{'fraction_non_alphanumeric': 0.04228533188339119, 'fraction_numerical': 0.02785561922288678, 'mean_word_length': 4.603468479025076, 'pattern_counts': {'":': 0, '<': 7, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling

Hedgehog signaling involves the dynamic movement of receptors and effectors in and out of cilia. Desai et al. found that the dynamics of Smo are regulated by ubiquitination, which modulates its interaction with the intraflagellar transport system to control ciliary levels of this receptor.


Introduction
Primary cilia play critical roles in development by monitoring the extracellular environment and transmitting this information to the cell body and nucleus, allowing the cell to coordinate its physiology with surrounding cells. In mammals and other vertebrates, ciliary dysfunction leads to a variety of structural birth defects in the brain, heart, lungs, kidneys, and other organs, along with craniofacial and other skeletal abnormalities. While numerous receptors are localized in the cilia, Hedgehog signaling is the best studied ciliary pathway. This pathway plays fundamental roles during development, and many of the developmental defects caused by ciliary dysfunction can be attributed to abnormal Hedgehog signaling. All the key components of the Hedgehog pathway are enriched in the cilium (Corbit et al., 2005;Haycraft et al., 2005;Ocbina and Anderson, 2008;Rohatgi et al., 2007), and their localization changes dynamically in response to the activity of the pathway. In the off state, patched-1 (Ptch1), the Hedgehog ligand receptor, accumulates in cilia and prevents ciliary accumulation and activation of smoothened (Smo). Upon binding of the ligand, Ptch1 is removed from the cilium, and Smo is derepressed and now accumulates in the cilium. Smo subsequently activates downstream signaling, which results in the accumulation of the Gli transcription factors at the ciliary tip before their modification and translocation to the nucleus, where they modulate expression of target genes.
The mechanisms underlying the trafficking of Hedgehog components to and within the cilium are not well understood. Part of the movement is facilitated by intraflagellar transport (IFT; Eguether et al., 2018;Eguether et al., 2014;Keady et al., 2012), and perturbing IFT disrupts Hedgehog signaling (Huangfu et al., 2003;Liem et al., 2012). IFT, which is critical for the assembly and maintenance of cilia, involves motor-driven transport of large complexes called IFT particles along the cilia. These particles are composed of at least 30 proteins organized in IFT-A, IFT-B, and BBSome subcomplexes. The IFT particles serve as motor adaptors connecting various proteins that need to be moved into or out of cilia to kinesin and dynein motors. We previously showed that Ift25 and Ift27, which are subunits of IFT-B, are not required for ciliary assembly. Instead, these two IFTs work with the adaptor protein Lztfl1 and the BBSome to regulate Hedgehog signaling by facilitating the removal of Smo from cilia at the basal state and Ptch1 after pathway activation (Eguether et al., 2018;Eguether et al., 2014;Keady et al., 2012).
In this work, we explore the mechanism underlying the dynamics of Smo localization to cilia and find that the removal of Smo from cilia is regulated by ubiquitination. Ubiquitination involves the covalent attachment of the 76-amino acid peptide ubiquitin (Ub) to cellular proteins. Ub is usually added to lysine residues, and it can be further ubiquitinated on one of its seven lysine residues to produce polyubiquitinated proteins. The type of Ub modification on a protein determines its cellular fate. For instance, K48 polyubiquitination drives degradation by the proteasome, K11 polyubiquitination drives degradation during specific points in the cell cycle, and K63 polyubiquitination often regulates complex formation and signaling (Swatek and Komander, 2016). The addition of Ub results from a cascade of activity whereby an E1 enzyme activates Ub and passes it to an E2 enzyme, which then either passes the Ub to an E3 enzyme for attachment to the protein of interest or works with an E3 to modify that protein. The process is reversible by the action of deubiquitinating enzymes (Swatek and Komander, 2016). Prior work identified components of the Ub system in cilia (Huang et al., 2009;Pazour et al., 2005;Raman et al., 2015), where it has been implicated in disassembly (Huang et al., 2009;Wang et al., 2019). The Ub system is also known to regulate a number of steps in Hedgehog signaling . In this work, we found that ubiquitination of Smo facilitates its interaction with the IFT system for efficient ciliary removal/export when the Hedgehog pathway is suppressed.

