Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis

Abstract

Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis.

Fig. 1

Itch ubiquitylates and binds SuFu. a, b HEK293T cells were transfected with plasmids expressing HA-ubiquitin (HA-Ub) in the presence of different E3 ubiquitin ligases (a) or increasing amount of Flag-Itch (b). Cell lysates were immunoprecipitated with an anti-SuFu antibody, and ubiquitylated forms were revealed with an anti-HA antibody. c HEK293T cells were co-transfected with Flag-Itch and HA-SuFu as indicated. Interaction between Itch and SuFu was detected by immunoprecipitation (IP) followed by immunoblot (IB) analysis with the indicated antibodies. d Interaction between endogenous Itch and SuFu was detected in HEK293T cells by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. e GST-Itch was bound to glutathione-sepharose beads and used for in vitro pull-down assay. In vitro translated ³⁵S-labelled SuFu was incubated with free GST control or GST-Itch. After GST pull-down, the protein complex was detected by fluorography. Coomassie blue staining shows the expression levels of recombinant proteins GST-Itch or GST only. f GST-SuFu was bound to glutathione-sepharose beads and used for in vitro pull-down assay. Untagged Itch recombinant protein was incubated with free GST control or GST-SuFu. After GST pull-down, the protein–protein interaction was detected by IB with an anti-Itch antibody. Coomassie blue staining shows the expression levels of recombinant proteins GST-SuFu or GST only. g Schematic representation of Itch and its interaction with SuFu. h, i GST-HECT (h) or GST-4WWs (i) were bound to glutathione-sepharose beads and used for in vitro pull-down assay with in vitro translated ³⁵S-labelled SuFu. After GST pull-down, protein complexes were analysed by IB. j A GST pull-down assay with GST-WW1, -WW2, -WW3, or -WW4 and in vitro translated ³⁵S-labelled SuFu was carried out as described in e

Fig. 2

Itch ubiquitylates SuFu through K63 linkage. a HEK293T cells were co-transfected with HA-Ub in the presence or absence of Flag-Itch or Flag-C830A. Cell lysates were immunoprecipitated with an anti-SuFu antibody, followed by immunoblotting with an anti-HA antibody to detect ubiquitylated forms. b Itch^−/− MEFs were transfected with HA-Ub in the presence or absence of Flag-Itch or Flag-C830A. The assay was carried out as described in a. Wild-type (WT) MEFs were used as control to evaluate the basal ubiquitylation of endogenous SuFu. c, d In vitro translated ³⁵S-labelled SuFu WT (c, d) or SuFu K-less (d) was incubated alone or in combination with GST-Itch for the indicated times. The ubiquitylated forms were detected by fluorography. e Schematic representation of SuFu protein showing its lysine residues involved in Itch-dependent ubiquitylation. f, g Flag-SuFu WT or Flag-SuFu mutants were co-transfected in HEK293T cells with HA-Ub in the presence or absence of Myc-Itch. The assay was carried out as described in a. h HEK293T cells were transfected with HA-Ub and Flag-SuFu in the presence or absence of Myc-Itch. Transfected cells were treated with MG132 (50 µM for 4 h) to enrich for ubiquitylated proteins. The assay was carried out as described in a. i HEK293T cells were transfected with Flag-SuFu in the presence or absence of increasing amount of Myc-Itch. Total protein levels were analysed by immunoblotting. j HEK293T cells were transfected in the presence or absence of increasing amount of Myc-Itch. Total protein levels were analysed by immunoblotting. k Immunoblotting analysis of SuFu and Itch proteins in HEK293T cells transfected with control (siCTR) or Itch siRNAs (siItch). β-Actin is shown as a control for loading (*non-specific bands). l SuFu protein levels in WT MEF or Itch^−/− MEF cells treated with cycloheximide (CHX, 100 µg/ml) at different time points. m Purified recombinant proteins wild-type Ub or Ub mutants K48 only (K48O), K48R, K63 only (K63O), K63R, or K-less were incubated with GST-Itch and in vitro translated ³⁵S-labelled SuFu for the indicated times. Ubiquitylated SuFu was detected by fluorography

