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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">02555</article-id><article-id pub-id-type="doi">10.7554/eLife.02555</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Requirement of Smurf-mediated endocytosis of Patched1 in sonic hedgehog signal reception</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-11863"><name><surname>Yue</surname><given-names>Shen</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-11872"><name><surname>Tang</surname><given-names>Liu-Ya</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-11873"><name><surname>Tang</surname><given-names>Ying</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-14873"><name><surname>Tang</surname><given-names>Yi</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-11875"><name><surname>Shen</surname><given-names>Qiu-Hong</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-11876"><name><surname>Ding</surname><given-names>Jie</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-11877"><name><surname>Chen</surname><given-names>Yan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-11878"><name><surname>Zhang</surname><given-names>Zengdi</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-11862"><name><surname>Yu</surname><given-names>Ting-Ting</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-11879"><name><surname>Zhang</surname><given-names>Ying E</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-11187"><name><surname>Cheng</surname><given-names>Steven Y</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Developmental Genetics, School of Basic Medical Sciences</institution>, <institution>Nanjing Medical University</institution>, <addr-line><named-content content-type="city">Nanjing</named-content></addr-line>, <country>China</country></aff><aff id="aff2"><institution content-type="dept">Laboratory of Cellular and Molecular Biology</institution>, <institution>Center for Cancer Research, National Cancer Institute</institution>, <addr-line><named-content content-type="city">Bethesda</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Krumlauf</surname><given-names>Robb</given-names></name><role>Reviewing editor</role><aff><institution>Stowers Institute for Medical Research</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>sycheng@njmu.edu.cn</email> (SYC); </corresp><corresp id="cor2"><label>*</label>For correspondence: <email>zhangyin@mail.nih.gov</email> (YEZ)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>12</day><month>06</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02555</elocation-id><history><date date-type="received"><day>16</day><month>02</month><year>2014</year></date><date date-type="accepted"><day>10</day><month>06</month><year>2014</year></date></history><permissions><license xlink:href="http://creativecommons.org/publicdomain/zero/1.0/"><license-p>This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">Creative Commons CC0 public domain dedication</ext-link>.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02555.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02555.001</object-id><p>Cell surface reception of Sonic hedgehog (Shh) must ensure that the graded morphogenic signal is interpreted accordingly in neighboring cells to specify tissue patterns during development. Here, we report endocytic sorting signals for the receptor Patched1 (Ptch1), comprising two ‘PPXY’ motifs, that direct it to degradation in lysosomes. These signals are recognized by two HECT-domain ubiquitin E3 ligases, Smurf1 and Smurf2, which are induced by Shh and become enriched in Caveolin-1 lipid rafts in association with Ptch1. Smurf-mediated endocytic turnover of Ptch1 is essential for its clearance from the primary cilium and pathway activation. Removal of both Smurfs completely abolishes the ability of Shh to sustain the proliferation of postnatal granule cell precursors in the cerebellum. These findings reveal a novel step in the Shh pathway activation as part of the Ptch1 negative feedback loop that precisely controls the signaling output in response to Shh gradient signal.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.001">http://dx.doi.org/10.7554/eLife.02555.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02555.002</object-id><title>eLife digest</title><p>Sonic hedgehog protein fulfils many vital roles in establishing the body plan of multicellular organisms during development. And in adult organisms it regulates the stem cells that maintain organs and tissues. In the embryo, Sonic hedgehog is secreted by certain cells to create a concentration gradient; cells then measure this concentration to work out where they are, which allows them to develop into the right sort of cells. However, many details of this process are not completely understood.</p><p>At the core of this process are the interactions between the Sonic hedgehog protein, a receptor called Patched1 that is found on plasma membranes, and another membrane protein called Smoothened. The job of Smoothened is to activate proteins that enter the cell nucleus and ‘switch on’ the pathway's target genes, which encode Patched1 and a number of other proteins. The role of Patched1, on the other hand, is to repress Smoothened. However, when sonic hedgehog binds to Patched1, the latter is unable to repress Smoothened.</p><p>Increasing the production of Patched1 is thought to serve two main roles: it prevents activation of the Sonic hedgehog pathway, and it prevents the Sonic hedgehog protein spreading to neighboring cells (by binding to it). But how is the level of Patched1 itself regulated? Yue et al. now report that two proteins, called Smurf1 and Smurf2, perform this regulation role in mammalian cells.</p><p>Smurf1 and Smurf2 are enzymes that attach a molecule called ubiquitin to proteins, setting in train a series of events that leads to the degradation of the protein. Yue et al. now show that Smurf1 and Smurf2 recognize a signal on Patched1 and perform a similar modification, causing the Patched1 to be internalized through an alternate pathway and degraded in lysosomes. This series of events ultimately allow the Sonic hedgehog pathway to be activated.</p><p>The work of Yue et al. exposes a critical enzymatic step that sorts unbound Patched1 receptors from those that are bound to Sonic hedgehog proteins. Further research is needed to determine if this signaling pathway can be manipulated for therapeutic purposes.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.002">http://dx.doi.org/10.7554/eLife.02555.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>sonic Hedgehog</kwd><kwd>ubiquitination</kwd><kwd>Patched</kwd><kwd>endocytosis</kwd><kwd>Smurfs</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001809</institution-id><institution>National Natural Science Foundation of China (NSFC)</institution></institution-wrap></funding-source><award-id>81272238, 81261120386</award-id><principal-award-recipient><name><surname>Cheng</surname><given-names>Steven Y</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100002855</institution-id><institution>Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)</institution></institution-wrap></funding-source><award-id>2012CB945003, 2009CB918403</award-id><principal-award-recipient><name><surname>Cheng</surname><given-names>Steven Y</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health (NIH)</institution></institution-wrap></funding-source><award-id>ZIA BC 011168</award-id><principal-award-recipient><name><surname>Zhang</surname><given-names>Ying E</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001809</institution-id><institution>National Natural Science Foundation of China (NSFC)</institution></institution-wrap></funding-source><award-id>81101497</award-id><principal-award-recipient><name><surname>Yue</surname><given-names>Shen</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Smurf1 and Smurf2 have essential functions in the mammalian Shh signaling pathway, binding to and ubiquitylating Patched1, leading to its endocytosis and subsequent degradation in lysosomes.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The secreted Sonic hedgehog (Shh) protein specifies spatial tissue patterns during development by providing positional cues embedded in its concentration gradient (<xref ref-type="bibr" rid="bib31">Jiang and Hui, 2008</xref>; <xref ref-type="bibr" rid="bib61">Robbins et al., 2012</xref>; <xref ref-type="bibr" rid="bib64">Ryan and Chiang, 2012</xref>). During embryogenesis, neighboring progenitor cells in a developing field are able to discern incremental changes in the Shh signal strength and adopt their respective fate accordingly (<xref ref-type="bibr" rid="bib60">Ribes and Briscoe, 2009</xref>; <xref ref-type="bibr" rid="bib3">Balaskas et al., 2012</xref>). This ability requires a cell surface reception system that can transform the graded Shh signal into different levels of signaling output, but how this is accomplished is poorly understood. In the adult, Shh plays a crucial role in guiding the differentiation of tissue-specific stem cells (<xref ref-type="bibr" rid="bib30">Jaks et al., 2008</xref>; <xref ref-type="bibr" rid="bib67">Shin et al., 2011</xref>; <xref ref-type="bibr" rid="bib2">Arwert et al., 2012</xref>), and inappropriate activation of Shh signaling could be the culprit that underlines neoplastic growth in the gut epithelium (<xref ref-type="bibr" rid="bib50">Nielsen et al., 2004</xref>) or lead to outright cancers (<xref ref-type="bibr" rid="bib66">Scales and de Sauvage, 2009</xref>; <xref ref-type="bibr" rid="bib68">Stecca and Ruiz, 2010</xref>; <xref ref-type="bibr" rid="bib51">Northcott et al., 2012</xref>).</p><p>At the cell surface, whereas a network of membrane proteins, including Hip1 (<xref ref-type="bibr" rid="bib13">Chuang et al., 2003</xref>), Gas1 (<xref ref-type="bibr" rid="bib39">Lee et al., 2001</xref>), Boc/iHog, and Cdo/Boi (<xref ref-type="bibr" rid="bib53">Okada et al., 2006</xref>; <xref ref-type="bibr" rid="bib73">Tenzen et al., 2006</xref>; <xref ref-type="bibr" rid="bib77">Yao et al., 2006</xref>; <xref ref-type="bibr" rid="bib4">Beachy et al., 2010</xref>), bind Shh and control the range and competence of its receiving cells, the core of Shh signal reception consists of Patched1 (Ptch1), a 12-pass membrane receptor that acts negatively on Smoothened (Smo), a G-protein-coupled, receptor-like signal transducer (<xref ref-type="bibr" rid="bib63">Rohatgi and Scott, 2007b</xref>). Binding of Shh to Ptch1 alleviates the Ptch1 inhibition of Smo, allowing the signal to propagate to three Gli proteins, the transcriptional effectors of the pathway, and activate the expression of target genes, including pathway components Ptch1 and Gli1 themselves. Since Gli1 is a potent activator of Shh target genes, its induction by the ligand ensures that pathway activation will attain the intended effect in a positive feedback loop. On the other hand, induction of the inhibitory Ptch1 amounts to a negative feedback control, which was regarded crucial to the interpretation of the Shh gradient signal (<xref ref-type="bibr" rid="bib60">Ribes and Briscoe, 2009</xref>). In effect, Ptch1 serves two roles in Shh signaling: first, it acts cell autonomously in suppressing the downstream pathway, and second, the excessive Ptch1 induced by Shh acts as a sink in limiting the spread of the ligand, thereby affecting the neighboring cells in a non-cell autonomous fashion (<xref ref-type="bibr" rid="bib11">Chen and Struhl, 1996</xref>; <xref ref-type="bibr" rid="bib74">Torroja et al., 2004</xref>). However, it is not clear what counteracts the induction of Ptch1 to achieve the precision of the regulation.</p><p>For many years, Ptch1 and Smo have been seen in punctate intracellular vesicles in both <italic>Drosophila</italic> and mammalian cells (<xref ref-type="bibr" rid="bib8">Capdevila et al., 1994</xref>; <xref ref-type="bibr" rid="bib58">Ramirez-Weber et al., 2000</xref>; <xref ref-type="bibr" rid="bib80">Zhu et al., 2003</xref>; <xref ref-type="bibr" rid="bib41">Li et al., 2012</xref>), and their trafficking between the cytoplasmic membrane and intracellular vesicles found to be crucial to the activation of the Hedgehog pathway (<xref ref-type="bibr" rid="bib17">Denef et al., 2000</xref>; <xref ref-type="bibr" rid="bib29">Incardona et al., 2000</xref>; <xref ref-type="bibr" rid="bib80">Zhu et al., 2003</xref>; <xref ref-type="bibr" rid="bib48">Nakano et al., 2004</xref>; <xref ref-type="bibr" rid="bib42">Lu et al., 2006</xref>; <xref ref-type="bibr" rid="bib45">Milenkovic et al., 2009</xref>; <xref ref-type="bibr" rid="bib41">Li et al., 2012</xref>). It is known that ligand engagement of <italic>Drosophila</italic> receptor Ptc triggers its internalization and membrane presentation of Smo, but membrane trafficking of Ptch1 and Smo in mammalian cells has an added complexity in that Shh signals through the primary cilium (<xref ref-type="bibr" rid="bib27">Huangfu et al., 2003</xref>; <xref ref-type="bibr" rid="bib14">Corbit et al., 2005</xref>; <xref ref-type="bibr" rid="bib21">Goetz and Anderson, 2009</xref>), a microtubule-based membrane protrusion that emanates from the interphase centrioles (<xref ref-type="bibr" rid="bib40">Lefebvre and Rosenbaum, 1986</xref>; <xref ref-type="bibr" rid="bib54">Pazour and Witman, 2003</xref>; <xref ref-type="bibr" rid="bib47">Nachury et al., 2010</xref>). The prevailing model for mammalian Shh activation entails Ptch1 exiting from and Smo translocating into the primary cilium (<xref ref-type="bibr" rid="bib62">Rohatgi et al., 2007a</xref>; <xref ref-type="bibr" rid="bib36">Kovacs et al., 2008</xref>). Some data suggest that Smo trafficking through membranous compartments is controlled by small lipids and the sterol-sensing domain of Ptch1 (<xref ref-type="bibr" rid="bib43">Martin et al., 2001</xref>; <xref ref-type="bibr" rid="bib5">Bijlsma et al., 2006</xref>; <xref ref-type="bibr" rid="bib15">Corcoran and Scott, 2006</xref>; <xref ref-type="bibr" rid="bib78">Yavari et al., 2010</xref>). Since the structural framework of Ptch1 resembles that of bacterial amino acid transporters (<xref ref-type="bibr" rid="bib9">Carstea et al., 1997</xref>), it is conceivable that Ptch1 controls Smo activity or trafficking through such a small molecular intermediate. However, little evidence is available to account for how Ptch1 internalization through endocytosis is regulated, and it is unclear whether ciliary trafficking and endocytosis are obligatorily coupled (<xref ref-type="bibr" rid="bib47">Nachury et al., 2010</xref>).