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.
    Full-text · Article · Jun 2014 · eLife Sciences
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    • "Using an antibody uptake assay, we found that Hh treatment inhibited Smo endocytosis and reduced the ratio of early endosome-localized Smo [13]. In addition, ubiquitination promotes endocytosis of Smo, whereas deubiquitination prevents the process [13], [14]. A transmission electron microscopic study of Drosophila imaginal discs indicated that Smo is directed primarily to the lysosomes of A compartment cells, but is enriched at the plasma membrane of P compartment cells [15]. "
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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.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "More recently, our knowledge of their signaling functions has dramatically broadened to involve a wide variety of signaling pathways and modes of regulation (Kovacs et al. 2009). The Drosophila genome encodes a single ortholog of b-arrestins, Kurtz (Krz) (Roman et al. 2000), which has been implicated in the regulation of GPCR signaling, as well as Notch, Hedgehog, receptor tyrosine kinase, and Toll pathways (Mukherjee et al. 2005; Ge et al. 2006; Tipping et al. 2010; Molnar et al. 2011; Li et al. 2012). Mammalian b-arrestin proteins can down-regulate NF-kB signaling by binding and stabilizing IkBa (Gao et al. 2004; Witherow et al. 2004) and by preventing autoubiquitination of TRAF6 (Wang et al. 2006). "
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    ABSTRACT: The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine-tune its activity. One such factor is β-arrestin Kurtz (Krz) that we previously implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. In this study, we have uncovered a link between these two modes of regulation and show that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production, melanotic mass formation, nuclear accumulation of Toll pathway transcriptional effectors Dorsal and Dif, and expression of immunity genes such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, we found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. Our results demonstrate that β-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation.
    Preview · Article · Sep 2013 · Genetics
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