Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in Drosophila

Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
PLoS Biology (Impact Factor: 9.34). 01/2012; 10(1):e1001239. DOI: 10.1371/journal.pbio.1001239
Source: PubMed


Author Summary
The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in diverse species ranging from Drosophila to human. Hh signals across the cell surface membrane by regulating the subcellular location and conformation of a membrane protein called Smoothened (Smo). In Drosophila, Smo accumulates on the cell surface in response to Hh, whereas in the absence of Hh it is internalized and degraded. The molecular mechanisms that control this intracellular trafficking and degradation of Smo were unknown, but here we show that Smo is modified by attachment of several molecules of a small protein called ubiquitin, which tags it for internalization and degradation within the cell. Hh inhibits this ubiquitination of Smo by inducing another modification, phosphorylation, of its intracellular tail by two types of protein kinase enzymes. This loss of ubiquitination and gain of phosphorylation causes the accumulation of Smo at the cell surface. What's more, we find that another protein called Kurtz interacts with Smo and acts in parallel with the ubiquitination process to promote internalization of Smo, and that the deubiquitinating enzyme UBPY/USP8 counteracts ubiquitination of Smo to promote its cell surface accumulation. Our study demonstrates that reversible ubiquitination plays a key role in regulating Smo trafficking to and from the cell surface and thus it provides novel insights into the mechanism of Hh signaling from the outside to the inside of the cell.

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Available from: Qing Shi, Mar 12, 2014
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    • "However, it is not clear what counteracts the induction of Ptch1 to achieve the precision of the regulation. For many years, Ptch1 and Smo have been seen in punctate intracellular vesicles in both Drosophila and mammalian cells (Capdevila et al., 1994; Ramirez-Weber et al., 2000; Zhu et al., 2003; Li et al., 2012), and their trafficking between the cytoplasmic membrane and intracellular vesicles found to be crucial to the activation of the Hedgehog pathway (Denef et al., 2000; Incardona et al., 2000; Zhu et al., 2003; Nakano et al., 2004; Lu et al., 2006; Milenkovic et al., 2009; Li et al., 2012). It is known that ligand engagement of Drosophila 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 (Huangfu et al., 2003; Corbit et al., 2005; Goetz and Anderson, 2009), a microtubule-based membrane protrusion that emanates from the interphase centrioles (Lefebvre and Rosenbaum, 1986; Pazour and Witman, 2003; Nachury et al., 2010). "
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    ABSTRACT: 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.
    eLife Sciences 06/2014; 3(3):e02555. DOI:10.7554/eLife.02555 · 9.32 Impact Factor
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    • "We further carried out series immunoprecipitation experiment and mapped the domain in Smo that interacts with Hrs (see below). In this domain of Smo, mutation of eight lysine residues that are known for Smo ubiquitination [14] did not change the binding property (Fig. 4D, top panel). SmoK13R that contains 13 lysine residues supposed to be ubiquitinated [14] did not change Smo-Hrs interaction either (data not shown). "
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    ABSTRACT: In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation.
    PLoS ONE 11/2013; 8(11):e79021. DOI:10.1371/journal.pone.0079021 · 3.23 Impact Factor
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    • "Smo endocytosis is also promoted by Kurtz (Krz), the Drosophila non-visual arrestin87,88. Hh inhibits Smo ubiquitination and attenuates Smo/Krz interaction, thereby stabilizing Smo on the cell surface86,87. The E3 ligase(s) that catalyzes Smo ubiquitination has remained elusive but a deubiquitinating enzyme, UBPY/USP8, is required for Hh-induced deubiquitination of Smo86,87. "
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    ABSTRACT: Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis, and its deregulation leads to numerous human disorders including cancer. Binding of Hh to Patched (Ptc), a twelve-transmembrane protein, alleviates its inhibition of Smoothened (Smo), a seven-transmembrane protein related to G-protein-coupled receptors (GPCRs), leading to Smo phosphorylation and activation. Smo acts through intracellular signaling complexes to convert the latent transcription factor Cubitus interruptus (Ci)/Gli from a truncated repressor to a full-length activator, leading to derepression/activation of Hh target genes. Increasing evidence suggests that phosphorylation participates in almost every step in the signal relay from Smo to Ci/Gli, and that differential phosphorylation of several key pathway components may be crucial for translating the Hh morphogen gradient into graded pathway activities. In this review, we focus on the multifaceted roles that phosphorylation plays in Hh signal transduction, and discuss the conservation and difference between Drosophila and mammalian Hh signaling mechanisms.Cell Research advance online publication 22 January 2013; doi:10.1038/cr.2013.10.
    Cell Research 01/2013; 23(2). DOI:10.1038/cr.2013.10 · 12.41 Impact Factor
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