</p><p>Receptor endocytosis plays crucial roles in coordinating the strength and duration of many cell signaling systems (<xref ref-type="bibr" rid="bib55">Piddini and Vincent, 2003</xref>; <xref ref-type="bibr" rid="bib56">Polo and Di Fiore, 2006</xref>). At various steps of the endocytic pathway, from the plasma membrane to the endosomes, receptors can be sorted to the proteolytic lumens of lysosomes, leading to desensitization, or back to the plasma membrane for a rapid recovery of cellular responsiveness. In addition to the classical Clathrin-mediated endocytosis, recent advances indicate that membrane receptors are also internalized through lipid rafts (<xref ref-type="bibr" rid="bib38">Le Roy and Wrana, 2005</xref>; <xref ref-type="bibr" rid="bib37">Lajoie and Nabi, 2010</xref>), which are specialized membrane domains enriched in cholesterol and sphingomyelin and stabilized by Caveolin 1 (Cav-1) (<xref ref-type="bibr" rid="bib1">Allen et al., 2007</xref>). Unlike the Clathrin-mediated endocytosis, cargos of caveolae were shown to be unloaded to late endosomes, thereby bypassing early endosomes (<xref ref-type="bibr" rid="bib57">Quirin et al., 2008</xref>; <xref ref-type="bibr" rid="bib23">Hayer et al., 2010</xref>; <xref ref-type="bibr" rid="bib65">Sandvig et al., 2011</xref>). A major forward endocytic sorting signal is ubiquitination (<xref ref-type="bibr" rid="bib25">Hicke and Dunn, 2003</xref>; <xref ref-type="bibr" rid="bib46">Mukhopadhyay and Riezman, 2007</xref>; <xref ref-type="bibr" rid="bib23">Hayer et al., 2010</xref>), and many HECT-domain E3 ligases have been implicated in the Ubiquitin control of endocytosis, including Smurf2 (<xref ref-type="bibr" rid="bib18">Di Guglielmo et al., 2003</xref>; <xref ref-type="bibr" rid="bib44">Metzger et al., 2012</xref>), which was first identified as a negative regulator of TGF-β/BMP signaling (<xref ref-type="bibr" rid="bib34">Kavsak et al., 2000</xref>; <xref ref-type="bibr" rid="bib79">Zhang et al., 2001</xref>). Here, we present evidence that Smurf1 and Smurf2 are the Ubiquitin E3 ligases that promote Ptch1 movement from lipid rafts to late endosomes for subsequent degradation in lysosomes. This movement is essential for Ptch1's clearance from primary cilia, Shh pathway activation, and the role of Shh in sustaining the proliferation of cerebellar granule cell precursors. In light of the negative feedback control of Shh signaling by Ptch1, this destruction system would allow the level of signaling output to be set precisely according to the level of the Ptch1 protein.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Both PPXY-motifs deletion and endocytosis blockade cause Ptch1 to accumulate in lipid rafts</title><p>The C-terminal tails of <italic>Drosophila</italic> Ptc and mouse Ptch1 play an important role in determining its membrane distribution and stability, possibly through the highly conserved ‘PPXY’ motif (<xref ref-type="bibr" rid="bib42">Lu et al., 2006</xref>; <xref ref-type="bibr" rid="bib35">Kawamura et al., 2008</xref>), which is recognized by the WW domain frequently found in HECT-domain E3 ligases (<xref ref-type="bibr" rid="bib44">Metzger et al., 2012</xref>). Mammalian Ptch1 contains an evolutionarily conserved C-terminal ‘PPXY’ motif and a second one in the third intracellular loop (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>), whereas Ptch2 does not and is quite stable (<xref ref-type="bibr" rid="bib35">Kawamura et al., 2008</xref>). Under a confocal microscope and in transfected murine embryonic fibroblasts (MEFs), Ptch1-GFP was primarily detected in punctate vesicles (<xref ref-type="fig" rid="fig1">Figure 1A</xref>), consistent with what was reported in COS and HeLa cells (<xref ref-type="bibr" rid="bib29">Incardona et al., 2000</xref>; <xref ref-type="bibr" rid="bib33">Karpen et al., 2001</xref>); a large proportion of these Ptch1-GFP vesicles were likely to be endosomes (see below and in <xref ref-type="bibr" rid="bib43">Martin et al., 2001</xref>; <xref ref-type="bibr" rid="bib28">Incardona et al., 2002</xref>). In light of the ubiquitination control of endocytosis, we suspected that the ‘PPXY’ motifs of Ptch1 might be the signal that regulates its turnover through endosomes and lysosomes. To test this hypothesis, we sought to determine how Ptch1 engages the endocytic pathway by focusing our attention at the rim of the plasma membrane, where treatment with conditioned medium (CM) from HEK293 cells expressing the N-terminal signaling fragment of Shh (ShhN) for 1 hr rendered some of the Ptch1-GFP vesicles also positive for Cav-1 (<xref ref-type="fig" rid="fig1">Figure 1A</xref>), a specific marker of lipid rafts. To quantify the colocalization, we sampled 10 randomly selected rim areas from different cells imaged for each data point and calculated the colocalization coefficient. The results indicated that ShhN almost doubled the colocalization coefficient between Ptch1-GFP and endogenous Cav-1 from 0.37 ± 0.04 to 0.68 ± 0.04 (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). Blocking late endosome/lysosome passage with chloroquine (Chlq) and concanavalin A (ConA) or lysosomal proteolysis with leupeptin (Leu) showed similar effects (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). In contrast, the mutant Ptch1Δ2PY-GFP that lacks both ‘PPXY’ motifs exhibited a higher level of colocalization with Cav-1 than its wildtype counterpart even without ShhN treatment (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). Some of the Ptch1-GFP vesicles at the plasma rim were also positive for Clathrin heavy chain that marks the Clathrin-coated pits, but in contrast to the ligand-inducible enrichment in Cav-1 lipid rafts, the fraction of Ptch1-GFP in Clathrin-coated pits was affected neither by ligand treatment nor deletion of the ‘PPXY’ motifs (<xref ref-type="fig" rid="fig1">Figure 1C,D</xref>). To complement the confocal imaging experiments, we conducted a co-sedimentation experiment in a discontinuous sucrose density gradient and found that both Ptch1-FLAG and Ptch1Δ2PY-FLAG co-sedimented with Cav-1 in 20% and 25% buoyancy fractions (<xref ref-type="fig" rid="fig1">Figure 1E</xref>), indicating that when expressed exogenously, Ptch1-FLAG can find its way into Cav-1 positive lipid rafts even without Shh induction. Thus, both deletion of the ‘PPXY’ motifs and blocking endocytosis cause Ptch1 to accumulate in lipid rafts.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.003</object-id><label>Figure 1.</label><caption><title>The PPXY motifs define sorting signals from lipid rafts to late endosomes.</title><p>(<bold>A</bold>) Confocal images showing colocalization of exogenously expressed Ptch1-GFP or Δ2PY (green) with native Cav-1 (red) at the rim of the plasma membrane, and (<bold>B</bold>) calculation of the colocalization coefficients in (<bold>A</bold>) in transfected MEFs. ShhN-CM and Chlq were added 1 hr prior to fixation. The chamber slides were chilled at 4°C for 20 min and then shifted to 37°C for another 20 min before fixation with 4% paraformaldehyde in PBS. The cells were then permeabilized with 0.5% Triton X-100 and stained with an antibody for Cav-1. (<bold>C</bold>) Representative images and (<bold>D</bold>) quantification of colocalization between Ptch1-GFP and clathrin heavy chain. MEFs were transfected with Ptch1-GFP or PtchΔ2PY-GFP, then treated with ShhN or Ctrl medium for 1 hr before fixation. (<bold>E</bold>) Western analyses of sucrose gradient fractions showing Ptch1-FLAG co-sedimented with Smurf2CG-Myc and Cav-1. Δ2PY was inefficient in bringing Smurf2CG-Myc into Cav-1 positive sedimentation fractions. (<bold>F</bold>) Western blot analyses of stabilities of Ptch1 and the ‘PPXY’ motif mutants in MEFs. Chlq or MG132 treatment was carried out for 4 hr. The confocal images were taken with a 63x objective, and the insets in 1A were digitally magnified. Bars represent mean ± standard deviation (SD). Statistical analyses were performed by two-tail Student's <italic>t</italic> test. ***p<0.001, and <italic>n.s</italic>., not statistically significant (p>0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.003">http://dx.doi.org/10.7554/eLife.02555.003</ext-link></p></caption><graphic xlink:href="elife02555f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Position and sequence alignment of ‘PPXY’ motifs.</title><p>(A) Schematic representation of Ptch1 constructs (left) and sequence alignments (right) of Drosophila and vertebrate Ptch1 surrounding the two evolutionarily conserved PPXY motifs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.004">http://dx.doi.org/10.7554/eLife.02555.004</ext-link></p></caption><graphic xlink:href="elife02555fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Lysosomal inhibitors cause Ptch1-GFP to accumulate in lipid rafts.</title><p>(<bold>A</bold>) Representative confocal images showing accumulation of Ptch1-GFP in Cav-1 positive lipid rafts after blocking endocytosis with lysosomal inhibitors Leu and ConA. (<bold>B</bold>) Calculation of colocalization coefficients in (<bold>A</bold>). The confocal images were taken with a 63x oil lens, and the insets were digitally magnified. Bars represent mean ± standard deviation (SD).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.005">http://dx.doi.org/10.7554/eLife.02555.005</ext-link></p></caption><graphic xlink:href="elife02555fs002"/></fig></fig-group></p><p>Since an end point of endocytosis is degradation in lysosomes, we further asked if wildtype Ptch1 and ‘PPXY’ motif mutants accumulate differently in the presence of proteasomal or lysosomal blocker. When expressed in MEFs, Ptch1-GFP was an unstable protein; the bulk of which appeared to turnover via proteasomes as Ptch1-GFP accumulated to a very high level in the presence of MG132 (<xref ref-type="fig" rid="fig1">Figure 1F</xref>, compare lanes 1 and 3). A small portion of Ptch1-FLAG appeared to turnover via lysosomes as indicated by the moderate level of accumulation in the presence of lysosomal inhibitor Chlq (<xref ref-type="fig" rid="fig1">Figure 1F</xref>, lanes 1 and 2). In contrast, Ptch1 mutants lacking either one of or both ‘PPXY’ motifs were relatively stable when expressed in MEFs, although they could be induced to accumulate further by MG132 but not Chlq (<xref ref-type="fig" rid="fig1">Figure 1F</xref>, lanes 4–12). These results suggest that Shh promotes turnover of at least a portion of ectopically expressed Ptch1 via endosomes and lysosomes, but the entry point is likely the Cav-1 positive lipid rafts rather than the conventional clathrin-coated pits.</p></sec><sec id="s2-2"><title>The ‘PPXY’ motifs define an endocytic sorting signals of Ptch1</title><p>To ascertain if the ‘PPXY’ motifs are the actual signal that sorts Ptch1 from lipid rafts to endosomes/lysosomes, we asked if Ptch1-GFP or Δ2PY-GFP could be identified in early endosomes, late endosomes, or lysosomes, which are marked by Rab5-RFP, Rab7-RFP, or Lamp1-RFP, respectively. In the absence of ShhN, Ptch1-GFP and Rab7-RFP could be readily detected together in punctate vesicles, and ShhN treatment drastically increased that colocalization as indicated by colocalization coefficient, which increased from 0.29 ± 0.03 to 0.51 ± 0.02 (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>). Similar colocalization between Ptch1-GFP and endogenous Rab7 was also observed under ShhN treatment using specific antibodies (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). We could not detect vesicles marked positively with both Ptch1-GFP and Lamp1-RFP or Ptch1-GFP and endogenous Lamp1-RFP without blocking lysosomal enzymes by leupeptin (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2A</xref>), but colozalization between Ptch1-GFP and endogenous Lamp1 was revealed with the use of leupeptin (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). We did not see Ptch1-GFP colocalizing with either transfected Rab5-RFP (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2B</xref>) or endogenous Rab5 (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3</xref>) without or with ShhN treatment. These observations are consistent with the notion that endocytic cargos of caveolae are unloaded directly to late endosomes, bypassing early endosomes (<xref ref-type="bibr" rid="bib57">Quirin et al., 2008</xref>; <xref ref-type="bibr" rid="bib23">Hayer et al., 2010</xref>; <xref ref-type="bibr" rid="bib65">Sandvig et al., 2011</xref>). In contrast to Ptch1-GFP, Δ2PY-GFP was never found together with any of the three endosome/lysosome markers and ShhN treatment caused no statistically significant change thereof (<xref ref-type="fig" rid="fig2">Figure 2A–C</xref>, <xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1 and 2</xref>), indicating that Shh is not able to induce Δ2PY to move beyond lipid rafts to enter late endosomes.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.006</object-id><label>Figure 2.</label><caption><title>PPXY motifs are required for Shh-induced endocytosis of Ptch1.</title><p>(<bold>A</bold>) Confocal images showing colocalization of Ptch1-GFP or Δ2PY (green) with Rab7-RFP (red), and (<bold>B</bold>) calculation of the colocalization coefficients in (<bold>A</bold>) in transfected MEFs. (<bold>C</bold>) Confocal images showing localization of Ptch1-GFP or Δ2PY (green) in vesicles marked anti-Lamp1 (red) in the presence of 1 mg/ml leupeptin. (<bold>D</bold>) Confocal imaging and (<bold>E</bold>) calculation of colocalization coefficient of Ptch1-GFP and Rab7-RFP in Kif3a<sup>−/−</sup> and control MEFs. ShhN treatment was for 1 hr and the cells were processed as in <xref ref-type="fig" rid="fig1">Figure 1A</xref>. Statistical analyses were performed by two-tail Student's <italic>t</italic> test. ***p<0.001, and <italic>n.s</italic>., not statistically significant (p>0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.006">http://dx.doi.org/10.7554/eLife.02555.006</ext-link></p></caption><graphic xlink:href="elife02555f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Shh promotes colocalizaiton of Ptch1-GFP with endogenous Rab7 in late endosomes.</title><p>Representative confocal images showing ShhN treatment promotes colocalization of Ptch1-GFP in late endosomes visualized by anti-Rab7. Transfected MEFs were treated with ShhN-CM or control conditioned medium for 1 hr, followed by incubations at 4°C for 20 min and 37°C for 20 min. The close-up images were digitally amplified.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.007">http://dx.doi.org/10.7554/eLife.02555.007</ext-link></p></caption><graphic xlink:href="elife02555fs003"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.008</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Lack of colocalization of Ptch1-GFP or Δ2PY-GFP with exogenous Rab5-RFP and Lamp1-RFP without blocking lysosomal turnover.</title><p>Representative Confocal images and quantification of colocalization coefficients showing that Ptch1-GFP or Δ2PY-GFP does not colocalize with Lamp1-RFP (red) (<bold>A</bold>) or Rab5-RFP (<bold>B</bold>). Transfected MEFs were treated ShhN or control conditioned medium without Leupeptin for 2 hr, and then the cells were processed as in <xref ref-type="fig" rid="fig2">Figure 2A</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.008">http://dx.doi.org/10.7554/eLife.02555.008</ext-link></p></caption><graphic xlink:href="elife02555fs004"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.009</object-id><label>Figure 2—figure supplement 3.</label><caption><title>Ptch1-GFP or Δ2PY-GFP was not found in early endosomes marked by anti-Rab5 immunofluorescence staining.</title><p>Representative Confocal images showing Ptch1-GFP or Δ2PY-GFP and endogenous Rab5 in non-overlapping green or red channel, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.009">http://dx.doi.org/10.7554/eLife.02555.009</ext-link></p></caption><graphic xlink:href="elife02555fs005"/></fig></fig-group></p><p>The current paradigm stipulates that Shh induces Ptch1 exit from the primary cilium during signaling (<xref ref-type="bibr" rid="bib62">Rohatgi et al., 2007a</xref>; <xref ref-type="bibr" rid="bib36">Kovacs et al., 2008</xref>). This prompted us to ask if ciliary export or its structural integrity is prerequisite to endocytosis of Ptch1 by comparing the abilities of Ptch1-GFP to associate with Rab7-RFP in <italic>Kif3a</italic><sup><italic>−/−</italic></sup> or otherwise isogenic control MEFs. Although <italic>Kif3a</italic><sup><italic>−/−</italic></sup> MEFs do not make cilia (<xref ref-type="bibr" rid="bib12">Chen et al., 2011</xref>), Ptch1-GFP could still proceed to late endosomes/lysosomes under the influence of ShhN unabatedly (<xref ref-type="fig" rid="fig2">Figure 2D,E</xref>), implying that Ptch1 endocytosis is downstream from or independent of ciliary trafficking.