Smo ubiquitination is inhibited by Hh and PKA/CK1-mediated phosphorylation.

<p>Smo ubiquitination is inhibited by Hh and PKA/CK1-mediated phosphorylation.</p

Smo accumulates in cells defective in the endocytic machinery.

<p>(A–A') Low (A) and high (A') magnification view of a wing imaginal disc carrying <i>hrs</i> mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. <i>hrs</i> mutant clones are marked by the lack of GFP staining (arrows). (B–E) A wild type wing disc (C) or wing discs expressing <i>UAS-Tsg101-RNAi</i> (B), <i>UAS-Avl-RNAi</i> (D), or <i>UAS-Rab5-RNAi</i> (E) with the <i>MS1096</i> Gal4 driver were immunostained to show the expression of Smo (red), Ci (green), and <i>dpp-lacZ</i> (blue). Arrows indicate Smo and Ci accumulation (B, D, E) as well as ectopic <i>dpp-lacZ</i> expression (D, E) in A-compartment cells situated distantly from the A/P boundary. Of note, <i>UAS-Dicer2</i> was coexpressed with <i>UAS-Tsg101-RNAi</i> and <i>UAS-Avl-RNAi</i> to enhance the RNAi effect.</p

Uba1 regulates Smo ubiquitination and cell surface expression.

<p>(A–B') Low (A, B) and high (A', B') magnification view of wing imaginal discs carrying <i>Uba1^H33</i> mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. <i>Uba1^H33</i> mutant clones are marked by the lack of GFP staining. Arrows and arrowheads indicate anterior and posterior clones, respectively. (C) The efficiency of Uba1 RNAi was evaluated by Western blot analysis of transfected Myc-Uba1. (D) S2 cells stably expressing a Myc-tagged Smo under the control of <i>metallothionein</i> promoter were treated with Uba1 dsRNA or control (Luciferase) dsRNA in the absence or presence of the E1 inhibitor PYR41. After treatment with MG132, cells extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with an anti-Ub antibody to visualize ubiquitinated Smo (top) or anti-Myc antibody to visualize Myc-Smo (bottom). Loading was normalized by the amount of Myc-Smo monomer. IP, immunoprecipitation; IB, immunoblot. (E) S2 cells stably expressing Myc-Smo were treated as in (D). Cells were immunostained with anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal.</p

A model for ubiquitin regulation of Smo.

<p>In the absence of Hh, Ptc inhibits Smo phosphorylation. Unphosphorylated or under-phosphorylated Smo is effectively ubiquitinated at multiple sites. In addition, Krz binds Smo and acts in parallel with Smo ubiquitination to promote Smo endocytosis. Smo is further ubiquitinated in the endocytic pathway and degraded by both proteasome and lysosome. In the presence of Hh, binding of Hh to Ptc inhibits its activity and promotes its degradation, allowing Smo phosphorylation by PKA and CK1. Phosphorylation inhibits Smo ubiquitination and its association with Krz, thereby inhibiting its internalization. UBPY catalyzes Smo deubiquitination in both signal “off” and “on” states and may facilitate Smo recycling back to the cell surface. See text for details.</p

UBPY regulates Smo ubiquitination and cell surface expression.

<p>(A) Myc-Smo expressing cells were treated with or without Hh-conditioned medium in the presence of UBPY or Luc dsRNA. After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub or anti-Myc antibody. Of note, shorter exposure was used for Western blot analysis of samples derived from cells not treated with Hh (left). (B) S2 cells were transfected with Myc-Smo and HA-tagged Ub (HA-Ub) and with or without Flag-tagged UBPY (Fg-UBPY). After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-HA or anti-Myc antibody. (C–D) S2 cells were transfected with Fg-UBPY and Myc-tagged wild type Smo or the indicated Smo variants and treated with or without Hh-conditioned medium. Western blot analyses were carried out on cell lysates or immunoprecipitates using the indicated antibodies. Asterisks indicate monomeric forms of Myc-Smo and Myc-Smo^ΔCT. (E–E”) Large magnification view of a wing disc carrying <i>UBPY</i> mutant clones and immunostained to show the expression of Smo (red channel) and GFP (green channel). <i>UBPY</i> mutant clones are marked by the lack of GFP expression. Posterior <i>UBPY</i> mutant clones had reduced cell surface accumulation of Smo (arrows). (F–J”) Wild type wing discs (F–F”, I–I”) or wing discs expressing <i>UAS-UBPY</i> alone (G–G”, J–J”) or together with <i>UAS-Smo-RNAi</i> (H–H”) under the control of <i>MS1096</i> were immunostained to show the expression of Smo (red), Ci (green), and <i>dpp-lacZ</i> or <i>ptc-lacZ</i> (blue). (K) Confocal images of S2 cells expressing Myc-Smo (red) alone or together with Fg-UBPY (green). Top panels show cell surface staining while bottom panels show regular staining.</p

The SAID domain promotes ubiquitination and endocytosis of a heterologous protein.

