Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system

<div><p>Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (Gli^A) in wild-type but not in <i>Sufu</i> mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.</p></div

Karyopherinβ2 (Kapβ2) regulates Gli ciliary localization and activation.

<p>Kapβ2 binds the PY-type nuclear localization signal (PY-NLS) in Gli to promote its ciliary localization. In the absence of Hedgehog (Hh), Patched (Ptc) inhibits Smoothened (Smo), allowing Gli to be processed into Gli^R that translocates to the nucleus to inhibit Hh target gene expression. Hh binding to Ptc leads to ciliary accumulation of the activated form of Smo, which converts ciliary-localized Gli into Gli activator (Gli^A) that translocates to the nucleus to activate the expression of Hh target genes, including Kapβ2. Kapβ2 up-regulation may further promote ciliary localization and activation of Gli.</p

The C-terminal region of Gli2 is required for its ciliary localization.

<p>(<b>A</b>) Diagram of wild-type mGli2 and Gli2/Ci chimeric proteins. (<b>B–C</b>) Ciliary localization (<b>B</b>) and its quantification (<b>C</b>) of the indicated Gli2/Ci constructs. Data are means ± SD from 2 independent experiments. **<i>P</i> < 0.01, NS: not significant. (<b>D</b>) Diagram of the Gli2/Ci chimeric protein and its C-terminal deletion constructs. (<b>E–F</b>) Ciliary localization (<b>E</b>) and its quantification (<b>F</b>) of Gli2/Ci and its deletion mutants. Data are means ± SD from 2 independent experiments. **<i>P</i> < 0.01, NS: not significant. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002063#pbio.2002063.s005" target="_blank">S1 Data</a>.</p

Karyopherinβ2 (Kapβ2) is required for Gli ciliary location and Hedgehog (Hh) pathway activation.

<p>(<b>A–B</b>) Ciliary localization of endogenous Gli2 in NIH3T3 cells infected with lentivirus containing short hairpin RNA (shRNA) targeting either green fluorescent protein (GFP) (Ctr) or mouse karyopherinβ2 (mKapβ2) (with or without reintroducing human karyopherinβ2 [hKapβ2] by a second round of lentiviral infection) and treated with or without sonic hedgehog (Shh). Quantitation of Gli2 ciliary localization is shown in (<b>B</b>). One hundred cells were examined for each condition. Data are means ± SD from 2 independent experiments. ***<i>P</i> < 0.001. (<b>C–E</b>) <i>Gli-luc</i> reporter assay (<b>C</b>), <i>Gli1</i> (<b>D</b>), or <i>Ptch1</i> (<b>E</b>) expression in NIH3T3 cells infected with lentivirus expressing GFP (Ctr) shRNA or mKapβ2 shRNA with or without hKapβ2 or mGli2 coexpression. Cells were treated with or without sonic hedgehog N-terminal fragment (Shh-N) (<b>C</b>) or SAG (<b>D–E</b>) as indicated. (<b>F</b>) <i>Gli-luc</i> reporter assay in the indicated cell lines treated with or without Shh. Data are means ± SD from 2 independent experiments. **<i>P</i> < 0.01, ***<i>P</i> < 0.001, NS: not significant. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002063#pbio.2002063.s005" target="_blank">S1 Data</a>.</p

Karyopherinβ2 (Kapβ2) regulates Hedgehog (Hh) signaling in zebrafish embryos.

<p>(<b>A–C′</b>) Zebrafish embryos injected with the indicated morpholinos (MOs) were immunostained at 24 hours post-fixation (hpf) with Engrailed (Eng) antibody (red) and DAPI (blue) to visualize nuclei. (<b>D–F</b>) Zebrafish embryos injected with the indicated MOs were immunostained at 24 hpf with F59 antibody (red) to visualize slow muscle fibers. (<b>G–I</b>) Zebrafish embryos injected with the indicated MOs were analyzed for Ptch2 expression at 10 hpf by in situ hybridization. (<b>J</b>) Relative mRNA levels of the indicated genes from 24 hpf zebrafish embryos injected with the indicated MOs were measured by quantitative reverse transcription PCR (RT-qPCR). Data are means ± SD from 3 independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.001, NS: not significant. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002063#pbio.2002063.s005" target="_blank">S1 Data</a>.</p

The PY-type nuclear localization signal (PY-NLS) is required for Gli activation.