</p><p>Based on results from the above several lines of investigation, we conclude that the ‘PPXY’ motifs constitute sorting signals that direct Ptch1 to move into late endosomes for turnover in lysosomes. This sorting event likely takes place in Cav-1 positive lipid rafts since Δ2PY accumulates there in the absence of this signal.</p></sec><sec id="s2-3"><title>Ptch1 endocytosis is required for the activation of Shh signaling</title><p>Ptc or Ptch1 endocytosis has been observed in cells from <italic>Drosophila</italic> to mammals for some time (<xref ref-type="bibr" rid="bib17">Denef et al., 2000</xref>; <xref ref-type="bibr" rid="bib29">Incardona et al., 2000</xref>, <xref ref-type="bibr" rid="bib28">2002</xref>; <xref ref-type="bibr" rid="bib43">Martin et al., 2001</xref>; <xref ref-type="bibr" rid="bib74">Torroja et al., 2004</xref>; <xref ref-type="bibr" rid="bib42">Lu et al., 2006</xref>), but its role was primarily attributed to ligand sequestration or clearing (<xref ref-type="bibr" rid="bib29">Incardona et al., 2000</xref>; <xref ref-type="bibr" rid="bib74">Torroja et al., 2004</xref>). In <italic>Drosophila</italic>, the role of Ptc in ligand sequestration has been shown to be separable from that of signaling based on analyses of certain mutants (<xref ref-type="bibr" rid="bib11">Chen and Struhl, 1996</xref>; <xref ref-type="bibr" rid="bib74">Torroja et al., 2004</xref>). However, we observed that when re-expressed in <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs, the ‘PPXY’ motif mutants accumulated in the primary cilium, in contrast to their wildtype counterpart; ShhN treatment effectively forced Ptch1-GFP to exit the primary cilium, but it was less effective against these mutants (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>). Ciliary accumulation of the ‘PPXY’ motif mutants is likely a consequence of their inability to endocytose, rather than a specific defect of ciliary export, since these mutants also accumulate in lipid rafts (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>) and blocking endocytosis with high concentration of leupeptin showed a similar effect without or with ShhN treatment (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Combined with results from the stability experiment (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), this observation indicated that these two ‘PPXY’ motifs play an equivalent role in regulating Ptch1 function in cilia. To support this notion, we made temporal measurements of endogenous Smo translocating into the primary cilium, which is an obligatory early event of Shh signaling and was reported as concurrent to the exit of Ptch1 therefrom (<xref ref-type="bibr" rid="bib62">Rohatgi et al., 2007a</xref>). In <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs, immunofluorescence staining showed that Smo was constitutively present in the primary cilium (<xref ref-type="fig" rid="fig3">Figure 3C</xref>), as expected (<xref ref-type="bibr" rid="bib14">Corbit et al., 2005</xref>; <xref ref-type="bibr" rid="bib62">Rohatgi et al., 2007a</xref>; <xref ref-type="bibr" rid="bib36">Kovacs et al., 2008</xref>). Re-introducing Ptch1-GFP cleared Smo out of the primary cilium, but ShhN treatment allowed Smo to move back in to nearly its full extent within 4 hr (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). Conversely, ShhN treatment triggered the ciliary export of Ptch1 at a rate comparable to that of Smo import (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, and compare <xref ref-type="fig" rid="fig3">Figure 3D,E</xref>). Re-introducing Δ2PY, on the other hand, only allowed a substantially lower level of Smo to be imported back into cilia after ShhN treatment and Δ2PY was itself resistant to Shh-induced export (<xref ref-type="fig" rid="fig3">Figure 3C,E</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.010</object-id><label>Figure 3.</label><caption><title>The ‘PPXY’ motifs regulate the opposing movements of Ptch1 out of and Smo into the primary cilium.</title><p>(<bold>A</bold>) Representative confocal images and (<bold>B</bold>) distribution of GFP fluorescence showing accumulation of the ‘PPXY’ motif mutants of Ptch1 in primary cilia in the absence or presence of ShhN. Two-tail Student's <italic>t</italic> test was used for statistical analysis. ***p<0.001, n.s., not significant (p>0.05). (<bold>C</bold>) Immunofluorescence of GFP as well as endogenous Smo (red) and acetylated tubulin (blue) staining in Ptch1<sup>−/−</sup> MEFs transfected with Ptch1-GFP or Δ2PY. (<bold>D</bold>) Quantification of anti-Smo staining and (<bold>E</bold>) GFP fluorescence as in (<bold>C</bold>). Only transfected GFP positive cells were counted for the ciliary localization of endogenous Smo. In all of the above experiments, transfected cells were grown to confluence and then serum-starved for 24 hr to allow for ciliogenesis. ShhN-CM treatment was for 24 hr, or as indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.010">http://dx.doi.org/10.7554/eLife.02555.010</ext-link></p></caption><graphic xlink:href="elife02555f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.011</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Inhibition of Lysosomal turnover dampens Shh-induced ciliary exit of Ptch1-GFP.</title><p>Representative confocal images and calculations thereof showing Ptch1-GFP fluorescence accumulated in primary cilia. ShhN and leupeptin (1 mg/ml) were added to the WT MEFs for 2 hr. Two-tail Student's <italic>t</italic> test was used for statistical analysis. **p<0.01, n.s., not significant (p>0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.011">http://dx.doi.org/10.7554/eLife.02555.011</ext-link></p></caption><graphic xlink:href="elife02555fs006"/></fig></fig-group></p><p>As a ligand-binding and inhibitory receptor, the functions of <italic>Drosophila</italic> Ptc are twofold; first acting through Smo, Ptc negatively regulates downstream pathway signaling cell autonomously, and second, through ligand sequestration Ptc suppresses Hh signaling in neighboring cells. To determine if Ptch1 endocytosis impinges on downstream pathway activation, we measured the ability of Ptch1-GFP or Δ2PY to confer Shh inducibility to the 8xGliBS-luc reporter in <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs. When co-transfected with Ptch1-GFP, the 8xGliBS-luc reporter showed a robust inductive response to ShhN, resulting in a dose–response curve typical of a substrate-enzyme relationship; however, this reporter was barely induced by ShhN when it was co-transfected with Δ2PY (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). The Shh signaling blockade imposed by Δ2PY could be by-passed by siRNA-mediated knockdown of Sufu (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), a downstream negative regulator, suggesting that the blockade is pathway-specific and occurs upstream of Sufu function. So far all our evidence points to inability of the ‘PPXY’ motif mutants to undergo Shh-induced endocytosis rather than a defect in their intrinsic activity. Indeed, in <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs, these mutants were equally effective as wildtype Ptch1 or cyclopamine in suppressing 8xGliBS-luc reporter independent of the Shh ligand (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). Finally to address the effect of the ‘PPXY’ motifs deletion on the non-cell-autonomous function of Ptch1, we designed a ‘mixing’ experiment, in which <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs re-expressing wildtype Ptch1-GFP or Δ2PY-GFP were mixed at 5 to 1 ratio with a line of stable NIH3T3 cells harboring the genomically integrated 8xGliBS-luc reporter (<xref ref-type="bibr" rid="bib12">Chen et al., 2011</xref>). In the presence of limiting amount of ShhN (1:64 dilution of the conditioned medium), Δ2PY showed a robust inhibition of the ligand-induced reporter activity in the neighboring cells; however this effect was nullified at high ShhN concentration (1:16 dilution) (<xref ref-type="fig" rid="fig4">Figure 4D</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.012</object-id><label>Figure 4.</label><caption><title>The ‘PPXY’ motifs are required for eliciting both cell and non-cell autonomous transcriptional responses to Shh.</title><p>(<bold>A</bold>) Luciferase assays for Ptch1 and the Δ2PY mutant in Ptch1<sup>−/−</sup> MEFs that were transfected together with the 8xGliBS-luc reporter construct. Each data point was obtained in triplicate and the error bars denote the standard error. (<bold>B</bold>) Rescuing Shh induction blockade imposed by Δ2PY using siSufu in <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs. The experiment was set up as in (<bold>A</bold>) except that Sufu was knocked down by siRNA at the same time as cDNA transfection and 1:16 dilution of ShhN-CM was used. (<bold>C</bold>) Relative activities of the GliBS-luc reporter that was co-expressed with Ptch1 or the ΔPY mutants in <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs without ShhN-CM treatment. The ΔPY mutants displayed same inhibitory effect as WT Ptch1. (<bold>D</bold>) Non-cell autonomous inhibition of GliBS-luc reporter in neighboring cells. <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs transfected with Ptch1-FLAG, Δ2PY, or the vector control were mixed at 5:1 ratio with NIH3T3:GliBS-luc reporter cells. The cells were given ShhN-CM for 24 hr, and two-tail Student's <italic>t</italic> test was used for statistical analyses. *p<0.05, **p<0.01, ***p<0.001, and <italic>n.s</italic>., not significant (p>0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.012">http://dx.doi.org/10.7554/eLife.02555.012</ext-link></p></caption><graphic xlink:href="elife02555f004"/></fig></p><p>In summary, our data indicate that whereas Ptch1 engagement to the ligand may have a nominal effect of internalizing Shh, it can be also regarded as an interaction that allows Shh to induce Ptch1 clearance from the primary cilium, the site of Shh signaling, and this regulation equally impinges on both cell and non-cell autonomous signaling functions of Ptch1.</p></sec><sec id="s2-4"><title>Smurf1 and Smurf2 are E3 ligases required for Shh signaling</title><p>Previously, the C-terminal domain of <italic>Drosophila</italic> Ptc was shown to be recognized by Nedd4 HECT-domain E3 ligase (<xref ref-type="bibr" rid="bib42">Lu et al., 2006</xref>). We expressed mouse Nedd4 and Nedd4l together with Ptch1-FLAG in HEK293 cells, and found that neither one promoted Ptch1 degradation, and several other HECT-domain E3 ligases including Wwp1, Wwp2, Huwe1, Herc1, Herc3, Herc4, Herc6, Hecw1, and Hecw2 also showed no effect, but co-expression of Smurf1 or Smurf2 did (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>). Consistent with a specific role, the ligase deficient Smurf1CA and Smurf2CG mutants failed to influence Ptch1 stability (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Since the ‘PPXY’ motif mutants accumulated in cilia, we asked if knockdown of either Smurf or both with siRNAs could augment the ciliary localization of Ptch1-GFP. We found this was the case in NIH3T3 cells without (<xref ref-type="fig" rid="fig5">Figure 5C,D</xref>) or with ShhN treatment (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Because Smurf2 is known to direct the TGF-β type I receptor and the μ opioid neuropeptide receptor to endocytic turnover (<xref ref-type="bibr" rid="bib18">Di Guglielmo et al., 2003</xref>; <xref ref-type="bibr" rid="bib24">Henry et al., 2012</xref>), we posited that Smurf1 and Smurf2 might be the enzymes that control Ptch1 endocytosis and chose them for further analysis.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.013</object-id><label>Figure 5.</label><caption><title>Smurf1 and Smurf2 are E3 ligases required for Shh signaling.</title><p>(<bold>A</bold>) Western analyses of Ptch1-FLAG in HEK293 cells that were co-transfected with cDNAs encoding a battery of HECT-domain E3 ligases as indicated, and (<bold>B</bold>) ligase deficient Smurf mutants. β-actin was used as a loading control. (<bold>C</bold>) Representative confocal images and (<bold>D</bold>) calculations of Ptch1-GFP fluorescence accumulated in primary cilia as the result of siRNA knockdown of <italic>Smurf1</italic>, <italic>Smurf2</italic>, or both in NIH3T3 cells. Primary cilia were marked by acetylated tubulin (red). (<bold>E</bold>) RT-PCR detection of Gli1, Smurf1, and Smurf2 mRNAs in wildtype (WT), <italic>Smurf1</italic><sup><italic>−/−</italic></sup> , and <italic>Smurf2</italic><sup><italic>−/−</italic></sup> MEFs transfected with non-silencing (NS) or Smurf-specific siRNAs as indicated. HPRT mRNA was used as an internal control. A representative gel image is shown here. (<bold>F</bold>) RT-qPCR quantification of fold induction of Gli1 mRNA from an experiment as in (<bold>E</bold>). Fold induction was calculated using Gli1 mRNA level normalized against that of Hprt for even loading and then against the normalized Gli1 mRNA level from cells transfected with NS siRNA and without ShhN treatment. (<bold>G</bold>) RT-qPCR analysis of relative levels of Smurf1 and Smurf2 mRNAs from the experiment in (<bold>F</bold>). (<bold>H</bold>) RT<bold>-</bold>qPCR detection of endogenous Gli1 mRNAs in <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs that were infected with either Ad-GFP (mock) or Ad-Cre for 12 hr, and then treated with either control or ShhN conditional medium for 72 hr. (<bold>I</bold>) Western analyses of endogenous Smurf2 in <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs from the experiment in (<bold>H</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.013">http://dx.doi.org/10.7554/eLife.02555.013</ext-link></p></caption><graphic xlink:href="elife02555f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.014</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Knockdown of Smurf1 and Smurf2 simultaneously dampens Shh-induced ciliary exit of Ptch1-GFP.