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. 2014 Mar 21;289(12):8390-401.
doi: 10.1074/jbc.M113.534024. Epub 2014 Feb 5.

Interaction With the Bardet-Biedl Gene Product TRIM32/BBS11 Modifies the Half-Life and Localization of Glis2/NPHP7

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Free PMC article

Interaction With the Bardet-Biedl Gene Product TRIM32/BBS11 Modifies the Half-Life and Localization of Glis2/NPHP7

Haribaskar Ramachandran et al. J Biol Chem. .
Free PMC article

Abstract

Although the two ciliopathies Bardet-Biedl syndrome and nephronophthisis share multiple clinical manifestations, the molecular basis for this overlap remains largely unknown. Both BBS11 and NPHP7 are unusual members of their respective gene families. Although BBS11/TRIM32 represents a RING finger E3 ubiquitin ligase also involved in hereditary forms of muscular dystrophy, NPHP7/Glis2 is a Gli-like transcriptional repressor that localizes to the nucleus, deviating from the ciliary localization of most other ciliopathy-associated gene products. We found that BBS11/TRIM32 and NPHP7/Glis2 can physically interact with each other, suggesting that both proteins form a functionally relevant protein complex in vivo. This hypothesis was further supported by the genetic interaction and synergist cyst formation in the zebrafish pronephros model. However, contrary to our expectation, the E3 ubiquitin ligase BBS11/TRIM32 was not responsible for the short half-life of NPHP7/Glis2 but instead promoted the accumulation of mixed Lys(48)/Lys(63)-polyubiquitylated NPHP7/Glis2 species. This modification not only prolonged the half-life of NPHP7/Glis2, but also altered the subnuclear localization and the transcriptional activity of NPHP7/Glis2. Thus, physical and functional interactions between NPHP and Bardet-Biedl syndrome gene products, demonstrated for Glis2 and TRIM32, may help to explain the phenotypic similarities between these two syndromes.

Keywords: Bardet-Biedl Syndrome; Ciliopathies; Genetic Diseases; Nephronophthisis; Protein Degradation; Protein Stability; Transcription Regulation; Ubiquitylation.