<p>(<b>A</b>) Western blot of cell lysates from the indicated cell lines indicated that Myc-Gli2^WT and Myc-Gli2^mPY were expressed at comparable levels that were slightly higher than that of endogenous Gli2. (<b>B–C</b>) Normalized mRNA levels of endogenous <i>Gli1</i> (<b>B</b>) or <i>Patch1</i> (<b>C</b>) measured by quantitative reverse transcription PCR (RT-qPCR) in control (green fluorescent protein [GFP] short hairpin RNA [shRNA]) or Gli2-depeleted NIH3T3 cells with or without lentiviral infection of the indicated Gli2 constructs. (<b>D</b>) Fractionation of Myc-Gli2^WT and Myc-Gli2^mPY from the indicated cell lines treated with or without SAG. Quantification of protein level is shown in the bottom panel. (<b>E–F</b>) Immunostaining (<b>E</b>) and quantification (<b>F</b>) of ciliary-localized Myc-Gli2^WT or Myc-Gli2^mPY in NIH3T3^mGli2-shRNA cells treated with or without sonic hedgehog (Shh). <i>N</i> = 50 cells were examined for each Gli construct. Data are means ± SD from 2 independent experiments. <i>**P < 0</i>.<i>01</i>. (<b>G</b>) <i>Gli-luciferase</i> assay was performed in NIH3T3 cells transfected with the indicated constructs. Data are means ± SD from 2 independent experiments. *<i>P</i> < 0.05, NS: not significant. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002063#pbio.2002063.s005" target="_blank">S1 Data</a>.</p

<i>gish</i> is required for high levels of Hh pathway activity and genetically interacts with <i>gprk2</i>.

<p>(A–J) Adult wings of the indicated genotypes. <i>MS>Smo</i>^<i>DN</i> caused a fusion between veins 3 and 4 (A). <i>MS>Smo</i>^<i>DN</i> wing carrying <i>gish</i>^<i>P</i> clones exhibited an enhanced phenotype (C). Heterozygosity for <i>gish</i>^<i>Df</i> (D) or <i>gish</i>^<i>Δ4</i> (E) but not <i>gish</i>^<i>P</i> (B) enhanced the fused wing phenotype caused by <i>MS>Smo</i>^<i>DN</i>. Overexpression of wild-type Gish (F) but not Gish^KD (G) or Gish^<i>Δ</i>C (H) rescued the fused wing phenotype caused by <i>MS>Smo</i>^<i>DN</i> in <i>gish</i> heterozygous background. Heterozygosity for <i>gprk2</i>^<i>Δ15</i> also enhanced the <i>MS>Smo</i>^<i>DN</i> phenotype (I). <i>gprk2</i>^<i>Δ15</i> and <i>gish</i>^<i>Δ4</i> double heterozygosity enhanced the <i>MS>Smo</i>^<i>DN</i> phenotype more dramatically than either <i>gprk2</i>^<i>Δ15</i> or <i>gish</i>^<i>Δ4</i> single heterozygosity (J compared with E and H). (K–N‴) Late third instar wild type wing discs (K, K', M, M') or wing discs carrying <i>gish</i>^<i>Δ4</i> mutant clones induced at 24–48 h after egg laying (AEL) (L–L‴, N–N'') were immunostained to show the expression of Ci (red), Gish (green), En (blue in K', L", L‴), or Smo (blue in M', N", N‴). <i>gish</i>^<i>Δ4</i> mutant cells abutting the A/P boundary (demarcated by dashed lines) exhibited reduced En and Smo staining (arrows in L", N").</p

Gish forms a complex with Smo regulated by Hh and PKA.