</title><p>Representative confocal images and calculations thereof showing Ptch1-GFP fluorescence accumulated in primary cilia. NIH3T3 cells were transfected with siRNAs specific for <italic>Smurf1</italic> and <italic>Smurf2</italic>, and then the cells were treated with control or ShhN conditioned medium before Ptch1-GFP was visualized in cilia and quantified. Two-tail Student's <italic>t</italic> test was used for statistical analysis. *p<0.05, ***p<0.001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.014">http://dx.doi.org/10.7554/eLife.02555.014</ext-link></p></caption><graphic xlink:href="elife02555fs007"/></fig></fig-group></p><p>Smurf1 and Smurf2 share redundant functions during development as individually knockout <italic>Smurf1</italic><sup><italic>−/−</italic></sup> or <italic>Smurf2</italic><sup><italic>−/−</italic></sup> mice are healthy and fertile, but the embryos lacking both genes were not able to develop to term (<xref ref-type="bibr" rid="bib76">Yamashita et al., 2005</xref>; <xref ref-type="bibr" rid="bib49">Narimatsu et al., 2009</xref>; <xref ref-type="bibr" rid="bib6">Blank et al., 2012</xref>). To assess the role of Smurfs in Shh signaling, we quantified the transcriptional responses of endogenous Gli1 by RT-PCR (<xref ref-type="fig" rid="fig5">Figure 5E</xref>) and RT-qPCR (<xref ref-type="fig" rid="fig5">Figure 5F</xref>) in MEFs with different Smurf genetic background, and found that silencing Smurf1 and Smurf2 simultaneously in wildtype MEFs completely abolished Shh induction of Gli1 (<xref ref-type="fig" rid="fig5">Figure 5E,F</xref>). MEFs that lack one of the two <italic>Smurf</italic> genes still mounted a considerable Gli1 transcriptional response to ShhN; however, silencing the remaining <italic>Smurf2</italic> allele in <italic>Smurf1</italic><sup><italic>−/−</italic></sup> or <italic>Smurf1</italic> allele in <italic>Smurf2</italic><sup><italic>−/−</italic></sup> MEFs, respectively, led to marked curtailment of Gli1 activation (<xref ref-type="fig" rid="fig5">Figure 5E,F</xref>). Expression of Smurfs showed a compensatory upregulation in response to the loss of the other Smurf in these MEFs as reported (<xref ref-type="bibr" rid="bib76">Yamashita et al., 2005</xref>; <xref ref-type="bibr" rid="bib72">Tang et al., 2011</xref>), but surprisingly, ShhN induced expression of both Smurfs (<xref ref-type="fig" rid="fig5">Figure 5E,G</xref>). During the course of this investigation, we generated <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> mice, which will be described in detail elsewhere. In <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs, Ad-cre infection-mediated ablation of conditional <italic>Smurf2</italic><sup><italic>fl</italic></sup> alleles severely dampened the Gli1 transcriptional response to ShhN (<xref ref-type="fig" rid="fig5">Figure 5H,I</xref>). Similarly, two other Shh signaling responses, namely Shh-induced ciliary import of Smo and Gli3, were also affected (<xref ref-type="fig" rid="fig6">Figure 6A–C</xref>). Since we could rescue Shh induction of GliBS-luc responses in Ad-cre infected <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs (<italic>Smurfs</italic> null) by reintroducing wildtype Smurf1 or Smurf2 but not mutant Smurf1CA or Smurf2CG cDNA (<xref ref-type="fig" rid="fig6">Figure 6D</xref>), or by siRNA-mediated knockdown of <italic>Suppressor of fused</italic> (<italic>Sufu</italic>) (<xref ref-type="fig" rid="fig6">Figure 6E</xref>), an essential downstream negative regulator of Shh signaling, the observed defects of GliBS-luc induction have to be Smurfs and Shh pathway specific. Taken together, the above results show that simultaneous inactivation of both <italic>Smurf</italic> genes and removal of the ‘PPXY’ motifs of Ptch1 have congruent effects on various Shh signaling events, and indicate that a common Smurf function is required at a step upstream from the control of the ciliary import of Smo.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.015</object-id><label>Figure 6.</label><caption><title>Smurf1 and Smurf2 are required For Shh signaling.</title><p>(<bold>A</bold>) Representative confocal images of Smo and Gli3 immunofluorescence staining in cilia of wildtype (WT), <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup>, or <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs infected with Ad-Cre viruses. (<bold>B</bold>) Quantification of Smo and (<bold>C</bold>) Gli3 immunofluorescence staining in cilia of (<bold>A</bold>). In the above experiments, ShhN treatment was carried out for 24 hr, and the means and standard deviation were calculated from two independent experiments (n = 20). (<bold>D</bold>) GliBS-luc assays in <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs showing the deficiency of Shh induction associated with genomic ablation of both Smurfs can be rescued by re-introducing either wildtype Smurf1 or Smurf2 but not their corresponding mutants. (<bold>E</bold>) GliBS-luc reporter assays for the ability of siSufu to by-pass the requirement of Smurfs in Shh signaling. <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs were infected with Ad-cre and then transfected with siSufu or ns control. The cells were then treated with a series of dilutions of ShhN-CM before luciferase activities were assayed. Error bars denote standard deviations. Statistical analyses were performed by two-tail Student's <italic>t</italic> test. *p<0.05, **p<0.01, ***p<0.001, and <italic>n.s</italic>., not significant (p>0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.015">http://dx.doi.org/10.7554/eLife.02555.015</ext-link></p></caption><graphic xlink:href="elife02555f006"/></fig></p></sec><sec id="s2-5"><title>Smurfs and Ptch1 colocalize and interact in lipid rafts</title><p>If Smurfs are the E3 ligases that recognize the endocytic sorting signals of Ptch1, these proteins should physically interact in lipid rafts. A number of evidence demonstrates that this is the case. First, in non-permeabilizing MEFs, we found exogenously expressed Ptch1-RFP colocalized with the ligase deficient, GFP-tagged Smurf2CG mutant in Cav-1 positive lipid rafts at the rim of the plasma membrane (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Although first identified as modulators of TGF-β/BMP signaling, Smurfs are preferentially localized in the nucleus (<xref ref-type="bibr" rid="bib34">Kavsak et al., 2000</xref>) and play a crucial function in maintaining genomic stability (<xref ref-type="bibr" rid="bib6">Blank et al., 2012</xref>). Serendipitously, we found that treatment with ShhN ligand or co-expression with Ptch1-RFP each caused Smurf2GFP to move from the nucleus to the cytoplasm (<xref ref-type="fig" rid="fig7">Figure 7B</xref>, <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). In light of the Shh induction (<xref ref-type="fig" rid="fig5">Figure 5E,F</xref>), these results indicate that Shh signaling could increase the cytoplasmic pool of Smurfs. Third, fluorescence resonance energy transfer (FRET) analysis showed that Ptch1-CFP was localized in close proximity with Smurf1-YFP or Smurf2-YFP at punctate intracellular vesicles in MEFs (<xref ref-type="fig" rid="fig7">Figure 7C,D</xref>), and ShhN treatment enhanced this colocalization (<xref ref-type="fig" rid="fig7">Figure 7E</xref>). However, Δ2PY-CFP failed to generate FRET with Smurf2-YFP (<xref ref-type="fig" rid="fig7">Figure 7C,D</xref>). Theses result were further corroborated in the discontinuous sucrose gradient sedimentation experiment described earlier, in which the ligase-deficient Smurf2CG-Myc co-sedimented in the Cav-1-containing 20–25% sucrose fractions readily with Ptch1-FLAG, whereas Δ2PY was inefficient in bringing Smurf2CG-Myc into these fractions (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Finally, using co-immunoprecipitation, we demonstrated that Ptch1 specifically binds either Smurf1 or Smurf2, and Ptch1 mutants lacking either PY-1 or PY-2 motif can still bind Smurfs, albeit with reduced affinity; however, Δ2PY completely lacks affinity for either Smurf1 or Smurf2 (<xref ref-type="fig" rid="fig7">Figure 7F</xref>).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.016</object-id><label>Figure 7.</label><caption><title>Colocalization and interaction between Ptch1 and Smurfs in Cav-1 positive lipid rafts.</title><p>(<bold>A</bold>) Confocal images showing colocalization of GFP-Smurf2CG and Ptch1-RFP in Cav-1 positive lipid rafts. The cells were not permeabilized before they were stained with anti-Cav-1, and the images were taken with a 63x oil lens. (<bold>B</bold>) Quantification of nuclear and cytoplasmic distribution of Smurf2GFP as in <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>. The percentage of mostly nuclear (N > C), even distribution (N = C), or mostly cytoplasmic (N < C) of Smurf2GFP pattern cells was calculated based images of 40 cells at each data point. (<bold>C</bold>) FRET analysis of Ptch1-CFP or Δ2PY-CFP interaction with Smurf1-YFP or Smurf2-YFP in transfected MEFs. Representative images of CFP, YFP, FRET fluorescence, and N-FRET are shown. (<bold>D</bold>) Quantification of N-FRET values using the sensitized emission method, which is expressed as means plus SD in the bar graph. (<bold>E</bold>) FRET analysis of Ptch1-CFP interaction with Smurf2-YFP in transfected MEFs that were treated with ShhN or control conditioned medium for 2 hr. Quantification of N-FRET values described in (<bold>D</bold>). (<bold>F</bold>) Co-immunoprecipitation analyses of FLAG-Ptch1 and the ‘PPXY’ motif mutants with Myc-tagged Smurf1CA or Smurf2CG ligase-deficient mutants. The immunocomplexes were precipitated using anti-FLAG, and blotted with anti-Myc.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.016">http://dx.doi.org/10.7554/eLife.02555.016</ext-link></p></caption><graphic xlink:href="elife02555f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.017</object-id><label>Figure 7—figure supplement 1.</label><caption><title>ShhN treatment and co-expression with Ptch1 caused Smurf2 to redistribute from the nucleus to the cytoplasm.</title><p>Representative fluorescent images showing subcellular localization of Smurf2GFP in MEFs co-transfected with empty vector or Ptch1-RFP and treated without or with ShhN conditioned medium.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.017">http://dx.doi.org/10.7554/eLife.02555.017</ext-link></p></caption><graphic xlink:href="elife02555fs008"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.018</object-id><label>Figure 7—figure supplement 2.</label><caption><title>Neither Smurf1 nor Smurf2 interact with Smo.</title><p>Western analyses of Ptch1-FLAG or SmoA1-FLAG immunoprecipitated with anti-FLAG in HEK293 cells that were also co-transfected with Myc-tagged Smurf1CA or Smurf2CG ligase-deficient mutants. Although Smurf1CA or Smurf2CG was readily detected in the anti-Ptch1-FLAG precipitates, they did not co-precipitate with Smo-FLAG.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.018">http://dx.doi.org/10.7554/eLife.02555.018</ext-link></p></caption><graphic xlink:href="elife02555fs009"/></fig></fig-group></p></sec><sec id="s2-6"><title>Smurfs are required for Ptch1 turnover and ubiquitin modification</title><p>To delineate the requirement of Smurfs for Shh-induced Ptch1 turnover, we took the advantage of the conditional <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs, and quantified the turnover rate of exogenously expressed Ptch1-FLAG following cyclohexamide treatment without or with removal of the <italic>Smurf2</italic> alleles following Ad-cre infection. The results indicated that Ptch1-FLAG was indeed rendered stable against ShhN induced degradation by the removal of the <italic>Smurf2</italic><sup><italic>fl</italic></sup> conditional alleles whereas the stability of Δ2PY was resistant to change in response to either ShhN treatment or eradication of <italic>Smurf</italic>’s function (<xref ref-type="fig" rid="fig8">Figure 8A–D</xref>). The induction by Shh is likely a function of ligand-binding, rather than a signaling outcome, as the loop2 mutant Ptch1 that lacks the ability to bind Shh (<xref ref-type="bibr" rid="bib7">Briscoe et al., 2001</xref>) completely lost the capacity to respond to ShhN treatment in wildtype MEFs, although it was more stable in <italic>Smurf</italic>-null MEFs (<xref ref-type="fig" rid="fig8">Figure 8E,F</xref>). We further found that Shh-induced endocytic turnover of Ptch1 was not affected in <italic>Smo</italic> null MEFs (<xref ref-type="fig" rid="fig8">Figure 8G,H</xref>), suggesting that it is an upstream signaling event, independent of Smo function.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.019</object-id><label>Figure 8.</label><caption><title>Smurfs are required for the Shh-induced endocytic turnover of Ptch1.</title><p>Western analysis of Ptch1-FLAG and Δ2PY-FLAG turnover rates (<bold>A</bold>) and quantification thereof (<bold>B</bold>) in WT MEFs. ShhN and CHX were added for duration as indicated. (<bold>C</bold>) Western analysis of Ptch1-FLAG and Δ2PY-FLAG turnover rates (<bold>C</bold>) and quantification thereof (<bold>D</bold>) in <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs infected with Ad-cre. (<bold>E</bold>) Western analysis of Ptch1-Δloop2-FLAG turnover rate and quantification thereof (<bold>F</bold>) in WT (upper) and <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs infected with Ad-cre (lower). (<bold>G</bold>) Western analysis of Ptch1-FLAG turnover rate and quantification thereof (<bold>H</bold>) in WT (upper) and <italic>Smo</italic><sup><italic>−/−</italic></sup> MEFs (lower). Each data point denotes mean ± standard deviation from two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.019">http://dx.doi.org/10.7554/eLife.02555.019</ext-link></p></caption><graphic xlink:href="elife02555f008"/></fig></p><p>To demonstrate the Ubiquitin E3 ligase activity of Smurfs on Ptch1, we assayed for the ability of Ptch1-FLAG or Δ2PY to be ubiquitinated by HA-tagged Ubiquitin (HA-Ub) in <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs. In these cells, Ptch1-FLAG was readily ubiquitinated, but the level of ubiquitination of Ptch1Δ2PY-FLAG was diminished (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). More importantly, neither of the two forms of Ptch1 was ubiquitinated after the conditional <italic>Smurf2</italic><sup><italic>fl</italic></sup> alleles were removed with Ad-cre, (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). We were also able to demonstrate ubiquitination of Ptch1-FLAG that was produced and isolated from HEK293 cells in an in vitro reconstituted system, in which the level of ubiquitinated species was greatly enhanced by His6-Smurf2, but not the ligase-inactive His6-Smurf2CG purified from the insect expression system (<xref ref-type="fig" rid="fig9">Figure 9B</xref>), indicating a direct enzyme and substrate relationship. Although we were not able to detect mono-ubiquitination, the poly-ubiquitin chains on Ptch1 are likely of both K48 and K63 linkage, as re-expression of Smurf2-Myc in <italic>Smurf2</italic> null cells enhanced Ptch1 ubiquitination in the presence of wt, KO, K48, or K63 ubiquitin (<xref ref-type="fig" rid="fig9">Figure 9C</xref>). Finally, ShhN treatment enhanced the level of high molecular weight ubiquitinated Ptch1 species in wildtype MEFs (<xref ref-type="fig" rid="fig9">Figure 9D</xref>), consistent with the ability of Shh to induce Ptch1 turnover (<xref ref-type="fig" rid="fig8">Figure 8</xref>).<fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.020</object-id><label>Figure 9.</label><caption><title>Smurfs are required for ubiquitin modification of Ptch1.