Results
Ubiquitinated Smo is retained in Ift27 −/− , Lztfl1 −/− , and Bbs2 −/− cilia but not in wild-type cilia Our previous work indicated that Ift25 and Ift27 are required for the regulated removal of Smo from cilia (Eguether et al., 2014;Keady et al., 2012). This suggests that Hedgehog signaling regulates the interaction between Smo and the IFT particle, but the underlying mechanism is unknown. Structurally, Smo is a member of the G protein-coupled receptor (GPCR) seven transmembrane protein family. Many GPCRs cycle between the cell surface and the endomembrane system depending upon ligand binding. The dynamics are often driven by β-arrestin binding to activated receptor, causing the receptor to associate with clathrin for internalization (Tian et al., 2014). Alternatively, the receptor may be ubiquitinated and internalized via the endosomal sorting complexes required for transport (ESCRT) machinery (Skieterska et al., 2017). While a published report indicates that Smo delivery to cilia required β-arrestin (Kovacs et al., 2008), we were not able to observe any Smo trafficking defects in mouse embryonic fibroblasts (MEFs) lacking β-arrestin 1 and 2 (Barr1/2; Fig. 1, A and B).
To explore the role of Ub in regulating the ciliary localization of Smo, we fused a single Ub open reading frame to the intracellular C-terminal end of Smo. This approach has been used to study the role of Ub in trafficking of GPCRs (Shih et al., 2000;Terrell et al., 1998). Cells expressing Smo-Flag from a cytomegalovirus (CMV) promoter show ∼25% Flag-positive cilia without pathway activation, and the numbers are elevated to ∼90% positive cilia upon pathway activation by Smoothened Agonist (SAG). The addition of Ub to the C-terminus greatly reduces the amount of ciliary Smo under basal conditions and notably also blocks the accumulation usually associated with pathway activation by addition of SAG (Fig. 1,C and D) or Sonic Hedgehog (SHH)-conditioned medium (Fig. S1, A and B). As we and others have previously observed for endogenous Smo (Eguether et al., 2014;Seo et al., 2011), Smo-Flag is retained in Ift27 −/− , Lztfl1 −/− , and Bbs2 −/− MEF cilia at both the basal state and after SAG induction. In contrast to the depletion of Smo-Flag-Ub from cilia that we saw in control cells, Smo-Flag-Ub is highly enriched in Ift27 −/− , Lztfl1 −/− , and Bbs2 −/− cilia (Fig. 1,C and D;and Fig. S2). Importantly, the expression of Smo-Flag-Ub does not interfere with the trafficking of endogenous Smo (Fig. S1 C). These data suggest that the IFT/Bardet-Biedl syndrome (BBS) system targets ubiquitinated Smo for ciliary removal.
To confirm our finding that Smo-Ub was retained in Ift27 −/− mutant cilia but not in control cilia, we examined IMCD3 control and IMCD3 Ift27−/− cells. In these kidney epithelial cells, Smo-Flag is retained in wild-type cilia without pathway activation (Fig. 2,  A and B). Confirming the MEF studies, Smo-Flag-Ub does not accumulate in control cilia but does accumulate in IMCD3 Ift27−/− cilia (Fig. 2, A and B).
To determine how other GPCRs behave, we tagged the somatostatin receptor Sstr3 with Flag and Ub. Sstr3-Flag is highly enriched in both control and IMCD3 Ift27−/− cilia, while Sstr3-Flag-Ub is not highly enriched in cilia of either cell line (Fig. 2,C and D). Similar results were obtained for the Ddr1 dopamine receptor (Fig. 2, E and F), suggesting that an Ift27-independent mechanism exists for the removal of some ubiquitinated cargos from cilia.