<p>(A–D) Confocal images of S2 cells transfected with CFP-tagged Fz2 (A), Fz2-SAID fusion (FS in B), Fz2-SAID with either the phospho-mimetic (FS-SD in C), or the phosphorylation deficient (FS-SA in D) mutations together with YFP-Rab5. Addition of the wild type or phosphorylation deficient but not the phospho-mimetic form of SAID to Fz2 increased its endocytosis and colocalization with Rab5. (E) Myc-tagged Fz2, FS-SA, and FS-SD were transfected into S2 cells with HA-Ub. Cell lysates were immuno-precipitated (IP) with anti-Myc antibody, followed by Western blot with anti-HA (top panel) and anti-Myc (bottom panel) antibodies.</p

Krz interacts with Smo and downregulates its cell surface expression.

<p>(A–B) A wing disc expressing <i>UAS-GFP</i> alone (A) or together with <i>UAS-Krz</i> (B) under the control of <i>ap-Gal4</i> was immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. Krz overexpression cells are marked by GFP in (B). Excessive Krz blocked Smo accumulation in P-compartment cells (arrows in B). (C) A wing imaginal disc carrying <i>krz</i> mutant clones was immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. <i>krz</i> mutant clones are marked by the lack of GFP staining. Anteriorly situated <i>krz</i> mutant clones did not accumulate Smo (arrows). (D–E) S2 cells were transfected with Krz-YFP and Myc-tagged wild type Smo or the indicated Smo variants and treated with or without Hh-conditioned medium. Western blot analyses were carried out on cell lysates or immunoprecipitates using the indicated antibodies. Asterisks indicate monomeric forms of Myc-Smo and Myc-Smo^ΔCT. (F) Confocal images of S2 cells transfected with CFP-Smo^SD, CFP-Smo^ΔCT, or CFP-Smo^WT either alone (left) or together with Krz-YFP (right). Overexpression of Krz-YFP internalized CFP-Smo^SD but not CFP-Smo^ΔCT. (G) S2 cells transfected with Myc-Smo or Myc-Smo^K13R in the presence of Krz RNAi or Luc RNAi were immunostained with anti-SmoN antibody prior to (top panels) or after (bottom panels) membrane permeabilization. Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal. (H) Krz RNAi efficiency was evaluated by Western blot analysis of transfected Krz-YFP. (I) S2 cells were transfected with Myc-Smo^K13R alone or together with Krz-YFP with or without Hh treatment, followed by immunostaining to visualize cell surface Myc-Smo^K13R (green) and Krz-YFP (red).</p

Smo is regulated by both multi- and polyubiquitination.

<p>(A) S2 cells were transfected with HA-Ub^K0 and Myc-Smo or indicated KR variants and treated with NH₄Cl. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. (B) S2 cells were transfected with Myc-Smo and HA-Ub^K0 or HA-Ub and treated with or without Hh-conditioned medium and/or MG132. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. The cell lysates were also immunoblotted with anti-HA antibody. (C) Myc-Smo expressing S2 cells or control cells were mock treated, or treated with either MG132 or NH₄Cl. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc antibody or a Lys 48-linkage specific polyubiquitin antibody (K48). Of note, Loading was normalized by the amount of Myc-Smo monomer.</p

Smo is internalized and degraded by multi-site ubiquitination.

<p>(A) Cell extracts from S2 cells transfected with Myc-Smo, Myc-Smo^K6R, Myc-Smo^K7R, or Myc-Smo^K13R were immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub (top) or anti-Myc antibody (bottom). (B) S2 cells were transfected with Myc-Smo or Myc-Smo^K13R together with Myc-CFP (as internal control) and treated with cycloheximide (CHX) for the indicated time. Cell extracts were subjected to Western blot analysis with anti-Myc antibody. Quantification of the Western blot analysis is shown at bottom. (C) S2 cells were transfected with Myc-Smo or Myc-Smo^K13R together with Myc-CFP and treated without or with MG132 and/or NH₄Cl. Cell extracts were subjected to Western blot analysis with anti-Myc antibody. (D) S2 cells transfected with Myc-Smo or Myc-Smo^K13R and treated with or without Hh-conditioned medium were immunostained with anti-SmoN antibody prior to (top panels) or after (bottom panels) membrane permeabilization. Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the ratio of cell surface Smo signal versus total Smo signal.</p

Smo is stabilized by both lysosome and proteasome inhibitors.

<p>(A) S2 cells stably expressing Myc-Smo were treated with MG132 and/or NH₄Cl alone or in combination, followed by Western blot analysis with an anti-Myc antibody. (B) S2 cells stably expressing Myc-Smo treated with or without MG132 and/or Hh-conditioned medium were immunostained with anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). MG132 treatment stabilized Smo in intracellular vesicles whereas Hh treatment led to cell surface accumulation of Smo. (C) Myc-Smo expressing S2 cells were transfected with YFP tagged Rab5 or Rab7, treated with or without MG132 and immunostained to show the expression of Myc-Smo (green) and Rab5/Rab7 (red).</p