</title><p>(<bold>A</bold>) Western analysis of ubiquitinated Ptch1-FLAG and Ptch1Δ2PY-FLAG in <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs infected with Ad-GFP or Ad-Cre. These MEFs were first infected with adenoviruses and then transfected with HA-Ub and the Ptch1 plasmids as marked. The exogenously expressed Ptch1 proteins were immunoisolated using anti-FLAG beads prior to analysis. (<bold>B</bold>) Western analysis of Ptch1-FLAG ubiquitination in vitro in a reconstituted system comprising purified recombinant His<sub>6</sub>-Smurf2 or the ligase-deficient His<sub>6</sub>-Smurf2CG from the baculovirus, HA-Ub, and an ATP regeneration system. Ptch1-FLAG was immunoisolated from HEK293 cells and the ubiquitination reaction was carried out on beads. The proteins were eluted prior to Western blot analysis. (<bold>C</bold>) Western analysis of ubiquitinated Ptch1-FLAG in Smurf2<sup>−/−</sup> MEFs that were also transfected with Wt, KO, K48, or K63 ubiquitin in the absence or presence of Myc-Smurf2. (<bold>D</bold>) Western analysis of ubiquitinated Ptch1-FLAG in WT MEFs treated with ShhN or control conditioned medium. Ptch1-FLAG in A-C was resolved by 6% SDS-PAGE, but a 4–12% gradient gel was used in <bold>D</bold>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.020">http://dx.doi.org/10.7554/eLife.02555.020</ext-link></p></caption><graphic xlink:href="elife02555f009"/></fig></p></sec><sec id="s2-7"><title>Requirement of Smurfs in sustaining the proliferation of cerebellar granule cell precursors by Shh</title><p>Mice deficient in both <italic>Smurf1</italic> and <italic>Smurf2</italic> were reported embryonic lethal due to absence of planar cell polarity among other pleiotropic defects (<xref ref-type="bibr" rid="bib49">Narimatsu et al., 2009</xref>). More than half of the double null embryos that we generated failed to gastrulate and those rare embryos that did escape seldom passed Theiler stage 13, thus precluding a thorough analysis of the neural tube phenotype where Shh function is well characterized. To address the physiological relevance of Smurf regulation of Ptch1 endocytosis, we examined the role of Smurfs in sustaining the proliferation of cerebellar granule cell precursors (GCPs), which has an absolute requirement for Shh. For this purpose, we cut cerebellar slices from P7 <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> pups and cultured them for 12 days in vitro as described (<xref ref-type="bibr" rid="bib32">Kapfhammer, 2010</xref>). Anti-NeuN immunofluorescence staining revealed that the number of post-mitotic granule cells were severely reduced in slices infected with Ad-cre viruses (<xref ref-type="fig" rid="fig10">Figure 10A</xref>), suggesting that Shh signaling was compromised there. We also isolated GCPs from cerebella of normal P7 pups of the C57/B6 strain, and cultured them in vitro. In the presence of ShhN, GCPs grew healthily for at least 5 days, but siRNA knockdown of <italic>Smurf1</italic> and <italic>Smurf2</italic> simultaneously blocked GCP proliferation (<xref ref-type="fig" rid="fig10">Figures 10B</xref>, <xref ref-type="fig" rid="fig10s1">Figure 10—figure supplement 1A,C</xref>). To ascertain that the effect of Smurf knockdown was Shh-pathway specific, we repeated the above experiment using IGF1, which is capable of sustaining the proliferation of GCPs in lieu of Shh (<xref ref-type="bibr" rid="bib59">Rao et al., 2004</xref>; <xref ref-type="bibr" rid="bib19">Fernandez et al., 2010</xref>), and found that knockdown of <italic>Smurfs</italic> had no effect on IGF-1-induced GCP growth (<xref ref-type="fig" rid="fig10">Figure 10C</xref>, <xref ref-type="fig" rid="fig10s1">Figure 10—figure supplement 1B</xref>). Thus, these data unequivocally demonstrated that Smurf1 and Smurf2 share a critical role in supporting Shh signaling during cerebellar organogenesis.<fig-group><fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.021</object-id><label>Figure 10.</label><caption><title>Requirements of Smurfs for Shh-induced organogenesis.</title><p>(<bold>A</bold>) Immunostaining of P7 cerebellar slices cultured in vitro with anti-calbindin (red) and anti-NeuN (green). The slices were first infected with control or cre-expressing adenoviruses for 24 hr and then continuously cultured for 12 days. Quantification of EdU incorporated GCPs in cerebellar slices cultured in the presence of ShhN from <xref ref-type="fig" rid="fig10s1">Figure 10—figure supplement 1A</xref> (<bold>B</bold>) or IGF-1 from <xref ref-type="fig" rid="fig10s1">Figure 10—figure supplement 1B</xref> (<bold>C</bold>), respectively. The data at each time point were derived from four separate fields, and the bars denote standard deviation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.021">http://dx.doi.org/10.7554/eLife.02555.021</ext-link></p></caption><graphic xlink:href="elife02555f010"/></fig><fig id="fig10s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02555.022</object-id><label>Figure 10—figure supplement 1.</label><caption><title>Smurfs are required for ShhN but not IGF-1 induced GCP proliferation.</title><p>EdU incorporation by GCPs growing in medium containing (<bold>A</bold>) ShhN or (<bold>B</bold>) IGF-1. Freshly isolated GCPs from normal C57/B6 mice were seeded in chamber slides that were coated with poly-D-lysine and Matrigel. The cells were then transfected with non-silencing (NS) control or Smurf1- and Smurf2-specific siRNAs. 12 hr later, Shh-N or IGF-1 conditioned medium was added to the culture and began the time point 0 hr. EdU was given to cells for 12 hr. (<bold>C</bold>) RT-PCR detection of Smurf1 and Smurf2 mRNAs for monitoring the siRNA knockdown efficiency.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.022">http://dx.doi.org/10.7554/eLife.02555.022</ext-link></p></caption><graphic xlink:href="elife02555fs010"/></fig></fig-group></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Shh plays a fundamental role in setting up the body plan during embryogenesis, and is also critical in guiding stem cell differentiation for maintaining tissue homeostasis in the adult. Cell surface reception of Shh signaling is a multistep process that entails, but is not limited to, ligand engagement, reciprocal movements of Ptch1 exiting from and Smo translocating into the primary cilium, and activation of the G-protein-coupled Smo by still-controversial mechanisms (<xref ref-type="bibr" rid="bib52">Ogden et al., 2008</xref>). The central task of this process is to sense and convert incremental changes in the Shh gradient into corresponding levels of signaling output, thereby allowing the positional cues to be executed. In this study, we extended our knowledge of the Shh signaling activation process by revealing a ubiquitination switch that regulates Ptch1 endocytosis, which is essential in clearing Ptch1 from its site of action in the primary cilium, and to ligand sequestration, as previously described (<xref ref-type="bibr" rid="bib29">Incardona et al., 2000</xref>). Our data demonstrate that ubiquitination of Ptch1 mediated by the two ‘PPXY’ motifs is controlled by HECT-domain E3 ligases Smurf1 and Smurf2, which are induced by Shh (<xref ref-type="fig" rid="fig5">Figure 5E,G</xref>) and redistributed into the cytoplasm under Shh influence (<xref ref-type="fig" rid="fig7">Figure 7B</xref>, <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). Shh also promotes the association of Ptch1 and Smurfs in intracellular vesicles (<xref ref-type="fig" rid="fig7">Figure 7E</xref>), most likely the Cav-1 positive lipid rafts (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>), as well as ubiquitination (<xref ref-type="fig" rid="fig9">Figure 9D</xref>) and endosomal entry (<xref ref-type="fig" rid="fig2">Figure 2A–C</xref>), leading to lysosomal turnover (<xref ref-type="fig" rid="fig1 fig8">Figures 1F, 8A–D</xref>). So, an increase in the Shh signal strength would cause a corresponding increase in both the production of Ptch1 and its rate of turnover en route from the primary cilium to the lipid rafts and to the endosomes/lysosomes. This regulatory scheme is reminiscent of an electronic amplification circuit, in which a feedback loop added to an open-loop amplifier has the effect of stabilizing the gain and increasing the linearity of the output signal to a given range, which can be controlled by adjusting the feedback strength. By analogy, Shh induction of Gli1 can be viewed as the open-loop amplifier, with Ptch1 providing the negative feedback. In this wiring logic, the graded Shh morphogenic signal can be stably transformed into stepwise output responses tailored for a predetermined cell fate specification. Without endocytosis, Ptch1 would accumulate in the primary cilium (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>), thus hampering Smo import and function. More importantly, without Ptch1 removal/degradation, the amplitude of Shh signaling would be restricted by the accumulation of newly synthesized inhibitory Ptch1. Oversupplied Ptch1 could also impact on signaling in neighboring cells through non-cell autonomous inhibition. So, Ptch1 endocytosis plays a crucial role in setting the output range of Shh signaling.</p><p>The presence of Ptc in membranous vesicles has long been noted in <italic>Drosophila</italic> and mammalian cells (<xref ref-type="bibr" rid="bib8">Capdevila et al., 1994</xref>; <xref ref-type="bibr" rid="bib17">Denef et al., 2000</xref>; <xref ref-type="bibr" rid="bib58">Ramirez-Weber et al., 2000</xref>; <xref ref-type="bibr" rid="bib80">Zhu et al., 2003</xref>), but its significance was not fully appreciated and regulation unknown. Ptc or Ptch1 is a 12-pass transmembrane protein, whose internal sequence spanning from IV to X transmembrane domains resembles the resistance, nodulation, division (RND) family of bacterial proton-driven transporter and the sterol-sensing domain found in SREBP and NPC1 (<xref ref-type="bibr" rid="bib9">Carstea et al., 1997</xref>; <xref ref-type="bibr" rid="bib71">Taipale et al., 2002</xref>). Substantial evidence in the literature suggests that Ptch1 inhibition of Smo occurs by way of small molecular intermediates that may be transported by Ptch1 through the membrane (<xref ref-type="bibr" rid="bib18">Di Guglielmo et al., 2003</xref>; <xref ref-type="bibr" rid="bib5">Bijlsma et al., 2006</xref>; <xref ref-type="bibr" rid="bib78">Yavari et al., 2010</xref>). Perhaps it is not a coincidence that we found Ptch1 exits the primary cilium and enters the endocytic pathway via cholesterol and sphingomyelin-rich lipid rafts, whereas Smo was shown previously to enter the primary cilium via Clathrin-coated pits when induced by Shh (<xref ref-type="bibr" rid="bib10">Chen et al., 2004</xref>; <xref ref-type="bibr" rid="bib36">Kovacs et al., 2008</xref>). It is possible that Ptch1 and Smo are required to be sorted into different membranous compartments and to keep a mutually exclusive presence in the primary cilium, so that a cross-membrane imbalance of the small molecular intermediates is attained. The RND/sterol-sensing domain is critical to Ptch1 function as multiple inactivating mutations in this region have been found in <italic>Drosophila</italic> as well as in Gorlin syndrome patients (<xref ref-type="bibr" rid="bib43">Martin et al., 2001</xref>; <xref ref-type="bibr" rid="bib70">Strutt et al., 2001</xref>; <xref ref-type="bibr" rid="bib71">Taipale et al., 2002</xref>). However, although certain RND mutants of <italic>Drosophila</italic> Ptc accumulate in endosomes (<xref ref-type="bibr" rid="bib43">Martin et al., 2001</xref>; <xref ref-type="bibr" rid="bib70">Strutt et al., 2001</xref>), this domain may be more important to Ptch1 function than to its endocytic turnover, since we found that combining a RND mutation with the 2-PY deletion did not alter the latter's impact on Ptch1 stability (data not shown).</p><p>Through cDNA-mediated screens, we have identified Smurf1 and Smurf2 as the E3 ligases responsible for generating the sorting signal for Ptch1 endocytosis. Although subsequent experiments indicated that deletion of one <italic>Smurf</italic> gene was not sufficient to inactivate Shh signaling, siRNA-mediated knockdown of either <italic>Smurf1</italic> or <italic>Smurf2</italic> was enough to dampen the 8xGliBS reporter response in transfected MEFs. This apparent discrepancy is likely to be reconciled by the mutual, compensatory upregulation of either of the two <italic>Smurf</italic> genes upon the loss of the other, resulting in the adaptation of single-<italic>Smurf</italic>-knockout MEFs for a robust Shh signaling response. On the other hand, such an adaptive response might not have been established in time under the conditions found in transiently transfected MEFs in response to siRNA-mediated knockdown. The observation of Shh induction of Smurf expression (<xref ref-type="fig" rid="fig5">Figure 5E,G</xref>) and cytoplasmic pivoting (<xref ref-type="fig" rid="fig7">Figure 7B</xref>, <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>) further implicated Smurfs in Shh signaling. Previously, <italic>Drosophila</italic> Ptc was shown to interact with and regulated by Nedd4 (<xref ref-type="bibr" rid="bib42">Lu et al., 2006</xref>), another HECT-domain E3 ligase. In addition, the mouse Ptch1 was also shown to bind Nedd4, but this interaction triggers apoptosis through ubiquitination of Caspase 9 (<xref ref-type="bibr" rid="bib20">Fombonne et al., 2012</xref>). It is likely that Ptch1 is regulated by multiple E3 ligases with different functional outcomes. Recently, <italic>Drosophila</italic> DSmurf was identified as a Ptc-interacting partner in a yeast 2-hybrid screen, and shown subsequently as a specific E3 ligase that regulates Ptc stability (<xref ref-type="bibr" rid="bib26">Huang et al., 2013</xref>). However, DSmurf was shown to promote Ptc turnover in the presence of activated Smo<sup>SD</sup>, bind Smo, and prefer ligand-unbound Ptc as a substrate (<xref ref-type="bibr" rid="bib26">Huang et al., 2013</xref>). We did not observe interaction between mammalian Smurfs and Smo by Co-IP experiments (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2</xref>), and found that Shh induction of Ptch1 turnover proceeded unabatedly even in the absence of Smo (<xref ref-type="fig" rid="fig8">Figure 8G,H</xref>). In Huang et al., when ectopically expressed in the anterior compartment of the wing disc, activated Smo<sup>SD</sup> induced massive amount of Ptc; these two proteins could form a complex at the high levels, much like their mammalian counterparts do when overexpressed in HEK293 cells (<xref ref-type="bibr" rid="bib69">Stone et al., 1996</xref>; <xref ref-type="bibr" rid="bib71">Taipale et al., 2002</xref>). Perhaps, DSmurf could recognize this unnatural complex and triggers a proteasomes-mediated degradation, even specifically.</p><p>Smurf2 was shown previously to function in lipid rafts (<xref ref-type="bibr" rid="bib18">Di Guglielmo et al., 2003</xref>), and the necessity of removing both <italic>Smurf1</italic> and <italic>Smurf2</italic> to reveal their requirement in Shh signaling strongly argues that this shared function has a deep root in evolution. In any event, our work presents a rather comprehensive view of the Shh pathway activation process. Considering two neighboring cells in a given Shh influence field (<xref ref-type="fig" rid="fig11">Figure 11</xref>), the cell that receives lower Shh input (upper cell) encounters a stronger feedback inhibition due to lower endocytic turnover of Ptch1, resulting in a lower level of Shh signaling output represented by Gli1. In the cell that receives higher Shh input (lower cell), although the synthesis of Ptch1 is induced, upregulation of Smurfs and the induction of colocalization in lipid rafts ensure a faster Ptch1 turnover such that the level of Ptch1 feedback inhibition is actually low, resulting in higher pathway activity. The endocytic turnover also has impact on the ligand sequestration role of Ptch1 through controlling the availability of the ligand ‘sink’ on cell surface. In this regard, the Smurf-mediated endocytosis of Ptch1 is an essential signaling event, and it is theoretically possible to block Shh function both cell and non-cell autonomously using Smurf inhibitors, thus opening a new route for Shh-targeted cancer treatment.<fig id="fig11" position="float"><object-id pub-id-type="doi">10.7554/eLife.02555.023</object-id><label>Figure 11.</label><caption><title>A model for the role of Smurf-mediated Ptch endocytosis in Shh signaling.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02555.023">http://dx.doi.org/10.7554/eLife.02555.023</ext-link></p></caption><graphic xlink:href="elife02555f011"/></fig></p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Animals</title><p>All mice were maintained and handled according to protocols approved by the Animal Care and Use Committee of the National Cancer Institute, NIH. The conditional <italic>Smurf2</italic> knockout allele, <italic>Smurf2</italic><sup><italic>fl</italic></sup> was generated by insertion of two loxP sites into introns flanking Exon 9 and 10 through homologous recombination. Further details of the construction will be described elsewhere.