SmoM2 is an oncogenic form of Smo that contains a Trp to Leu mutation (MmSmo W539L ) at the cytoplasmic face of the last transmembrane domain (Xie et al., 1998). This mutation constitutively activates Hedgehog signaling, and SmoM2 accumulates in cilia regardless of whether Hedgehog ligand is present or not. Tagging SmoM2 with Ub removed it from wild-type cilia as expected, but unexpectedly SmoM2-Ub was observed to be barely above background in Ift27 −/− , Lztfl1 −/− , and Bbs2 −/− MEF cilia (Fig. 3, A and B). Similar results were obtained in Ift27 −/− IMCD3 cells (Fig. 3, F and G). One possibility for the failure of SmoM2-Ub to accumulate in Ift27 −/− , Lztfl1 −/− , and Bbs2 −/− mutant cells is that perhaps SmoM2-Ub is highly unstable and degraded before it is able to traffic to cilia. To compare the relative stability of Smo-Ub and SmoM2-Ub, we treated cells expressing these proteins with MG132 for 4 h to build up levels of Smo-Ub or SmoM2-Ub, then added cycloheximide to prevent new synthesis, and removed the MG132 to allow proteasomal degradation to proceed. MG132 treatment caused significant buildup of Smo-Ub and SmoM2-Ub in cells, which decayed with similar rates, suggesting that it is not the stability of SmoM2-Ub that prevents its accumulation in Ift27 −/− cilia (Fig. S3 A). The large buildup of SmoM2-Ub caused by MG132 treatment was not sufficient to cause SmoM2-Ub to accumulate in Ift27 −/− cilia (Fig. 3 C). Recently, ectocytosis was proposed as an alternative pathway for clearing some GPCRs from cilia (Nager et al., 2017). This pathway is activated in Bbs mutants, suggesting that perhaps ectocytosis can remove SmoM2-Ub from cilia on Ift27 −/− , Lztfl1 −/− , and Bbs2 −/− mutant cells. However, ectocytosis does not appear to be the explanation, as SmoM2-Ub did not accumulate when ectocytosis was blocked with cytochalasin D (Fig. 3 C). β-arrestin-driven retrieval is another possible mechanism for removal of SmoM2-Ub, but this does not appear to be the mechanism as loss of Barr1/2 did not cause accumulation of SmoM2-Ub in cilia (Fig. 3 C).
The observation that SmoM2 is retained in cilia in spite of the repressive activity of Ptch1 suggests that SmoM2 is in a conformation that is not a substrate for ubiquitination and removal was quantitated from the cells described in C. N is 3 replicates with at least 100 cells counted per condition. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. For the mutants, significance is shown with respect to same condition in wild-type by three-way ANOVA. Error bars indicate SD.