<p>(A–G) Western blots of coimmunoprecipitation experiments from lysates of S2 cells transfected with the indicated constructs. Cells were grown in the presence or absence of Hh stimulation for 24 h. In A–D and G, cells were exposed to MG132 (50 μM) for 4 h before harvest. Myc-Smo coimmunoprecipitated with HA-Gish but not HA-Gish^CS or HA-Gish^CS, and Smo/Gish association was enhanced by Hh stimulation (C). Preventing Smo cell surface accumulation by artificially conjugating an Ub moiety to the C-terminus of Smo (Myc-Smo-Ub) diminished its interaction with Gish (D). HA-Gish interacted with Myc-Smo, Myc-SmoΔ650, and Myc-SmoC but not with Myc- SmoΔ570 (E). Arrow indicates IgG and asterisks indicate monomeric forms of SmoΔ570 and SmoΔ650 (E). Hh stimulation increased the association between HA-Gish and Myc-SmoΔ650 (F). The SA mutation reduced while the SD mutation enhanced Smo/Gish association (G).</p

Gish positively regulates Hh signaling.

<p>(A–H) Control wing (A) or wings expressing a dominant negative Smo using <i>MS1096</i> (<i>MS>Smo</i>^<i>DN</i>) either alone (B) or together with <i>UAS-Gish-RNAi</i> (C), <i>UAS-Myc-Gish</i> (D), <i>UAS-Myc-Gish</i>^<i>KD</i> (E), <i>UAS-Gish</i>^<i>ΔC</i> (F), <i>UAS-Flag-CK1α</i> (G), or <i>UAS-Flag-CK1ε</i> (H). <i>MS>Smo</i>^<i>DN</i> caused a fusion between veins 3 and 4 (arrow in B), which was enhanced by Gish RNAi (C) but partially suppressed by Gish overexpression (arrow in D). Overexpression of Gish^KD, Gish^<i>Δ</i>C, CK1<i>α</i>, or CK1ε failed to suppress the “fused wing” phenotype caused by <i>MS>Smo</i>^<i>DN</i>. (A'-D') Late third instar wing discs of the indicated genotypes immunostained with an anti-Ptc antibody. <i>MS>Smo</i>^<i>DN</i> diminished <i>ptc</i> expression near the A/P boundary (arrow in B'). Gish RNAi further reduced while Gish overexpression restored <i>ptc</i> expression (arrows in C', D'). In this and the following figures, images are representatives of five or more adult wings or wing discs for each genotype.</p

Hh promotes CL-II phosphorylation through Gish.

<p>(A) Diagram of Smo with PKA, CK1, and Gprk2 sites indicated by different colors. Black and gray boxes denote the seven transmembrane domains and the Smo auto-inhibited domain (SAID), respectively. (B) In vitro kinase assay using recombinant CK1 and GST-Smo fusion proteins carrying wild-type (GST-Smo_601-700) or mutated CL-II site (GST-Smo_601-700CL-IISA). (C, D) Western blots of lysates from S2 cells transfected with the indicated constructs, treated with or without the indicated dsRNA and Hh-conditioned medium. (E, F, L) Western blots of immunoprecipitation experiments from lysates of S2 cells transfected with the indicated constructs. Cells were grown in the presence or absence of Hh stimulation for 24 h and exposed to MG132 (50 μM) for 4 h before harvest. (G, H) S2 cells transiently (G) or stably (H) expressing Myc-Smo were treated with control RNAi, Gish RNAi, or D4476. Cells were grown in the presence or absence of Hh stimulation for 24 h and exposed to MG132 (50 μM) for 4 h, followed by immunoprecipitation and western blot analysis.</p