Fig. 3

Itch-dependent K63-linked ubiquitylation of SuFu leads to Gli3R formation. a Gli3/SuFu proteins interaction by NanoBiT technology. Itch^−/− MEFs were transfected with indicated plasmids. *P < 0.05, Gli3+SuFu WT versus Gli3; **P < 0.05, Gli3+SuFu WT+Itch versus Gli3+SuFu WT; ***P < 0.05, Gli3+SuFuK321/457R versus Gli3+SuFu WT. b, c Association between endogenous Gli3 and Flag-SuFu WT or Flag-SuFuK321/457R assayed by determining the amount of SuFu that co-precipitated with anti-Gli3 antibody or control goat antisera (IgG) from MEFs lysates (b). The ratio of the SuFu signal to the Gli3FL signal from b was plotted (c). *P < 0.05. d–g Gli3/SuFu, interaction, assessed as in (b), in WT MEFs transfected with siItch or siCTR (d) or in Itch^−/− MEFs transfected with the indicated plasmids (f). The ratio of the SuFu signal to the Gli3FL signal from (d) and (f) was plotted (respectively (e) and (g)). *P < 0.05. h WT MEFs were co-transfected with indicated plasmids in the presence or absence of Itch. Cell lysates were immunoprecipitated with anti-Flag agarose beads (1st IP). After two elutions with Flag peptide, cell lysates were re-immunoprecipitated with anti-HA agarose beads (2nd IP), followed by immunoblotting as indicated. i WT MEFs were co-transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting as indicated. j Gli3FL and Gli3R protein levels in SuFu^−/− MEFs before and after expression of SuFu WT or SuFuK321/457R. k Gli3 half-life in SuFu^−/− MEFs after cycloheximide treatment. Gli3FL and Gli3R protein levels were analysed by immunoprecipitation from whole-cell lysates. The graph shows densitometric analysis. *P < 0.05, SuFu WT versus empty vector; **P < 0.05, SuFuK321/457R versus SuFu WT. l Subcellular fractions generated from WT MEFs transfected with Flag-SuFu WT or Flag-K321/457R. Lamin B and Tubulin were used as nuclear and cytoplasmic markers, respectively. m Gli3FL and Gli3R protein levels in WT MEFs transfected with siCTR or siItch. n Gli3 half-life in Itch^−/− MEFs transfected as indicated and then treated with cycloheximide for the indicated times. The graph shows densitometric analysis. *P < 0.05, Itch versus pcDNA; **P < 0.05, C830A versus Itch. o The graphs show the mRNA levels of the indicated Hh target genes in SuFu^−/− MEFs transfected with Gli3 alone or in combination with Flag-SuFu WT or Flag-K321/457R. *P < 0.05, Gli3+SuFu WT versus Gli3; **P < 0.05, Gli3+SuFuK321/457R versus Gli3+SuFu WT. *Non-specific band. Each experiment was performed three times independently. Error bars indicate SD. P-values were determined using Student’s t-test

Fig. 4

Itch-dependent ubiquitylation of SuFu is reverted by Hh pathway activation. a Cerebellum lysates from CD1 mice killed at 2d, 4d, 7d, 10d, 12d, and 15d postpartum (P2, P4, P7, P10, P12, P15) were immunoprecipitated with an anti-SuFu antibody and immunoblotted with an anti-Ub antibody. Gli3 proteins were only detected after enriching its levels by immunoprecipitation with an anti-Gli3 antibody. b The graph shows the mRNA levels of Gli1 gene, as a control of pathway activation, in the cerebella described in a. Error bars indicate SD from three independent experiments. *P < 0.05 (Student’s t-test). c Association between endogenous Gli3 and SuFu from cell lysates of CD1 mice cerebellum (P3, P7, P10, P15) immunoprecipitated with an anti-Gli3 antibody. d Itch-dependent SuFu ubiquitylation is inhibited by activation of the Hh pathway. NIH3T3 cells were transfected with HA-Ub in the presence or absence of Myc-Itch and treated with SAG (200 nM for 6 h). Cell lysates were immunoprecipitated with an anti-SuFu antibody and immunoblotted with an anti-HA antibody

Fig. 5

β-arrestin2 increases the Itch-dependent SuFu ubiquitylation. a WT MEFs were transfected with Flag-Ub in the presence or absence of HA-β-arrestin1 (HA-βArr1) or HA-β-arrestin2 (HA-βArr2). Cell lysates were immunoprecipitated with an anti-SuFu antibody and ubiquitylated forms were revealed with an anti-Flag antibody. b HEK293T cells transfected with the indicated plasmids were immunoprecipitated with an anti-SuFu antibody. Ubiquitylated forms were revealed with an anti-HA antibody. c In vitro translated ³⁵S-labelled SuFu was incubated alone or in combination with untagged recombinant Itch protein in the presence or absence of recombinant β-arrestin2 protein for the indicated times. Levels of ubiquitylated ³⁵S-labelled SuFu were detected by fluorography. d SuFu ubiquitylation in WT MEFs transfected with HA-Ub in the presence or absence of Myc-Itch and with specific siRNA for β-arrestin2 (siβArr2) or non-specific control (siCTR). Immunoprecipitation and immunoblotting were performed as in (b). e HEK293T cells were co-transfected with the indicated plasmids. Interaction of β-arrestin2 with SuFu and Itch was detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. f SuFu, Itch, and β-arrestin2 form a trimeric complex. WT MEFs were transfected with different combinations of Flag-Itch, GFP-β-arrestin2, and HA-SuFu constructs. Protein lysates were immunoprecipitated with anti-Flag agarose beads. One-third of immunocomplexes was probed with antibodies to the indicated proteins (1st IP), whereas two-thirds were subjected to two elutions with Flag peptide and re-immunoprecipitated with HA-agarose beads followed by immunoblotting as indicated (2nd IP). g β-Arrestin2^−/− MEF cells were transfected with HA-β-arrestin2 plasmid. Interaction of Itch with SuFu and β-arrestin2 was detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. h Association of endogenous Itch with SuFu and β-arrestin2 from cell lysates of CD1 mice cerebellum (P3, P7, P10, P15) immunoprecipitated with an anti-Itch antibody. i, j NIH3T3 cells transfected with the indicated plasmids were treated with SAG (200 nM for 6 h) or vehicle only. Interaction between Flag-Itch and HA-SuFu (i) or HA-β-arrestin2 (j) was detected by anti-Flag immunoprecipitation, followed by immunoblot analysis with the indicated antibodies. k Association of endogenous Itch with SuFu and β-arrestin2 from cell lysates of CD1 mice cerebellum (P10) and from MB Ptch^+/− tissue both immunoprecipitated with an anti-Itch antibody