Blocking ubiquitination causes Smo to accumulate in cilia
The ligation of Ub to a substrate requires a cascade starting with an E1 Ub-activating enzyme, followed by an E2 Ub-conjugating enzyme, and lastly an E3 Ub ligase. Pyr41 inhibits E1 enzymes (Yang et al., 2007), and we reasoned that if ubiquitination of Smo was required for its removal from cilia, Pyr41 should cause Smo to accumulate in cilia. While blocking an E1 activating enzyme is expected to affect many processes, we found that cells treated with 3.5 µM Pyr41 appeared healthy, remained ciliated, and accumulated Smo in cilia. The Smo accumulation in cilia was detectable within 6 h of treatment and at 24 h was similar to the accumulation driven by SAG treatment (Fig. 4, A and C). Pyr41 did not appear to affect general IFT, as Ift27 staining was similar in Pyr41-treated cells and controls ( Fig. 4 B).  (Fig. 5, C and D). However, drug selection did not work well for this construct, so the percentage of transfection was variable. To circumvent the problem of variable transfection, we ratioed the percentage of Smo-Flag positive without SAG treatment to the percentage green; arrows), and nuclei (DAPI; blue). Left image of each pair is a three-color composite; right image shows only the red Flag channel. Scale bar is 5 μm and applies to all images in the panel. (B) Presence of SmoM2 or SmoM2-Ub in cilia was quantitated from the cells described in A. N is 3 replicates with at least 100 cells counted per condition. ****, P < 0.0001; ns, not significant. Mutants were not significantly different from the same condition in wild-type by two-way ANOVA. Error bars indicate SD. (C) Wild-type, Ift27, and Barr1/2 mutant SmoM2-Ub cells treated as in B except that 1 µM MG132 for 4 h or 500 nM cytochalasin D (CytoD) was added for 24 h before fixation. **, P < 0.01; *, P < 0.05; ns, not significant. No differences in CytoD treatment were significant by two-way ANOVA. Error bars indicate SD. positive after SAG treatment (Fig. 5 D). Confirming the results with the CMV promoter (Fig. 5, A and B), Smo noK expressed from the GgCryD1 promoter (GP805) accumulated in cilia without pathway activation (Fig. 5, C and D). Further analysis showed that the lysines in the C-terminal tail and loop two are not needed for removal of Smo-Flag from cilia, but at least one of the three lysines in loop three (GP803) is critical. Further mutation of loop three indicated that when both Lys444 and Lys448 are mutated to arginine (GP826), Smo accumulates in cilia without pathway activation. Mutation of either of these lysines on its own was not enough to retain Smo (Fig. 5 D), suggesting that either residue can serve the regulatory role. This region of loop three is highly conserved in vertebrates, with no substitutions in any of the main model organisms. In Drosophila, the region is not conserved except that it contains three lysines (Fig. 5 F).   (Fig. 6 A). Probing for HA showed a smear up the gel as expected for ubiquitinated proteins and showed that significantly more HA-Ub precipitated with Smo from control cells compared with cells treated with SHH-conditioned medium (Fig. 6, A and B). No HA-Ub was detected after immunoprecipitation of Smo noK , indicating that the HA signal was due to ligation of Ub onto Smo (Fig. 6, A and C). Interestingly, we did not detect SmoM2 ubiquitination at either basal or induced state (Fig. 6, A and D). This suggests that the ubiquitination of SmoM2 is blocked by the conformation change that accompanies its activation.  (Fig. 7). The removal of ubiquitinated GPCRs from cilia appears to be a general phenomenon as the addition of Ub to Sstr3 prevented it from accumulating in cilia. However, in contrast to Smo-Ub, the removal of Sstr3-Ub was not dependent on Ift27. Unexpectedly, oncogenic SmoM2 behaved like Sstr3 rather than nononcogenic Smo, as SmoM2-Ub did not accumulate in cilia lacking Ift27. This suggests that an alternative mechanism exists to remove certain GPCRs from cilia. The removal was not blocked by loss of β-arrestin or by inhibiting ectocytosis, making it likely to be an IFT-dependent process. If it is IFT dependent, then subunits of either IFT-A or IFT-B (besides Ift25 and Ift27) must be able to bind ubiquitinated GPCRs to remove them from cilia.    (Seo et al., 2011). This region contains the amphipathic helix that is shifted and partially unwound by the activation of Smo (Huang et al., 2018). It is possible that the SmoM2 and SmoPi mutations change the structure of this helix and the tail so it can be bound by other components of the IFT system for removal from cilia. The role of Ub in ciliary biology and Hedgehog signaling has not been extensively studied, although several studies indicate that Ub is critical to both cilia and Hedgehog signaling. In Chlamydomonas reinhardtii, Ub and isoforms of E1, E2, and E3 ligases are present in the cilium (Huang et al., 2009;Long et al., 2016;Pazour et al., 2005). Ciliary proteins become ubiquitinated while cilia are disassembling, and ubiquitinated cargos accumulate in cilia when IFT is disrupted (Huang et al., 2009). In mammalian cells, valosin-containing protein (VCP), which uses ATP to segregate Ub from binding partners, is complexed with IFT-B though Ubxn10. Both VCP and Ubxn10 are required for ciliogenesis (Raman et al., 2015). In Trypanosoma brucei, Ub copurifies with the BBSome, and BBSome mutations affect cellular localization of ubiquitinated receptors (Langousis et al., 2016). In Caenorhabditis elegans, attachment of Ub to the ciliary membrane protein Pkd2 reduced its level in cilia, and similar to our findings, the BBSome appeared to be required (Hu et al., 2007;Xu et al., 2015).

