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. 2012 Jan;10(1):e1001239.
doi: 10.1371/journal.pbio.1001239. Epub 2012 Jan 10.

Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila

Shuang Li  1 , Yongbin Chen, Qing Shi, Tao Yue, Bing Wang, Jin Jiang
Affiliations

Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila

Shuang Li et al. PLoS Biol. 2012 Jan.

Abstract

Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Uba1 regulates Smo ubiquitination and cell surface expression.
(A–B') Low (A, B) and high (A', B') magnification view of wing imaginal discs carrying Uba1H33 mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. Uba1H33 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 metallothionein 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.
Figure 2
Figure 2. Smo accumulates in cells defective in the endocytic machinery.
(A–A') Low (A) and high (A') magnification view of a wing imaginal disc carrying hrs mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. hrs mutant clones are marked by the lack of GFP staining (arrows). (B–E) A wild type wing disc (C) or wing discs expressing UAS-Tsg101-RNAi (B), UAS-Avl-RNAi (D), or UAS-Rab5-RNAi (E) with the MS1096 Gal4 driver were immunostained to show the expression of Smo (red), Ci (green), and dpp-lacZ (blue). Arrows indicate Smo and Ci accumulation (B, D, E) as well as ectopic dpp-lacZ expression (D, E) in A-compartment cells situated distantly from the A/P boundary. Of note, UAS-Dicer2 was coexpressed with UAS-Tsg101-RNAi and UAS-Avl-RNAi to enhance the RNAi effect.
Figure 3
Figure 3. Smo is stabilized by both lysosome and proteasome inhibitors.
(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).
Figure 4
Figure 4. Smo ubiquitination is inhibited by Hh and PKA/CK1-mediated phosphorylation.
Figure 5
Figure 5. The SAID domain promotes ubiquitination and endocytosis of a heterologous protein.
(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.
Figure 6
Figure 6. Smo is internalized and degraded by multi-site ubiquitination.
(A) Cell extracts from S2 cells transfected with Myc-Smo, Myc-SmoK6R, Myc-SmoK7R, or Myc-SmoK13R 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-SmoK13R 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-SmoK13R 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-SmoK13R 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.
Figure 7
Figure 7. Krz interacts with Smo and downregulates its cell surface expression.
(A–B) A wing disc expressing UAS-GFP alone (A) or together with UAS-Krz (B) under the control of ap-Gal4 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 krz mutant clones was immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. krz mutant clones are marked by the lack of GFP staining. Anteriorly situated krz 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-SmoΔCT. (F) Confocal images of S2 cells transfected with CFP-SmoSD, CFP-SmoΔCT, or CFP-SmoWT either alone (left) or together with Krz-YFP (right). Overexpression of Krz-YFP internalized CFP-SmoSD but not CFP-SmoΔCT. (G) S2 cells transfected with Myc-Smo or Myc-SmoK13R 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-SmoK13R alone or together with Krz-YFP with or without Hh treatment, followed by immunostaining to visualize cell surface Myc-SmoK13R (green) and Krz-YFP (red).
Figure 8
Figure 8. UBPY regulates Smo ubiquitination and cell surface expression.
(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-SmoΔCT. (E–E”) Large magnification view of a wing disc carrying UBPY mutant clones and immunostained to show the expression of Smo (red channel) and GFP (green channel). UBPY mutant clones are marked by the lack of GFP expression. Posterior UBPY mutant clones had reduced cell surface accumulation of Smo (arrows). (F–J”) Wild type wing discs (F–F”, I–I”) or wing discs expressing UAS-UBPY alone (G–G”, J–J”) or together with UAS-Smo-RNAi (H–H”) under the control of MS1096 were immunostained to show the expression of Smo (red), Ci (green), and dpp-lacZ or ptc-lacZ (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.
Figure 9
Figure 9. Smo is regulated by both multi- and polyubiquitination.
(A) S2 cells were transfected with HA-UbK0 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-UbK0 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.
Figure 10
Figure 10. A model for ubiquitin regulation of Smo.
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.
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