</p></sec><sec id="s4-2"><title>Cells, plasmids, and siRNAs</title><p><italic>Smurf1</italic><sup><italic>−/−</italic></sup>, <italic>Smurf2</italic><sup><italic>−/−</italic></sup>, and <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> MEFs were isolated from E14.5 embryos and cell immortalization was carried out according to the 3T3 protocol. NIH3T3:Gli-Luc-3T3 and <italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs were described previously (<xref ref-type="bibr" rid="bib12">Chen et al., 2011</xref>). Full-length mouse Ptch1 cDNA was obtained from ATCC, and the FLAG, GFP, or RFP-tagged variants of which were generated by PCR and subcloned into the pRK5 vector. The ΔPY mutants of Ptch1 were generated using a PCR-based strategy. All PCR-amplified fragments were sequence verified. Plasmids for Myc-tagged Smurf1, Smurf1CA, Smurf2, Smurf2CG, GFP-tagged Smurf2, HA-tagged Ub, UbKO, UbK63 and UbK48 were described previously (<xref ref-type="bibr" rid="bib79">Zhang et al., 2001</xref>; <xref ref-type="bibr" rid="bib76">Yamashita et al., 2005</xref>, <xref ref-type="bibr" rid="bib75">2008</xref>; <xref ref-type="bibr" rid="bib72">Tang et al., 2011</xref>; <xref ref-type="bibr" rid="bib6">Blank et al., 2012</xref>). RFP-tagged Rab5, Rab7, and Lamp1 were acquired from Addgene. siRNAs specific for the mouse HECT family of E3 ligases and cDNAs encoding human HECT E3 ligases were purchased from QIAGEN (Germantown, MD).</p></sec><sec id="s4-3"><title>Immunofluorescence staining</title><p>Approximately 0.6 × 10<sup>5</sup> cells per well were seeded in Lab-Tek chambered slides and cultured for 24 hr. The cells were transfected, allowed to recover for 24 to 36 hr, and then treated with ShhN-CM or other compounds, as indicated. For visualizing ciliary proteins, the transfected cells were starved in DMEM containing 0.5% FBS for 24 hr before other treatments. The cells were fixed with 4% paraformaldehyde for 10 min at 4°C, and standard procedures for immunostaining were followed. The primary antibodies used were rabbit anti-Caveolin-1 (1:1000; Sigma-Aldrich (St. Louis, MO)), rabbit anti-Clathrin heavy chain (1:200; Cell Signaling Technology (Danvers, MA)), rabbit anti-Rab5 (1:150, Cell Signaling Technology), rabbit anti-Rab7 (1:50, Cell Signaling Technology), rabbit anti-Lamp1 (1:150; Sigma), mouse anti-acetylated Tubulin (1:2000; Sigma), rabbit anti-Gli3 (1:500; R&D (Minneapolis, MN)), and rabbit anti-Smo (1:500; a gift from Dr Rajat Rohatgi). Alexa-coupled secondary antibodies were purchased from Life Technologies Corp.</p></sec><sec id="s4-4"><title>Confocal microscopy and FRET</title><p>Confocal images were acquired on a Carl Zeiss LSM710 microscope. Colocalization coefficient was calculated using Zeiss ZEN2011 program, and quantification of the fluorescence intensity of Ptch1-GFP, Smo, and Gli3 in primary cilia was carried out using Image-Pro as described previously (<xref ref-type="bibr" rid="bib12">Chen et al., 2011</xref>). For FRET analysis, MEFs were transfected with the plasmids encoding Ptch1-CFP or Δ2PY-CFP together with Smurf1-YFP or Smurf2-YFP. Confocal images were acquired with a 40 × objective lens. In track I, cells were excited with a 405-nm laser, and CFP signals were collected in channel II at 470–500 nm. FRET signals were collected in channel III at >530 nm. In track II, cells were excited with a 514-nm laser line, and YFP signals were collected in channel III at >530 nm. FRET efficiency between CFP and YFP, shown as N-FRET, was calculated using Zeiss ZEN2011 program, and the sensitized emission crosstalk coefficients were determined using control cells that expressed only CFP or YFP.</p></sec><sec id="s4-5"><title>GliBS-luc reporter assay for non-cell autonomous inhibition of Ptch1</title><p><italic>Ptch1</italic><sup><italic>−/−</italic></sup> MEFs were transfected with Ptch1-GFP or Ptch1Δ2PY-GFP along with the Rellina control (15:1) using Lipofectamine Plus (Life technologies, Grand Island, NY)). These cells were then re-seeded with NIH3T3:GliBS-luc reporter cells at 5:1 ratio. After 24 hr, the cells were treated with ShhN-CM in different dilutions for additional 36 hr before the luciferase activities were assayed using the luciferase assay system on a GloMax-96 luminometer (Promega, Madison, WI). The firefly luciferase activity from the indicator cells was normalized against the Rellina luciferase activity to correct for transfection efficiency of Ptch1 constructs in the testing <italic>Ptch</italic><sup><italic>−/−</italic></sup> MEFs as the measurement of non-cell autonomous inhibition by Ptch1.</p></sec><sec id="s4-6"><title>Immunoprecipitation and immunoblotting</title><p>Transfected cells were lysed in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 1% n-Dodecyl β-D-maltoside, 0.25% wt/vol sodium deoxycholate, 1 mM DTT, and 1 × Roche cOmplete Protease Inhibitor Cocktail) for 1 hr at 4°C. The lysate was clarified by centrifugation for 1 hr at 20,000×<italic>g</italic>. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 6 × SDS loading buffer, incubated at room temperature for 1 hr, subjected to SDS-PAGE, followed by western blot analysis. To assay for interactions between exogenous Ptch1-FLAG and the Myc-Smurfs, transfected Ptch1-FLAG was immunopurified with anti-FLAG M2 agarose beads (Sigma) and subjected to SDS-PAGE, followed by western blotting with anti-Myc (Santa Cruz Biotechnology, Dallas, TX).</p></sec><sec id="s4-7"><title>Ubiquitination assays</title><p>To assay for Ptch1 ubiquitination in vivo, <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>/Smurf2</italic><sup><italic>flox/flox</italic></sup> MEFs were infected with either Ad-GFP or Ad-Cre adenovirus for 24 hr, then transfected with Ptch1-FLAG or Ptch1Δ2PY-FLAG along with HA-Ub using Lipofectamine Plus (Invitrogen). The cells were lysed 24 hr later and Ptch1 and its mutant were isolated with anti-FLAG agarose beads and resolved by SDS-PAGE on 6% or 4–12% gradient gels. The ubiquitinated Ptch1 was then detected with anti-HA (Roche-Shanghai, China). To assay for Ptch ubiquitination in vitro, an ubiquitination assay was modified from a previously described procedure (<xref ref-type="bibr" rid="bib72">Tang et al., 2011</xref>). Ptch1-FLAG was captured from transfected HEK293 cell lysates using anti-FLAG agarose. After a thorough wash, the Ptch1-bound agarose was divided into three aliquots. Empty anti-FLAG agarose was used as a control. The in vitro ubiquitination assay was performed by incubating either Ptch1-bound agarose or control agarose at 37°C for 1 hr with ubiquitin-activating enzyme UBE1, E2-conjugating enzyme UbcH5c, HA-Ub and ATP (all from Boston Biochem, Cambridge, MA) in the presence or absence of purified His6-Smurf2 or His6-Smurf2CG. After the incubation, the supernatant was removed, the agarose thoroughly washed, and the Ptch1-FLAG eluted using the FLAG peptide (Sigma). The eluted fraction was then subjected to Western blot analysis.</p></sec><sec id="s4-8"><title>Sucrose gradient sedimentation</title><p>Sucrose equilibrium density gradient sedimentation experiments were performed as described (<xref ref-type="bibr" rid="bib16">Coulombe et al., 2004</xref>). Briefly, HEK293 cells grown in 10 cm plates were transiently transfected with Ptch1-FLAG or Δ2PY-FLAG along with Myc-Smurf2CG. 48 hr after transfection, the cells were lysed in pre-chilled 2 ml MES buffer, which contains 25 mM MES (2-[N-morpholino]ethanesulfonic acid), pH 6.5, 150 mM NaCl, 1% Triton X-100, supplemented with 1 × Roche cOmplete Protease Inhibitor Cocktail and was set on ice for 1 hr. The lysates were mixed with equal volume of 80% (wt/vol) sucrose/MES solution and placed at the bottom of an ultracentrifuge tube. Tube was then overlaid in consecutive order with 2 ml each of 30%, 25%, 20%, and 4 ml of a 5% (wt/vol) sucrose/MES buffer. After centrifugation at 39,000 rpm for 16 hr at 4°C in an SW 41 Ti rotor on Beckman Optima L-100 XP ultracentrifuge, the gradient was separated into twelve 1 ml fractions taken from the top for Western blot analysis.</p></sec><sec id="s4-9"><title>RT-PCR and quantitative real-time PCR</title><p>Total RNA was isolated from cultured cells using the RNAiso reagent (TaKaRa, Shiga, Japan), and reverse transcription was carried out using the PrimeScript RT reagent Kit (TaKaRa). Standard RT-PCR was carried out with the following primers: mouse Gli1 (5′-TCCAGCTTGGATGAAGGACCTTGT-3′ and 5′-AGCATATCTGGCACGGAGCATGTA-3′), mouse Smurf1 (5′-CTACCAGCGTTTGGATCTAT-3′ and 5′-TTCATGATGTGGTGAAGCCG-3′), mouse Smurf2 (5′-TAAGTCTTCAGTCCAGAGACC-3′ and 5′-AATCTCTTCCCTAGACACCTC-3′), and mouse HPRT (5′-TATGGACAGGACTGAAAGAC-3′ and 5′-TAATCCAGCAGGTCAGCAAA-3′). Real-time PCR was carried out using the FastStart SYBR Green Master mix (Roche) on a 7500 Real-Time PCR System (Applied Biosystems, Grand island, NY) with primers for mouse Gli1 (5′-GCTTGGATGAAGGACCTTGTG-3′ and 5′-GCTGATCCAGCCTAAGGTTCTC-3′) and mouse HPRT (5′-TATGGACAGGACTGAAAGAC-3′ and 5′-TAATCCAGCAGGTCAGCAAA-3′). Experiments were repeated at least three times, and samples were analyzed in triplicate.</p></sec><sec id="s4-10"><title>Cerebellar slice culture</title><p>Cerebellar slice cultures were prepared as described (<xref ref-type="bibr" rid="bib32">Kapfhammer, 2010</xref>). Briefly, sagittal sections (350 µm) were cut from cerebella of P7 <italic>Smurf1</italic><sup><italic>−/−</italic></sup><italic>;Smurf2</italic><sup><italic>fl/fl</italic></sup> pups using a McIlwain tissue cutter under septic condition. Slices were transferred onto a permeable membrane (Millicell-CM, Millipore-China, Beijing, China) in a 6-well plate with 0.8 ml of culture medium (Neurobasal A medium with B27 supplement) and incubated at 37°C, 5% CO2. For adenovirus infection, the viral stock (3 × 10<sup>10</sup> pfu/ml) was mixed with equal volume of type I collagen gel and applied as a drop on top of each slice, and 5 × 10<sup>7</sup> pfu of virus was also added in the culture medium. After 24 hr, the infected slices were washed and maintained in culture medium. The medium was changed every 2–3 days for a total of 12 days. Slices were then fixed in 4% paraformaldehyde overnight at 4°C and immunostained with anti-calbindin (1:500; Sigma) and anti-NeuN (1:100; Millipore).</p></sec><sec id="s4-11"><title>GCP isolation and proliferation assay</title><p>Mouse cerebellar GCPs were isolated from 7-day-old pups according to a published protocol (<xref ref-type="bibr" rid="bib22">Hatten and Shelanski, 1988</xref>). Briefly, cerebella were removed aseptically and incubated at 37°C for 5 min in trypsin/DNase buffer. Tissues were then triturated with fine Pasteur pipettes to obtain a single-cell suspension, overlaid on top of a step gradient of 35% and 65% Percoll (Pharmacia, GE Health-China, Shanghai, China) and centrifuged at 2,000×<italic>g</italic> for 10 min at 4°C. GCPs harvested from the 35% and 65% Percoll interface were further purified by depleting adherent cells with two consecutive 1-hr incubations in tissue culture dishes, then seeding them in Lab-Tek chambered slides coated with poly-D-lysine and Matrigel, and incubating them at 35°C, 5% CO<sub>2</sub>. GCPs were transfected with siRNAs using FugeneHD Transfection Reagent (Promega) after 1 hr incubation. Proliferation of transfected GCPs was evaluated using Click-iT EdU cell proliferation assays (Life Technologies) at different time points after ShhN-CM or IGF1 (100 ng/ml) treatment. GCPs were incubated with EdU (5-ethynyl-2′-deoxyuridine) for 12 hr before fixation and permeabilization. EdU detection was performed according to the manufacturer's instruction. Images were acquired on a Leica inverted fluorescence microscope (DMI 300B) with a 20 × objective lens. Quantification of EdU-positive GCPs was performed using the ImageJ software.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We wish to thank Rajat Rohatgi for the generous gift of the Smoothened antibody, and Tian Jin, Joseph Brzostowski and Valarie Barr for their assistance with confocal imaging. This work was supported by funding from the US-China Biomedical Collaborative Research program to SYC and YEZ; grants from the Chinese National Science foundation (81272238 and 81261120386) and the National Basic Research Program of China (973 Program) to SYC (2012CB945003 and 2009CB918403); and by funding from the intramural research program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research to YEZ. SY is supported by a young investigator grant from the Chinese National Science Foundation (81101497).</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>SY, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>L-YT, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>YT, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>Q-HS, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>JD, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>YC, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>ZZ, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>YT, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con9"><p>T-TY, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>YEZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con11"><p>SYC, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: All mice were maintained and handled according to protocols (ASP 13-214) approved by 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editor</role><aff><institution>Stowers Institute for Medical Research</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Requirement of Smurf-mediated endocytosis of Patched1 in Sonic Hedgehog signal reception” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Robb Krumlauf (Reviewing editor); Jin Jiang and Ben Allen (peer reviewers).</p><p>The Reviewing editor (Robb Krumlauf) and the other reviewers (Jin Jiang, Ben Allen, and a third anonymous reviewer) discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The consensus view of all of the reviewers is that the work is potentially of significant interest and could represent an important advance in the field. However, each reviewer has substantial concerns that would need to be addressed before publication of the paper could be considered. This involves additional experimentation and major revisions to the text. There are also issues raised over interpretation of data and missing key citations. Under normal circumstances requests for such substantial revisions would lead to a decision to reject the paper, but in this case because the reviewers would like to see the paper published if their concerns are met we wish to offer the opportunity for a revision.</p><p>To aid the revision process in this case we provide the specific comments of all three reviewers.</p><p><italic>Reviewer #1</italic>:</p><p>In this manuscript, Yue et al investigated the role of Smurf-mediated Ptch1 ubiquitination in the regulation of Shh signaling. They provided evidence that Shh promotes Ptch1 enrichment in the lipid rafts and that the PPXY sorting signals (PY motifs) in Ptch1 promotes endocytosis and degradation of Ptch1. They further showed that the PY motifs are required for Shh-induced ciliary exit of Ptch1 and optimal Hh pathway activation. They identified Smurf 1 and 2 as two E3 critical ligases that promote Ptch1 ubiquitination and degradation through the PY motifs. Interestingly, they found that the expression of Smurf1/2 is upregulated in response to Shh. By using FRET and CoIP, they provided evidence that Smurf and Ptch1 physically interact depending on the PY motifs. Finally, they showed that genetic ablation of Smurf1 and 2 specifically affected Shh-induced proliferation of GCPs. Overall, the experiments were well executed and the data are convincing. The work is complementary to a recent publication that mainly described a role of Smurf in targeting Drosophila Ptc (Huang et al., PLOS Bio 2013), and represents an important advance in the field, allowing one to compare and contrast the fly and mammalian systems. However, the authors should address the following concerns either by discussion or by additional experiments before publication is recommended.