IndraLab

Statements

SMO binds USP8. 36 / 40
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"In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation."
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"The finding that USP8 interacted with Smo led to the hypothesis that Hh might control the accessibility of Smo to the DUB."
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"Overall, these results together imply that Rack1 stabilizes Smo and increases Smo cell membrane localization through promoting Smo–Usp8 interaction."
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"These data suggest that phosphorylation of Smo promotes the formation of a Smo and USP8 complex, which may amplify the Hh stimulation and lead to the activation of Smo."
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"However, we found that UBPY decreases Smo ubiquitination regardless of the Hh signaling states and that the association between UBPY and Smo is not significantly affected by either Hh stimulation or Smo phosphorylation, suggesting that Smo deubiquitination by UBPY is unlikely to be a major mechanism by which Hh inhibits Smo ubiquitination, although we can not rule out the possibility that Hh regulates UBPY binding to Smo in a subtle way that escaped the detection by our coimmunoprecipitation assay."
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"Furthermore we show that Hh can enhance the interaction between Smo and USP8."
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"In addition, we found, in an immunoprecipitation experiment, that a substantial amount of Smo SD123 interacted with USP8 ( xref , lane 2, top panel)."
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"We then carried out coimmunoprecipitation assays to determine whether UBPY physically interacts with Smo."
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"However, we found that Hrs blocks Smo phosphorylation (XREF_FIG), whereas USP8 does not XREF_BIBR, suggesting that Hrs and USP8 bind Smo in different conformation."
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"Intriguingly, with Hh stimulation, Rack1 dissociated from Ci–Cos2 complex and formed a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation."
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"Even after de-sumoylation, UBPY already bound to a Smo C-tail could be " trapped " there by SUMO independent interaction with multiple Smo C-tails within a Smo multimer, thus exerting a long lasting effect on Smo ubiquitination."
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"The finding that USP8 interacted with Smo led to the hypothesis that Hh might control the accessibility of Smo to the DUB."
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"We also found that the interaction between Smo and USP8 was not changed by either overexpression or RNAi of Hrs (XREF_FIG), suggesting that Hrs regulates Smo ubiquitination not by preventing the binding of the deubiquitinase to Smo."
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"Even after de-sumoylation, UBPY already bound to a Smo C-tail could be “trapped” there by SUMO-independent interaction with multiple Smo C-tails within a Smo multimer, thus exerting a long-lasting effect on Smo ubiquitination."
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"In addition, we found that USP8 is required for Hh-induced cell surface accumulation of Smo, and that Hh promotes the interaction between Smo and USP8 (see xref )."
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"Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625–753, which covers the three PKA and CK1 phosphorylation clusters."
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"As shown in xref , Myc-Smo and Myc-Smo CT but not Myc-Smo ΔCT pulled down a flag-tagged UBPY (Fg-UBPY) when expressed in S2 cells, suggesting that UBPY interacts with Smo through its C-tail."
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"In contrast, Smo PKA123 barely interacted with USP8 ( xref , lane 3, top panel)."
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"Indeed, co-immunoprecipitation experiments revealed that Hh stimulated the binding of UBPY to Smo when NEM was added to the cell lysates to preserve SUMO conjugated Smo (XREF_FIG, lanes 1-2)."
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"However, we found that UBPY decreases Smo ubiquitination regardless of the Hh signaling states and that the association between UBPY and Smo is not significantly affected by either Hh stimulation or Smo phosphorylation, suggesting that Smo deubiquitination by UBPY is unlikely to be a major mechanism by which Hh inhibits Smo ubiquitination, although we cannot rule out the possibility that Hh regulates UBPY binding to Smo in a subtle way that escaped the detection by our coimmunoprecipitation assay."
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"UBPY binds Smo C-tail and antagonizes Smo ubiquitination."
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"In support of this notion, we found that Hh promotes the interaction between Smo and USP8 ( xref )."
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"We further figured out that Rack1 could enhance the affinity between Usp8 and Smo, suggesting that Rack1 may acts as a scaffold to pull Usp8 and Smo together."
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"These data suggest that phosphorylation of Smo promotes the formation of a Smo-USP8 complex, which may amplify the Hh stimulation and lead to the activation of Smo."
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"In support of this notion, we found that Hh promotes the interaction between Smo and USP8 (XREF_FIG)."
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"In the presence of Hh, Hh dissociates Ci–Rack1 interaction, and promotes Rack1 forming a trimeric complex with Usp8 and Smo, resulting in Smo deubiquitination and cell surface accumulation (Fig.  xref )."
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"As shown in XREF_FIG, Myc-Smo and Myc-Smo CT but not Myc-Smo DeltaCT pulled down a flag tagged UBPY (Fg-UBPY) when expressed in S2 cells, suggesting that UBPY interacts with Smo through its C-tail."
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"In addition, we found that USP8 is required for Hh induced cell surface accumulation of Smo, and that Hh promotes the interaction between Smo and USP8 (see XREF_SUPPLEMENTARY)."
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"Hh promotes the formation of a Smo-USP8 complex, and USP8 further promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes by deubiquitinating Smo, leading to increased Hh signaling activity."
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"Indeed, co-immunoprecipitation experiments revealed that Hh stimulated the binding of UBPY to Smo when NEM was added to the cell lysates to preserve SUMO-conjugated Smo ( xref , lanes 1–2)."
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"The association between UBPY and Myc-Smo was not significantly affected by Hh stimulation (XREF_FIG)."
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"We then carried out coimmunoprecipitation assays to determine whether UBPY physically interacts with Smo."
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"UBPY binds Smo C-tail and antagonizes Smo ubiquitination."
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"Several lines of evidence suggest that the ubiquitin pathway regulates Smo endocytic trafficking and degradation: (1) Smo was accumulated in mutant clones lacking the ubiquitin-activating enzyme Uba1 in wing imaginal discs, and inactivation of Uba1 in S2 cells inhibited Smo ubiquitination and promoted its cell surface accumulation; (2) Smo was accumulated when the activity of several endocytic components or lysosome was inhibited; (3) Hh and PKA/CK1-mediated Smo phosphorylation inhibited Smo ubiquitination and increased Smo cell surface expression; (4) the Smo autoinhibitory domain (SAID) promoted receptor ubiquitination and internalization; (5) Smo was ubiquitinated at multiple sites both inside and outside the SAID domain and mutating the ubiquitin acceptor sites in SAID increased Smo half-life and cell surface expression; and (6) Smo cell surface expression was promoted by the deubiquitinating enzyme UBPY that binds Smo and counteracts Smo ubiquitination."
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"We also found that the interaction between Smo and USP8 was not changed by either overexpression or RNAi of Hrs ( xref ), suggesting that Hrs regulates Smo ubiquitination not by preventing the binding of the deubiquitinase to Smo."
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"Hh promotes the formation of a Smo and USP8 complex, and USP8 further promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes by deubiquitinating Smo, leading to increased Hh signaling activity."
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