Discussion
Ub has been implicated in a number of steps of the Hedgehog pathway, ranging from the production of Hedgehog ligand to regulating the proteolytic conversion of Gli3 full length to Gli3 repressor . Less is known about the role of Ub in regulating the dynamics of Ptch1 and Smo, although previous studies support a function for Ub in this process. In Drosophila, Smo is ubiquitinated in the off state, and this drives sequestration in cytoplasmic vesicles (Ma et al., 2016;Xia et al., 2012;Zhou et al., 2018). The E3 ligase modifying Drosophila Smo is reported to be Herc4 (Jiang et al., 2019;Sun et al., 2019), and other studies suggest that the Usp8 and Uchl5 deubiquitinases regulate cellular localization of Smo. Herc4 was identified as a genetic modifier of Hedgehog signaling, and its loss stabilized Smo protein (Jiang et al., 2019;Sun et al., 2019). Usp8 was identified in an RNAi screen as a Smo deubiquitinase, and its loss increased Smo ubiquitination (Xia et al., 2012). Uchl5 stabilizes Drosophila Smo and promotes its localization on the cell surface. The human homologue UCH37 was reported to promote Smo localization to cilia (Zhou et al., 2018). However, we used CRISPR to knock out Herc4 along with its close paralog Herc3, Usp8, and Uchl5/  (1) is ubiquitinated by unknown E3 ligase (2) on lysine residues in intracellular loop three, making it cargo for removal from the cilium by the IFT system (3). Activation of the pathway by SHH binding to Ptch1 would suppress the activity of the E3 ligase or, if the E3 is ciliary localized, remove it from the cilium. This would allow Smo that enters the cilium to remain, become activated, and trigger downstream signaling events. A simple mechanism for regulating the ciliary localization of an E3 ligase could involve binding the ligase to Ptch1, which is known to bind E3 ligases. By attaching the ligase to Ptch1, the ligase would be ciliary localized at the basal state and be removed upon pathway activation. While we have no data on the role of Ub in regulating Ptch1, a similar mechanism whereby Ptch1 is ubiquitinated by pathway activation could serve to keep ciliary Ptch1 levels low in the activated state. Ptch1 is also likely to be regulated by ubiquitination, as the E3 ligases Smurf1 and Smurf2 ubiquitinate Ptch1 and promote its degradation (Yue et al., 2014). While this work focused on the degradation of Ptch1 by ubiquitination, it showed that knockdown of Smurf1 and Smurf2 increased the intensity of Ptch1-GFP in mammalian cilia (Yue et al., 2014). Another relevant study found two PY motifs, which are binding sites for HECT family ubiquitinases, in Ptch1 and showed that mutating these binding sites caused Ptch1 to remain in cilia after pathway activation (Kim et al., 2015). Proteomic analysis indicates that numerous E3 ligases bind Ptch1 (Yamaki et al., 2016).