</p><p>1) Whereas the evidence for Smurf/PY-mediated Ptch1 endocytosis and degradation is strong, it is not so clear how this process is promoted by Shh. Although Shh-induced upregulation of Smurf could contribute, other mechanisms may exist. Have the authors examined whether Shh promotes the binding of Smurf to Ptch1? For example, does Shh treatment increase the FRET between GFP-Smurf2CG and Ptch1-RFP shown in <xref ref-type="fig" rid="fig9">Figure 9A-C</xref>? On the other hand, Smurf-mediated degradation of Ptch1 could be Shh independent, as suggested by Casali (Science Signaling, 2010). For example, Ptch1Δ2, which has the Shh binding domain deleted (Briscoe et al., Mol Cell 2001), might still be regulated by Smurf/PY. Furthermore, Huang et al suggested that Smurf prefers degrading ligand-unbound Ptc (Huang et al., PLOS Bio 2013). How could the authors reconcile their finding that Shh promotes Ptch1 degradation? Could they examine whether Ptch1Δ2 is degraded more or less effectively by Smurf than Ptch1 in the presence of Shh?</p><p>2) Huang et al argued that Smurf-mediated ubiquitination and degradation of Ptc are promoted by activated forms of Smo (Smo<sup>SD</sup>) in Drosophila (Huang et al., PLOS Bio 2013). Have the authors examined whether Shh promotes Ptch1 degradation through Smo? For example, does overexpression of mammalian Smo<sup>SD</sup> promote Smurf-mediated ubiquitination/degradation of Ptch1?</p><p>3) The authors showed that mutating the PY motifs or Smurf1/2 affected both Ptch1 ciliary exit and endocytosis. Is the failure of Ptch1 ciliary exit the result of defective endocytosis? Or could Shh induce Ptch1 ubiquitination in the primary cilium, which may directly regulate ciliary exit of Ptch1? Is there any evidence that Smurf1/2 can be found in the primary cilium with or without Shh treatment? In <xref ref-type="fig" rid="fig6">Figure 6C</xref>, can Shh trigger ciliary exit of Ptch1 in the absence of Smurf1/2? Does pharmacological blockage of Ptch1 endocytosis/degradation affect Ptch1 ciliary exit?</p><p>4) The effect of Δ2PY-GFP on Smo ciliary localization presented in <xref ref-type="fig" rid="fig4">Figure 4</xref> does not match the quantification well, especially at 4 hours after Shh treatment where there is almost no difference in the ciliary Smo levels between Δ2PY-GFP and Ptch1-GFP (<xref ref-type="fig" rid="fig4">Figure 4C</xref>) while there is a 2-told difference in the quantification (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). The authors need to provide a better image reflecting the quantification. Of note, it has been shown that Shh/Ptch1 regulates both the ciliary localization and conformation of Smo (Zhao et al., nature 2007). Have the authors examined whether Δ2PY-GFP affect mSmo conformation using FRET analysis?</p><p><italic>Reviewer #2</italic>:</p><p>In this manuscript Yue et al. uncover a role for the Hect E3 ligases Smurf1 and Smurf2 in promoting Hedgehog-dependent changes in the subcellular localization of the Patched receptor that leads to their increased turnover. This Smurf1/2-mediated trafficking of Patched is also important for its exit from primary cilia during pathway activation. Using cultured cells including MEFs knockout for Smurf1&2, the authors show that this trafficking event is, in turn, important for the ciliary accumulation of Smoothened and for the activation of Gli-mediated transcription. The authors show that the Shh-promoted proliferation of granule cell progenitors requires Smurf1 and Smurf2, suggesting an important function of this regulatory mechanism in a well-characterized physiological context dependent on Hedgehog ligands.</p><p>This is an interesting manuscript that adds to our understanding of the molecular mechanisms underlying Hedgehog signaling. In particular, although it has been speculated that endocytic trafficking may be implicated in Patched and Smoothened localization, ciliary accumulation and signaling, the molecular mechanisms implicated in this process are poorly defined.</p><p>The strongest aspects of this manuscript are the loss of function experiments conducted with the Sf1-/-,Sf2fl/fl MEFs and GPCs. Indeed, the complete absence of Sf1 and Sf2 leads to a remarkable inhibition of Smo and Gli3 ciliary localization and blunting of Shh-promoted induction of Gli1 levels in MEFs. These results are strongly supported by the experiments in <xref ref-type="fig" rid="fig11">Figure 11</xref> showing a reduction of neural progenitors in cerebellar slice cultures knockout for Sf1 and Sf2 and an inability of Shh to promote the in vitro proliferation of granule cell progenitors when Sf1 and Sf2 are knocked out. These experiments strongly support an important functional requirement of Smurf proteins for Hedgehog signal transduction.</p><p>In terms of mechanisms describing the function of Smurf proteins, the evidence presented in the manuscript are however disappointing in that they are too often not convincing, confusing or incomplete. For example, according to their model, Hedgehog ligands are shown to promote the localization of Patched in caveolae, a transitory localization that promote the Smurf dependent ubiquitination of Patched and its endosomal routing to the lysosomes where it is degraded. First of all, although scattered evidence suggests that caveosomes and endosomes may physically interact in specific contexts, the authors present their evidence supporting a role of Rab proteins and endosomal trafficking in promoting Patched exit from caveolae as a well defined and accepted mechanism. However, caveolae-mediated endocytosis is most often described to be separate from endosomal sorting. Although this could represent a novel sorting mechanism for cell surface receptors, the characterization of this process needs to be strengthened and better discussed.</p><p>Moreover, all of the evidence supporting the localization of Ptch in different subcellular fraction relies on overexpression experiments and on colocalization with overexpressed markers tagged with fluorescent proteins (especially important for Rab7). These experiments should be repeated using endogenous proteins and images obtained at higher resolution to more precisely follow the fate of Ptch trafficking and more convincingly support the implication of caveolae and/or endosomal trafficking.</p><p>In addition in my opinion the biggest question that is left unanswered is how ubiquitination of Patched by Smurf proteins contributes to its function. Do Smurfs lead to Patched mono-ubiquitination or to K63 or K48 ubiquitin chain conjugations? Is ubiquitination involved in Patched endocytosis per se or in its sorting from endosomes to lysosomes? Does Hedgehog ligand promote the interaction of Patched with Smurfs? Do Hedgehog ligands promote Patched ubiquitination?</p><p>There also seems to be a disconnection between the results obtained using the Ptch-d2PY mutant (when rescuing the Ptch1-/- MEFs) and the results obtained in the Sf1, Sf2 double KO cells. Indeed, whereas the Shh-promoted accumulation of Smo and Gli1 activation are blunted in the dKO cells, Smo accumulation is only reduced when the d2PY mutant is expressed (4C,D). Since the interaction between the d2PY mutant and Smurf proteins seems to be completely abolished (9E) how is this explained? If there is more Ptch1-d2PY in cilia, why do Smo enters at all?</p><p><italic>Reviewer #3</italic>:</p><p>In the manuscript entitled “Requirement of Smurf-Mediated Endocytosis of Patched 1 in Sonic Hedgehog Signal Reception”, Yu et al. present evidence that Smurf1 and Smurf2 promote ubiquitination of PTCH1 resulting in endocytic turnover that is required for HH pathway activation. In particular, the authors provide significant experimental data examining the subcellular localization of PTCH1 and the role of two PPXY motifs in regulating PTCH1 localization turnover, and downstream effects on HH pathway function. While, overall the results appear to be of high quality, there are some issues with both interpretation of the data and proper acknowledgement of previous work that the authors must address.</p><p>Major comments:</p><p>1) There is an unfortunate lack of proper citation of previous work by other labs in this field. Two essential examples include the recent publication of work identifying a role for Smurfs in regulating Drosophila Ptc turnover (Huang et al., PLOS Biology, 2013), and work from Tom Kornberg that defined a role for the PPXY motif in regulating the turnover of vertebrate PTCH1 (Kawamura et al., JBC, 2008). These two papers directly impact the current study by Yue et al., and this work should be considered in the context of these previous studies.</p><p>2) In <xref ref-type="fig" rid="fig5">Figure 5</xref>, the authors utilize Ptch1-/- MEFs to address differences in the ability of PTCH1 and PTCH1Δ2PY to promote ligand-dependent signaling. However, the authors miss an opportunity to distinguish between the ligand-dependent and ligand-independent effects of PTCH1 in the HH pathway. They should use these cells and constructs to examine the ability of PTCH1 or PTCH1Δ2PY to antagonize SMO in the absence of ligand. That is, Ptch1-/- MEFs display constitutive HH pathway activation; however, re-expressing PTCH1 rescues this pathway activity. The question is whether PTCH1Δ2PY is equally effective? Do the authors observe equivalent antagonism of SMO in these cells? Or is PTCH1Δ2PY a more effective antagonist of SMO than wt PTCH1? These are straightforward questions to address since the authors have all the necessary tools and reagents in hand.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02555.025</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>Reviewer #1:</p><p><italic>In this manuscript, Yue et al investigated the role of Smurf-mediated Ptch1 ubiquitination in the regulation of Shh signaling. […] However, the authors should address the following concerns either by discussion or by additional experiments before publication is recommended</italic>.</p><p><italic>1) Whereas the evidence for Smurf/PY-mediated Ptch1 endocytosis and degradation is strong, it is not so clear how this process is promoted by Shh. Although Shh-induced upregulation of Smurf could contribute, other mechanisms may exist. Have the authors examined whether Shh promotes the binding of Smurf to Ptch1? For example, does Shh treatment increase the FRET between GFP-Smurf2CG and Ptch1-RFP shown in</italic> <xref ref-type="fig" rid="fig9"><italic>Figure 9A-C</italic></xref><italic>? On the other hand, Smurf-mediated degradation of Ptch1 could be Shh independent, as suggested by Casali (Science Signaling, 2010). For example, Ptch1Δ2, which has the Shh binding domain deleted (Briscoe et al., Mol Cell 2001), might still be regulated by Smurf/PY. Furthermore, Huang et al suggested that Smurf prefers degrading ligand-unbound Ptc (Huang et al., PLOS Bio 2013). How could the authors reconcile their finding that Shh promotes Ptch1 degradation? Could they examine whether Ptch1Δ2 is degraded more or less effectively by Smurf than Ptch1 in the presence of Shh?</italic></p><p>We thank this reviewer for raising these very important issues. Our previous and new data indicate that Shh promotes the Smurf-mediated endocytosis of Ptch1 in several ways. First, Smurfs are preferentially localized in the nucleus in normal cells (Kavsak et al., Mol Cell 6:1365-75, 2000) and play important roles in maintaining the genomic stability (Blank et al, Nature Medicine 18:227-34, 2012). In the revised manuscript, we show that Shh promotes a re-pivoting of Smurf2 from the nucleus to the cytoplasm (<xref ref-type="fig" rid="fig7">Figure 7B</xref>, and <xref ref-type="fig" rid="fig7s1">Figure 7–figure supplement 1</xref>). Second, our data also show that Shh induces Smurfs expression (<xref ref-type="fig" rid="fig5">Figure 5E, 5G</xref>). So, these two events should lead to an increase of the effective cytoplasmic concentration of Smurfs. Third, as requested, we conducted a new FRET experiment and found that Shh indeed promotes the colocalization of Ptch1 and Smurf2 (<xref ref-type="fig" rid="fig7">Figure 7E</xref>). Fourth, we further add new data showing that ShhN treatment enhances the ubiquitin modification of Ptch1 (<xref ref-type="fig" rid="fig9">Figure 9D</xref>), consistent with our data showing that Shh promotes Ptch1 turnover (<xref ref-type="fig" rid="fig8">Figure 8</xref>).</p><p>In Huang et al, the authors ectopically expressed activated Smo mutants, Smo<sup>SD</sup>, in the entire A-compartment, which drastically increased the level of Ptc (Huang et al, <xref ref-type="fig" rid="fig4">Figure 4D</xref>). They argue that DSmurf prefers the ligand-unbound Ptc as a substrate because ectopic expression of DSmurf reduced Ptc staining selectively in the A compartment. However, comparing their <xref ref-type="fig" rid="fig4">Figure 4D and 4E</xref>, one could find that the intensity of Ptc staining at the A/P boundary was also reduced by DSmurf, notwithstanding the fact that Ptc is normally high at the boundary. On the other hand, since the authors did not examine the distribution of Hh in the disc that received the ectopically expressed Smo<sup>SD</sup>, it would be an unsupported assumption that the elevated Ptc in the A compartment was still in the unbound form. After all, the Hh ligand is normally restricted to the compartmental border by the high level of Ptc there. If the border stripe of high level Ptc was made to expand, Hh zone should expand with it. Furthermore, it is well known in the field that Ptc and Smo, when over-expressed, tend to form a nonphysiological complex (Stone et al, Nature 384:129, 1996, and Taipale et al, Nature 418:892, 2002).This raises a possibility that the nonphysiological Ptc-Smo complex could trigger an “unfolded protein response” of some sort that leads to the DSmurf-mediated destruction. This type of degradation is very different from the one that we describe in our manuscript, although both could be mediated by the Smurf E3 ubiquitin ligases, even specifically.</p><p>Notwithstanding the above analysis, assuming DSmurf does prefer the ligand unbound form of Ptc for degradation, this would put the site of DSmurf action in the A compartment, where Ptc level is low and Smo is in an inactive state. However, their data indicated that Smo has to be activated in order to promote Ptc degradation. In Huang et al, there is no data that either indicate or imply the source of the activated Smo for activating the Smurf-mediated Ptc turnover or to explain this conspicuous conflict.</p><p>We measured the turnover rate of the loop2 mutant of Ptch1 in wt MEFs, and found that the effect of Shh ligand induction was abolished (<xref ref-type="fig" rid="fig8">Figure 8E, 8F</xref>). We further quantified the turnover rate of Ptch1 in Smo<sup>null</sup> cells, and found that Shh still promotes Ptch1 turnover there (<xref ref-type="fig" rid="fig8">Fig.8G, 8H</xref>). Moreover, we did not detect interaction between Smo and Smurfs by co-IP experiments, even though Smurf was shown to bind Ptch1 readily (<xref ref-type="fig" rid="fig7s2">Figure7–figure supplement 2</xref>). So, these results demonstrate that Smurfs likely promote Ptch1 endocytic turnover through direct binding, rather than using Smo as an intermediate, as suggested by Huang et al. However, Smo probably still has a long term feedback role through enhancing downstream Smurf gene expression.