Fig. 6

Human Daoy MB xenografts. a PET/CT images of representative CTR (n = 6), SuFu WT (n = 6), and SuFu K321/457R (n = 8) mice showing tumour FDG (F-18-fluorodeoxyglucose) uptake at 41 days after implantation. ROI (region of interest) drafts the tumour mass. Significant differences were observed between SuFu WT and SuFu K321/457R mutant mice. U urinary bladder, M femoral muscle. b Graphic representation of SUV (standard uptake value). For each tumour, the SUV as mean tumour FDG uptake normalised for animal body weight was calculated. Significant difference in tumour FDG uptake was observed between SuFu K321/457R and SuFu WT mice. *P < 0.05, SuFu WT versus CTR; **P < 0.05, SuFu K321/457R versus SuFu WT. c Images of xenografted NOD/SCID mice with bilateral MB xenograft tumours. Tumour volume in SuFu K321/457R mutant mice is visually bigger than SuFu WT (scale bars = 5 mm). d Tumour volumes were monitored over time by caliper measurements at the indicated times. *P < 0.01, SuFu WT versus CTR; **P < 0.01, SuFu K321/457R versus SuFu WT. e Tumour volumes were measured post explantation. *P < 0.01, SuFu WT versus CTR; **P < 0.01, SuFu K321/457R versus SuFu WT. f Immunohistochemistry of Ki67 and Gli1 stainings. Scale bars indicate 50 µm. g Quantification of Ki67 and Gli1 stainings from immunohistochemistry. *P < 0.01, SuFu WT versus CTR and **P < 0.01, SuFu K321/457R versus SuFu WT. h Western blot analysis shows protein expression levels. Error bars indicate SD. P-values were determined using Mann–Whitney U-test

Fig. 7

Human Daoy orthotopic MB xenografts. a Representative images of haematoxylin and eosin (H&E), Ki67, and Gli1 immunohistochemical stainings of a human Daoy MB cell-derived orthotopic tumour in NOD/SCID mice (n = 6 mice for each group, CTR, SuFu WT, and SuFu K321/457R). Scale bars represent 500 µm for H&E staining and 50 µm for Ki67 and Gli1 stainings. b Representative tumour average volumes after explantation. c, d Quantification of Ki67 (c) and Gli1 (d) stainings from immunohistochemistry shown in (a). *P < 0.01, SuFu WT versus CTR; **P < 0.01, SuFu K321/457R versus SuFu WT. Error bars indicate SD. P-values were determined using Mann–Whitney U-test

Fig. 8

Mouse Ptch1^+/− MB allografts. a Images of representative CTR, SuFu WT, and SuFu K321/457R flank allograft masses (n = 6 mice for each group) (scale bars = 5 mm). b Tumour volumes were monitored over time by caliper measurements at the indicated times. *P < 0.05, SuFu WT versus CTR; **P < 0.05, SuFu K321/457R mutant versus SuFu WT. c Tumour volumes were measured post explantation. *P < 0.05, SuFu WT versus CTR; **P < 0.05, SuFu K321/457R mutant versus SuFu WT. d Immunohistochemistry of Ki67 and Gli1 stainings. Scale bars indicate 50 µm. e Quantification of Ki67 and Gli1 stainings from immunohistochemistry. *P < 0.05, SuFu WT versus CTR and **P < 0.05, SuFu K321/457R mutant versus SuFu WT, for Ki67 staining quantification. *P < 0.05, SuFu WT versus CTR and **P < 0.01, SuFu K321/457R versus SuFu WT, for Gli1 staining quantification. Error bars indicate SD. P-values were determined using Mann–Whitney U-test

Fig. 9

Model of the Itch/β-arrestin2-dependent regulation of the SuFu/Gli3 complex function. When Hh pathway is OFF, Itch, coadjuvated by the protein adaptor β-arrestin2, ubiquitylates SuFu. This event does not lead to SuFu degradation, but increases the association between SuFu and Gli3. In this way Gli3 is protected from SPOP-dependent degradation and is cleaved into a repressor form (Gli3R) that inhibits Hh target gene transcription and cell growth. When Hh pathway is switched ON, β-arrestin2 dissociates from the SuFu/Itch complex, thus abrogating Itch-dependent SuFu ubiquitylation. This process induces the dissociation of the Gli3-SuFu complex and impairs Gli3R formation, thereby leading to Hh pathway activation and sustained cell growth. Alterations in this mechanism, caused by SuFu mutations that make it insensitive to Itch-dependent ubiquitylation, are responsible for MB tumorigenesis