Two recent large-scale CRISPR screens of genes involved in Hedgehog signaling identified a number of Ub-related enzymes regulating Hedgehog signaling (Breslow et al., 2018;Pusapati et al., 2018). Genes identified included E1, E2, and E3 ligases along with deubiquitinases and adaptor proteins. Pusapati et al. (2018)  In summary, our work uncovers a mechanism for regulating the dynamic distribution of Smo during Hedgehog signaling (Fig. 7). In this model, Hedgehog signaling regulates the activity or subcellular distribution of E3 ligases (and  (Seo et al., 2011). Upon activation of Hedgehog signaling, the E3 ligase is inhibited and/or removed from the cilium, or alternatively, the activation of Smo may change its confirmation such that it is no longer a substrate for ubiquitination. Thus, Smo that enters the cilium will not be ubiquitinated and will not interact with the IFT/BBS complex; instead, it will remain in the cilium. The simplest mechanism for regulating the ciliary localization of an E3 ligase is to bind it to Ptch1, which is known to be removed from cilia upon pathway activation. As discussed previously, Ptch1 contains two PY motifs and is known to bind HECT family E3 ligases, making this a plausible mechanism. In addition to regulating Smo distribution, Ub could more generally regulate the ciliary levels of other receptors, the most likely being Ptch1. Perhaps the binding of ligand to Ptch1 activates an E3 ligase that promotes Ptch1 removal from cilia.

Plasmids
Plasmids were assembled by Gibson assembly (NEB) into the pHAGE lentiviral backbone (Wilson et al., 2008). All inserts are derived from mouse unless otherwise stated. Mutations were generated by PCR amplification with mutated primers and the products Gibson assembled. For mutation of the 16 lysines in the C-terminal tail of Smo, the tail sequence was chemically synthesized (gBlock; IDT) and PCR amplified for Gibson assembly. All inserts were fully sequenced and match the National Center for Biotechnology Information reference sequence or expected mutant forms. Plasmids are described in Table 1 and Fig. S4. SnapGene files will be provided upon request.
For SAG experiments, MEFs were plated at near-confluent densities and serum starved (same culture medium as described above but with 0.25% fetal bovine serum) for 48 h before treatment to allow ciliation. SAG (Calbiochem) was used at 400 nM.
Sonic Hedgehog (SHH)-conditioned medium was generated from HEK cells transfected with a human (Hs) SHH expression construct (MS03). Cells stably expressing MS03 were grown to confluency in 90% DMEM (4.5 g/liter glucose), 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin; medium was then replaced with low serum medium (0.25% fetal bovine serum) and grown for 48 h. Medium was collected, and the filter was sterilized and titered for ability to relocate Smo to cilia. Dilutions similar in effect to 400 nM SAG were used for experiments.

Immunofluorescence
Cells were fixed with 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton-X-100 for 2 min, and stained as described (Follit et al., 2006). In some cases, fixed cells were treated with 0.05% SDS for 5 min before prehybridization to retrieve antigens. The primary antibodies are described in Table 2. Confocal images were acquired with an inverted microscope (TE-2000E2; Nikon) equipped with a Solamere Technology-modified spinning-disk confocal scan head (CSU10; Yokogawa). Threeimage Z stacks were acquired at 0.2-μm intervals and converted to single planes by maximum projection with MetaMorph software (MDS Analytical Technologies). Widefield images were captured using an Orca ER or a FLIR camera on a Zeiss Axiovert 200M microscope equipped with a 100× Zeiss objective. Contrast adjustment and cropping were done in ImageJ or Photoshop.
For immunoprecipitations, cells were serum starved for 48 h, and proteins were extracted with lysis buffer (Cell Lytic Solution; C2978; Sigma) with 0.1% CHAPSO, 0.1% NP-40, and protease inhibitor (Complete EDTA-Free; Roche). Insoluble components were removed by centrifugation at 20,000 g. Flag beads (Anti Flag M2 Affinity Gel; A2250; Sigma) were added to the cell extract and the mixture was incubated for 2 h at 4°C. After centrifugation, beads were washed 3X with 0.1% Tween20/TBS and 3X with TBS before elution with 3XFLAG peptide (F4799; Sigma). Gel loading buffer was added to the eluates for SDS-PAGE electrophoresis and Western blotting analysis.
Isolation of mRNA and quantitative mRNA analysis were performed as previously described (Jonassen et al., 2008) using the primers described in Table 3.
Online supplemental material