</p><p><italic>2) Huang et al argued that Smurf-mediated ubiquitination and degradation of Ptc are promoted by activated forms of Smo (Smo</italic><sup><italic>SD</italic></sup><italic>) in Drosophila (Huang et al., PLOS Bio 2013). Have the authors examined whether Shh promotes Ptch1 degradation through Smo? For example, does overexpression of mammalian Smo</italic><sup><italic>SD</italic></sup> <italic>promote Smurf-mediated ubiquitination/degradation of Ptch1?</italic></p><p>As stated above, we examined Ptch1 turnover in Smo<sup>null</sup> cells, and found that Shh still promotes Ptch1 turnover. We also found by Co-IP experiment that Ptch1 binds Smurf but Smo does not (<xref ref-type="fig" rid="fig7s2">Figure 7-figure supplement 2</xref>). These data strongly argue that Shh-induced, Smurfs-mediated Ptch1 endocytic turnover is independent of Smo.</p><p><italic>3) The authors showed that mutating the PY motifs or Smurf1/2 affected both Ptch1 ciliary exit and endocytosis. Is the failure of Ptch1 ciliary exit the result of defective endocytosis? Or could Shh induce Ptch1 ubiquitination in the primary cilium, which may directly regulate ciliary exit of Ptch1? Is there any evidence that Smurf1/2 can be found in the primary cilium with or without Shh treatment? In</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6C</italic></xref><italic>, can Shh trigger ciliary exit of Ptch1 in the absence of Smurf1/2? Does pharmacological blockage of Ptch1 endocytosis/degradation affect Ptch1 ciliary exit?</italic></p><p>It is our interpretation that Ptch1Δ2PY fails to exit cilia because of defective endocytosis. Despite an initial hypothesis, we found neither endogenous nor transfected Smurfs in the cilia with or without Shh treatment. Our data also show that the Shh-induced ciliary export of Ptch1 was compromised when Smurf1 and Smurf2 were knocked down with siRNAs (<xref ref-type="fig" rid="fig5s1">Figure 5–figure supplement 1</xref>). We further show that blocking Ptch1 endocytosis with Leupeptin also blocked its ciliary exit (this result were not included in the previous submission, but is now added as <xref ref-type="fig" rid="fig3s1">Figure 3–figure supplement 1</xref> in the revised manuscript).</p><p><italic>4) The effect of Δ2PY-GFP on Smo ciliary localization presented in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref> <italic>does not match the quantification well, especially at 4 hours after Shh treatment where there is almost no difference in the ciliary Smo levels between Δ2PY-GFP and Ptch1-GFP (</italic><xref ref-type="fig" rid="fig4"><italic>Figure 4C</italic></xref><italic>) while there is a 2-told difference in the quantification (</italic><xref ref-type="fig" rid="fig4"><italic>Figure 4D</italic></xref><italic>). The authors need to provide a better image reflecting the quantification. Of note, it has been shown that Shh/Ptch1 regulates both the ciliary localization and conformation of Smo (Zhao et al., nature 2007). Have the authors examined whether Δ2PY-GFP affect mSmo conformation using FRET analysis?</italic></p><p>We replaced the images in the old <xref ref-type="fig" rid="fig4">Figure 4C</xref> with better ones in the revision (new <xref ref-type="fig" rid="fig3">Figure 3C</xref>). By using a sophisticated FRET imaging approach, Zhao et al elegantly demonstrated that Hh induces phosphorylation and a conformational change of Smo c-tail that result in Smo dimerization and activation of downstream signaling. Their work also extended this observation to mammalian Smo. However, this regulation, albeit a likely key event in the Shh pathway activation, lies downstream to Ptch1 functions. Since we have demonstrated that Shh-induced Ptch1 endocytic turnover is independent of Smo, and analyzed extensively the ciliary trafficking of Smo, another well recognized key event of the Shh pathway activation, we felt that examining Δ2PY-GFP on mSmo conformation would be a repetition of an already well-addressed issue. In addition, setting up the FRET experiment on Smo conformation would not be a trivial endeavor, if one needs to do it properly. If this reviewer and the editors deem this FRET experiment absolutely essential, which we would respectfully disagree, we will perform as demanded, provided that we are granted additional time.</p><p>Reviewer #2:</p><p><italic>In this manuscript Yue et al. uncover a role for the Hect E3 ligases Smurf1 and Smurf2 in promoting Hedgehog-dependent changes in the subcellular localization of the Patched receptor that leads to their increased turnover</italic>. <italic>[…]</italic></p><p><italic>The strongest aspects of this manuscript are the loss of function experiments conducted with the Sf1-/-,Sf2fl/fl MEFs and GPCs. Indeed, the complete absence of Sf1 and Sf2 leads to a remarkable inhibition of Smo and Gli3 ciliary localization and blunting of Shh-promoted induction of Gli1 levels in MEFs. These results are strongly supported by the experiments in</italic> <xref ref-type="fig" rid="fig11"><italic>Figure 11</italic></xref> <italic>showing a reduction of neural progenitors in cerebellar slice cultures knockout for Sf1 and Sf2 and an inability of Shh to promote the in vitro proliferation of granule cell progenitors when Sf1 and Sf2 are knocked out. These experiments strongly support an important functional requirement of Smurf proteins for Hedgehog signal transduction</italic>.</p><p><italic>In terms of mechanisms describing the function of Smurf proteins, the evidence presented in the manuscript are however disappointing in that they are too often not convincing, confusing or incomplete. For example, according to their model, Hedgehog ligands are shown to promote the localization of Patched in caveolae, a transitory localization that promote the Smurf dependent ubiquitination of Patched and its endosomal routing to the lysosomes where it is degraded. First of all, although scattered evidence suggests that caveosomes and endosomes may physically interact in specific contexts, the authors present their evidence supporting a role of Rab proteins and endosomal trafficking in promoting Patched exit from caveolae as a well defined and accepted mechanism. However, caveolae-mediated endocytosis is most often described to be separate from endosomal sorting. Although this could represent a novel sorting mechanism for cell surface receptors, the characterization of this process needs to be strengthened and better discussed</italic>.</p><p>We agree with this reviewer that caveolae was a recently recognized alternative route for internalization of membrane-bound ligand-receptor complexes, but this phenomenon was actually noted more than two decades ago. At that time, a term of “potocytosis” was coined to distinguish it from the Clathrin-mediated endocytosis (Anderson RG, Science 255:410-1, 1992; Gleizes PE, Eur. J. Cell Biology 71:144-53, 1996), because the cargo of potocytosis was thought to be emptied directly into the cytosol. Later studies demonstrated that caveolae-mediated internalization actually feeds into the conventional endocytic pathway, and “caveosomes”, which were previously regarded as independent organelles distinct from endosomes, were actually late endosomes modified by the accumulated Caveolin-1 therein (Hayer et al, J Cell Biol 191:615-29, 2010; Sandvig et al, Curr Opin Cell Biol 23:413-420, 2011). To clarify this issue, we made modifications in the Introduction and cited several key references.</p><p><italic>Moreover, all of the evidence supporting the localization of Ptch in different subcellular fraction relies on overexpression experiments and on colocalization with overexpressed markers tagged with fluorescent proteins (especially important for Rab7). These experiments should be repeated using endogenous proteins and images obtained at higher resolution to more precisely follow the fate of Ptch trafficking and more convincingly support the implication of caveolae and/or endosomal trafficking</italic>.</p><p>Antibodies again mouse Ptch1 are not commercially available, precluding a direct visualization of the endogenous Ptch1, which is present at extremely low level in cells (Rohatgi et al Science). Fluorescence labeled Rab5, Rab7, and Lamp1 are widely used for marking early endosomes, late endosomes, and lysosomes, and the data in question were generated through confocal imaging on a newly acquired Zeiss LSM710 microscope. We have repeated the experiments in question using Ptch1GFP and antibodies against endogenous Rab5, Rab7, and Lamp1, respectively. The data are displayed in new <xref ref-type="fig" rid="fig2">Figure 2</xref> and <xref ref-type="fig" rid="fig2s1 fig2s3">Figure 2–figure supplements 1 and 3</xref>. Signals from antibody staining of endogenous proteins were quite low, probably reflecting the low abundance of the interacting species or the low avidity of this commercial antibody, nevertheless, colocalization between Ptch1GFP and Rab7 poitive late endosomes was confirmed. We also showed colocalization between Ptch1GFP and Lamp1 positive lysosomes using leupeptin to block proteolysis. However, we were unable to detect colocalization between Ptch1GFP and early endosomes (Rab5) without or with ShhN treatment, confirming our previous finding that Ptch1 traverses from lipid rafts directly to late endosomes, bypassing early endosomes. Finally, the d2PY mutant was not colocalized with any of these vesicles. We want to emphasize that these confocal images presented were taken in z-stack using a 63x oil lens. Some images may appear fuzzy, particularly in colocalizing areas/vesicles. This is likely because only a very small fraction of cytoplasmic Ptch1 is channeled to the endocytic pathway; the bulk of forced expressed Ptch1 still turns over via proteasomes (<xref ref-type="fig" rid="fig1">Figure 1F</xref>).</p><p><italic>In addition in my opinion the biggest question that is left unanswered is how ubiquitination of Patched by Smurf proteins contributes to its function. Do Smurfs lead to Patched mono-ubiquitination or to K63 or K48 ubiquitin chain conjugations? Is ubiquitination involved in Patched endocytosis per se or in its sorting from endosomes to lysosomes? Does Hedgehog ligand promote the interaction of Patched with Smurfs? Do Hedgehog ligands promote Patched ubiquitination?</italic></p><p>In our humble opinion, elucidation of the type of Smurfs-mediated ubiquitin modification of Ptch1is certainly informative, but is nevertheless a mechanistic detail in our investigation. It is also extremely difficult to visualize monoubiquitination of Ptch1 under natural settings, given the size of this protein. We did however use mutant forms of ubiquitin and found that Smurf2 promotes Ptch1 to undergo both K63 and K48 ubiquitin chain-mediated ubiquitination (new <xref ref-type="fig" rid="fig9">Figure 9C</xref>). We further show that Shh-N promotes interaction of Ptch1 with Smurfs (new <xref ref-type="fig" rid="fig7">Figure 7E</xref>) and Ptch1 polyubiquitination (new <xref ref-type="fig" rid="fig9">Figure 9D</xref>). Because Ptch1 ΔPY is accumulated in Caveolin-positive lipid raft but not in late endosome (<xref ref-type="fig" rid="fig1 fig2">Figs.1A, 1B, and 2A, 2B</xref>), we believe that Smurf-mediated Ptch1 ubiquitination is involved in sorting of Ptch1 from lipid raft to late endosomes.</p><p><italic>There also seems to be a disconnection between the results obtained using the Ptch-d2PY mutant (when rescuing the Ptch1-/- MEFs) and the results obtained in the Sf1, Sf2 double KO cells. Indeed, whereas the Shh-promoted accumulation of Smo and Gli1 activation are blunted in the dKO cells, Smo accumulation is only reduced when the d2PY mutant is expressed (4C,D). Since the interaction between the d2PY mutant and Smurf proteins seems to be completely abolished (9E) how is this explained? If there is more Ptch1-d2PY in cilia, why do Smo enters at all?</italic></p><p>We replaced the d2PY images in old <xref ref-type="fig" rid="fig4">Figure 4C</xref> as well as those in old <xref ref-type="fig" rid="fig8">Figure 8B</xref> with new ones that better reflect the corresponding statistic graphs. We apologize for those images that may have exaggerated the difference. Judging from the data graphs, it is clear that the reduction in Smo ciliary localization and Gli1 activation caused by d2PY deletion is clearly in line with that by Smurfs knockdown (compare <xref ref-type="fig" rid="fig3">Figure 3D</xref>, time point 1-4 hours vs. <xref ref-type="fig" rid="fig6">Figure 6B, 6C</xref>).</p><p>With regard to the last question, the current paradigm of Ptch1 inhibiting Smo by preventing the latter entry into cilia is based on the observation that Smo moves in whereas Ptch1 moves out of cilia under the influence of Shh (Rohatgi et al, Science 317:372-8, 2007). However, there is no evidence to indicate that the presence of these two membrane receptors in the cilium is mutually exclusive. To the contrary, there are published studies reporting cyclopamine actually promotes Smo entry into the cilium, suggesting that Smo and Ptch1 can co-exist in cilia.</p><p>Reviewer #3:</p><p><italic>In the manuscript entitled “Requirement of Smurf-Mediated Endocytosis of Patched 1 in Sonic Hedgehog Signal Reception”, Yu et al. present evidence that Smurf1 and Smurf2 promote ubiquitination of PTCH1 resulting in endocytic turnover that is required for HH pathway activation. In particular, the authors provide significant experimental data examining the subcellular localization of PTCH1 and the role of two PPXY motifs in regulating PTCH1 localization turnover, and downstream effects on HH pathway function. While, overall the results appear to be of high quality, there are some issues with both interpretation of the data and proper acknowledgement of previous work that the authors must address</italic>.</p><p><italic>Major comments:</italic></p><p><italic>1) There is an unfortunate lack of proper citation of previous work by other labs in this field. Two essential examples include the recent publication of work identifying a role for Smurfs in regulating Drosophila Ptc turnover (Huang et al., PLOS Biology, 2013), and work from Tom Kornberg that defined a role for the PPXY motif in regulating the turnover of vertebrate PTCH1 (Kawamura et al., JBC, 2008). These two papers directly impact the current study by Yue et al., and this work should be considered in the context of these previous studies</italic>.</p><p>We have cited these two papers and discussed extensively the Huang’s recent publication.</p><p><italic>2) In</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>, the authors utilize Ptch1-/- MEFs to address differences in the ability of PTCH1 and PTCH1Δ2PY to promote ligand-dependent signaling. However, the authors miss an opportunity to distinguish between the ligand-dependent and ligand-independent effects of PTCH1 in the HH pathway. They should use these cells and constructs to examine the ability of PTCH1 or PTCH1Δ2PY to antagonize SMO in the absence of ligand. That is, Ptch1-/- MEFs display constitutive HH pathway activation; however, re-expressing PTCH1 rescues this pathway activity. The question is whether PTCH1Δ2PY is equally effective? Do the authors observe equivalent antagonism of SMO in these cells? Or is PTCH1Δ2PY a more effective antagonist of SMO than wt PTCH1? These are straightforward questions to address since the authors have all the necessary tools and reagents in hand</italic>.</p><p>We did the experiment as requested and the results indicate that Δ2PY is equally effective as the wt Ptch1 in antagonizing Smo in Ptch1-/- MEFs (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). This is different from the results obtained from Shh-induced signaling events.</p></body></sub-article></article> |