Figures

FIGURE 1.
FIGURE 1.
Glis2 interacts with BBS1, BBS2, and BBS11/TRIM32. FLAG-tagged BBS proteins (BBS1–12) were transiently co-transfected with V5.Glis2 into HEK 293T cells. After 24 h of transfection, cells were lysed, and FLAG-tagged BBS proteins were immunoprecipitated with FLAG-M2 beads. The precipitates were loaded onto 10% SDS gel and analyzed by Western blotting using anti-V5 and anti-FLAG antibodies. V5.Glis2 interacted with F.BBS1, F.BBS2, and F. BBS11/TRIM32, but not with the other BBS proteins. Antibody heavy chains were marked with HC. IP, immunoprecipitation; WB, Western blotting.
FIGURE 2.
FIGURE 2.
TRIM32 interacts with the zinc finger domains of Glis2. A, FLAG-tagged TRIM32 (F.TRIM32) or F.BBS2 were transiently co-expressed in HEK 293T cells with V5-tagged Glis2 (V5.Glis2) or V5.NPHP5. Precipitation of F.TRIM32 immobilized V5.Glis2, but not V5.NPHP5. B, lysates of HEK 293T cells, transiently transfected with F.Glis2, were incubated with recombinant GST.TRIM32 or GST. F.Glis2 was pulled down by GST.TRIM32 but not by GST. Expression levels of GST.TRIM32 and GST were checked by Coomassie staining. C, truncations of Glis2, V5.Glis2 (amino acids 1–152), V5.Glis2 (amino acids 346–525), and V5.Glis2 (amino acids 146–359) were generated to map the interaction with TRIM32. F.TRIM32 interacted with the zinc finger-containing fragment of V5.Glis2, spanning amino acids 146–359. D, FLAG-tagged fragments of TRIM32 were co-expressed with V5.Glis2. Only the N-terminal fragment of TRIM32, spanning amino acids 1–256, interacted with Glis2. HC, antibody heavy chains; LC, antibody light chains; IP, immunoprecipitation; WB, Western blotting.
FIGURE 3.
FIGURE 3.
Combined depletion of zGlis2 and zTRIM32 enhances cyst formation of the zebrafish pronephros. A, WT1-GFP transgenic zebrafish embryos, highlighting the proximal pronephros, were injected with control or zTRIM32-specific MO and scored for cyst formation at 55 h postfertilization. Knockdown of zTRIM32 resulted in pronephric cyst formation in a dose-dependent manner. The pronephric cysts of zTRIM32-deficient zebrafish embryos are marked with asterisks in the GFP-labeled pronephric ducts. No cyst formation was observed in zebrafish embryos injected with control MO. B, zebrafish embryos were injected with different MO combinations as indicated. Low doses of zTRIM32 MO enhanced the cyst formation in zebrafish embryos injected with zGlis2 MO.
FIGURE 4.
FIGURE 4.
TRIM32 stabilizes Glis2. A, HEK 293T cells were transiently transfected with V5.Glis2 together with a control vector, F.TRIM32, or F.CD2AP. After 24 h of transfection, cells were lysed; proteins were resolved on SDS-PAGE gel and detected by Western blotting, using anti-V5 and anti-FLAG antibodies. Protein levels of Glis2 were quantified using LabImage 1D software and normalized to β-actin protein levels. The bars (right panel) represent the relative protein levels from three independent experiments. B, HEK 293T cells, transfected with V5.Glis2, were treated with DMSO or the proteasomal inhibitor MG132 (12 μm) for 2 h and co-transfected with F.TRIM32 as indicated. MG132 treatment increased Glis2 protein levels but had no apparent effect in the presence of TRIM32. The bars (right panel) represent the quantification of Glis2 protein levels normalized to β-actin levels from three independent experiments. C, HEK 293T cells were transfected with F.Glis2 and HA-tagged ubiquitin. After 24 h of transfection, cells were treated with MG132 or DMSO (control). F.Glis2 was precipitated using FLAG-M2 beads. Ubiquitylated Glis2 (Ubi-Glis2) was detected using an anti-HA antibody, demonstrating an increase of ubiquitylated Glis2 species in the presence of MG132. D, HEK 293T cells, transfected with Glis2 alone or together with TRIM32, were treated with cycloheximide (30 μg) to inhibit protein synthesis. Glis2 protein levels were followed over a time course of 9 h by Western blot analysis. Glis2 protein levels remained stable in the presence of TRIM32. Anti-β-actin staining was used to control for equal loading. E, the graph demonstrates the decline of Glis2 protein levels after inhibition of protein synthesis with cycloheximide in the absence (purple line) or presence (blue line) of TRIM32. The results represent the average of three experiments; β-actin was used as a loading control (see Fig. 3D). F, protein levels of V5.Glis3 remained unaffected in the presence of different levels of F.TRIM32. G, protein levels of Glis2 were not increased by the presence of BBS1. H, the N-terminal truncation of F.TRIM32 (1–256), interacting with Glis2, was sufficient to increase Glis2 protein levels, whereas noninteraction F.TRIM32 truncations failed to affect Glis2 protein levels. IP, immunoprecipitation; WB, Western blotting.
FIGURE 5.
FIGURE 5.
