da (brown circles). The mass difference may result from β‐mercaptoethanol.

3. MS spectrum of SPOP assemblies under partial denaturing conditions. SPOP was analyzed by performing MS after adding of 0.1% formic acid (30 μM) to disturb the non‐covalent interactions. Under these conditions, trimers (red circles) and pentamers (orange circles) were generated. The fact that odd numbers of assemblies appear only under mild denaturing conditions indicates strong interactions between two monomers.

4. Size distribution of SPOP oligomers at 30 μM protomer concentration from seven independent measurements.

Figure EV5. The dynamic nature of higher‐order SPOP oligomers is mediated by the BACK domain

Samples were either mixed at 4°C and immediately injected onto the column or were incubated at 37°C for 90 min prior to injection. All SEC was performed at 4°C.

1. SEC chromatograms for individual SPOP constructs injected at a loading concentration of 533 μM (and SPOP ^28–359 at 1,066 μM) are shown. In agreement with CG‐MALS results, SPOP mutBTB and SPOP mutBACK predominantly form dimers (red and blue lines, respectively).

2. SEC chromatogram for mixtures of 533 μM SPOP ^28–359 with 533 μM of SPOP mutBACK is shown. When equal amounts of SPOP ^28–359 and one of the mutants were mixed at 4°C, we observed that SPOP mutBACK, which cannot participate in typical BACK–BACK interactions, did not increase the population of higher‐order oligomers relative to that of WT only, as evidenced by a similar elution peak from the oligomeric species. This result shows that BTB dimers do not dissociate on the timescale of SEC at low temperature and cannot form hetero‐oligomers with SPOP ^28–359. However, incubation at 37°C for 90 min renders BTB domain interactions dynamic enough to allow for BTB domain‐mediated exchange between WT and SPOP mutBACK (dark gray line). The hetero‐oligomers are smaller in size than oligomeric species observed for SPOP ^28–359 alone at 533 μM (orange line).

3. SEC chromatogram for mixtures of 533 μM SPOP ^28–359 with 533 μM of SPOP mutBTB is shown. Conversely, in mixtures of SPOP ^28–359 and SPOP mutBTB, which can effectively interact only through the BACK domain, the population of the higher‐order oligomeric species increased relative to that of SPOP ^28–359 alone, irrespective of incubation time or temperature (gray lines). However, the mixed oligomers are smaller than SPOP ^28–359 oligomers at an equimolar protein loading concentration (golden line).

Figure EV6. Higher‐order SPOP self‐association promotes ubiquitination in vitro

1. In vitro ubiquitination assays with CRL3^SPOP were performed as described previously (Zhuang et al, 2009) using His‐Gli3^1–455 as a substrate, SPOP ^28–359, and each of the self‐association‐incompetent mutants. The reaction was monitored for 30 min, and time points taken are indicated below the Western blot. Ubiquitination efficiency was monitored by immunoblotting with an anti‐His antibody.

2. Reactions were performed as in (A) and monitored for 30 min. Equal amounts of SPOP variants were used as shown in the SDS–PAGE gel.

Figure 9. The self‐association‐deficient SPOP mutant mutBTB has a substrate degradation defect in vivo

UAS transgenes were expressed under the control of an epithelial driver C765‐Gal4 in Drosophila melanogaster.

1. A

   UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1–5.

2. B

   Expression of SPOP WT resulted in a modest Hh loss‐of‐function phenotype with fusion of LV3 and LV4 (arrow).

3. C

   mutBTB acts in a dominant‐negative manner, as evidenced by a modest Hh gain‐of‐function phenotype with expansion of the LV3–LV4 intervein space.

4. D, E

   Expression of (D) mutBACK and (E) mutBTB–BACK does not induce a wing‐patterning defect.

Data information: ~50 animals each were analyzed from two independent crosses. Source data are available online for this figure.

Figure 10. Higher‐order SPOP oligomers localize to nuclear speckles

We propose a model in which the ability of SPOP to form higher‐order oligomers promotes its localization to nuclear speckles. Smaller oligomeric SPOP species are diffusely distributed, but higher‐order oligomers preferentially localize to speckles. Dynamic BACK domain association mediates exchange between higher‐order/punctate structures and the diffuse pool. SPOP interacts with a number of binding partners, including other components of the CRL and substrates, and can likely recruit them to punctate structures (Kwon et al, 2006; Chen et al, 2009). We speculate that nuclear speckles serve as hotspots of SPOP‐mediated ubiquitination by concentrating SPOP oligomers, substrates, and other components locally.