TRIM32 promotes the accumulation of ubiquitylated Glis2. A, HEK 293T cells were transfected with HA-tagged ubiquitin in combination with the plasmids as indicated. After 24 h of transfection, F.Glis2 was precipitated using FLAG-M2 beads; ubiquitylated Glis2 (Ubi-Glis2, top panel) was detected using an anti-HA antibody. Autoubiquitylation of TRIM32 was detectable except for the TRIM32.D487N mutation (lane 5, bottom panel). B, the Glis2-interacting, N-terminal domain of TRIM32 (aminio acid 1–256) was sufficient to enhance ubiquitylation of Glis2. C, the accumulation of ubiquitylated Glis2 was preserved in the presence of E3 ligase-deficient TRIM32 ΔRING mutant. F.Glis2 was co-transfected with V5.TRIM32 lacking E3 ligase activity (TRIM32 ΔRING) and V5.TRIM32 WT. Consistent with the elimination of the E3 ligase activity, no autoubiquitylation was detectable for the TRIM32 ΔRING mutant (lane 4, bottom panel). D, HEK 293T cells were transfected with the plasmids as indicated. Polyubiquitylation of F.Glis2 was detected in the presence of the E3 ligase-deficient TRIM32 ΔRING mutant, using anti-Fk1 antibody. IP, immunoprecipitation; WB, Western blotting.
FIGURE 6.
FIGURE 6.
TRIM32 promotes K63-linked ubiquitylation of Glis2. A, HECT E3 ubiquitin ligases did not increase Glis2 ubiquitylation. Ubiquitylation of F.Glis2 was monitored in the presence of different HECT E3 ubiquitin ligases, and ubiquitylated Glis2 was detected by an anti-HA antibody. B, HEK 293T cells were transfected with constructs as indicated, and ubiquitylation of F.Glis2 was compared in the presence of the ubiquitin K48R and K63R mutants. K48R ubiquitin reduced the ubiquitylation more efficiently, suggesting that in the absence of TRIM32, Glis2 is predominantly K48-ubiquitylated. C, in vivo ubiquitylation assay was performed to assess the ubiquitylation of Glis2 in the presence of wild-type HA.ubiquitin (Ubi-WT), HA.ubiquitin K48R (Ubi-K48R), and HA.ubiquitin K63R (Ubi-K63R). The TRIM32 ΔRING was used to prevent autoubiquitylation of TRIM32. Both K48R and K63R ubiquitin reduced Glis2 ubiquitylation to a similar degree, suggesting that TRIM32 shifts Glis2 ubiquitylation toward K63 ubiquitin chain species. IP, immunoprecipitation; WB, Western blotting.
FIGURE 7.
FIGURE 7.
TRIM32 facilitates the accumulation of K63 ubiquitin chain-containing Glis2. Glis2 was co-expressed with HA-tagged, wild-type ubiquitin (Ubi-WT), HA-tagged ubiquitin K48R (Ubi-K48R), or HA-tagged ubiquitin K63R (Ubi-K63R) in HEK 293T cells. In A, cells were incubated for the last 2 h with 12 μm MG132. In B, cells were co-transfected with V5-tagged TRIM32 (V5.TRIM32). K63-linked ubiquitin chain accumulated in the presence of V5.TRIM32, but not after incubation with MG132, using a K63 specific antibody. IP, immunoprecipitation; WB, Western blotting.
FIGURE 8.
FIGURE 8.
TRIM32 recruits Glis2 into subnuclear domains. A, immunofluorescence analysis of HEK 293T cells expressing YFP.Glis2 (green) alone or in the presence of TRIM32. Nuclei were stained with Hoechst blue. YFP.Glis2 alone showed a diffuse nuclear localization (left panel). The arrowheads point highlight the recruitment of YFP.Glis2 into specific subnuclear domains in the presence of TRIM32. Cells with Glis2 localizing nuclear dots were counted and quantified (right panel). DIC, differential interference contrast. B, the N-terminal domain of F.TRIM32, spanning the Glis2-interacting amino acids 1–256, is sufficient to drive the recruitment to nuclear aggregates (top panel), whereas the two noninteracting fragments (F.TRIM32 (amino acids 247–426) and F.TRIM32 (amino acids 400–653)) failed to alter the subcellular localization of Glis2. C, no recruitment of Glis2 was detected in the presence of TRIM28, another TRIM family member. D, YFP.Glis2 and F.TRIM32 partially co-localized within the nucleus, suggesting that Glis2 facilitates the nuclear import of TRIM32. YFP.Glis2 and FLAG-tagged TRIM32 were transiently co-expressed in HEK 293T cells. F.TRIM32 was detected using a polyclonal FLAG rabbit antibody followed by Cy3-conjugated donkey anti-rabbit IgG (red). E, only a subset of PML bodies (red) co-localized with the YFP.Glis2-positive nuclear aggregates in HEK 293T cells. Endogenous PML was detected using anti-PML mouse antibody followed by Cy3-conjugated donkey anti-mouse IgG.
FIGURE 9.
FIGURE 9.
Glis2 interacts with PML only in the presence of TRIM32. HEK 293T cells were transfected with the plasmids as indicated. Precipitation of F.PML did not immobilize V5.Glis2 (left panel). However, V5.Glis2 was detectable in the immunoprecipitates of F.PML in the presence of V5.TRIM32 (middle panel), but not in the presence of a control protein (V5.CD2AP) (right panel). IP, immunoprecipitation; WB, Western blotting.
FIGURE 10.
FIGURE 10.
TRIM32 modulates the transcriptional activity of Glis2. A, the repressive effect of Glis2 on β-catenin-dependent transcriptional activity (TOPflash) was reversed by the co-transfection of TRIM32. Four independent experiments were performed in triplicate, and the values were normalized to β-galactosidase. B, TRIM32 inhibits the transcriptional activation of mIns2 (mouse insulin-2 promoter) by Glis2. HEK 293T cells were transfected with the depicted constructs to quantify the effect of TRIM32 on the transcriptional activation of the mIns2 promoter by Glis2.

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