Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila.
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.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.PLoS Biology 06/2011; 9(6):e1001083. · 12.69 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.Nature 12/1996; 384(6605):129-34. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Here, we show that patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overproliferation of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner.Developmental Cell 12/2005; 9(5):699-710. · 12.86 Impact Factor
Hedgehog-Regulated Ubiquitination Controls
Smoothened Trafficking and Cell Surface Expression in
Shuang Li1, Yongbin Chen1, Qing Shi1, Tao Yue1, Bing Wang1, Jin Jiang1,2*
1Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 2Department of
Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America
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 b-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.
Citation: Li S, Chen Y, Shi Q, Yue T, Wang B, et al. (2012) Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in
Drosophila. PLoS Biol 10(1): e1001239. doi:10.1371/journal.pbio.1001239
Academic Editor: Konrad Basler, University of Zurich, Switzerland
Received June 17, 2011; Accepted November 23, 2011; Published January 10, 2012
Copyright: ? 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from NIH (GM61269) and Welch Foundation (I-1603) to J. Jiang. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: A, anterior; Avl, Avalanche; b2AR, b2-Adrenergic Receptor; CHX, cycloheximide; Ci, Cubitus interruptus; C-tail, carboxyl intracellular tail; dpp,
decapentaplegic; dsRNA, double-stranded RNA; FS, Fz2-SAID; Fz2, Frizzle 2; GPCR, G protein coupled receptor; Hh, Hedgehog; Hrs, HGF-regulated tyrosine kinase
substrate; Krz, Kurtz; mSmo, mammalian Smo; MVBs, multivesicular bodies; P, posterior; Ptc, Patched; RTK, receptor tyrosine kinase; SAID, Smo autoinhibitory
domain; Smo, Smoothened; Wg, Wingless
* E-mail: email@example.com
Hedgehog (Hh) signaling governs cell growth and patterning in
species ranging from insects to human [1,2]. Because of its pivotal
role in embryonic development and adult tissue homeostasis,
misregulation of Hh signaling activity has been linked to many
human disorders including birth defects and cancers [1,3,4]. Hh
exerts its biological influence through a largely conserved signaling
cascade that culminates at the activation of latent transcription
factors Cubitus interruptus (Ci)/Gli .
The core Hh reception system consists of a 12-transmembrane
protein Patched (Ptc) that acts as the Hh receptor and a seven-
transmembrane protein Smo that acts as the Hh signal transducer
[5,6]. Hh and Ptc reciprocally regulate the subcellular localization
and active state of Smo [7–10]. In Drosophila, Hh stimulation or
loss of Ptc leads to cell surface accumulation of Smo [7,11].
Increased cell surface expression and activation of Smo are
regulated by Hh-induced and PKA/CK1-mediated phosphoryla-
tion of Smo carboxyl intracellular tail (C-tail) [12–14].
Several observations suggest that Smo cell surface expression is
controlled by endocytic trafficking. A transmission electron
microscopic study of Drosophila imaginal discs indicated that
Smo is localized primarily in the lysosome of anterior compart-
ment cells but is enriched on the plasma membrane of posterior
compartment cells . In Drosophila salivary gland cells,
blocking endocytosis promotes Smo cell surface accumulation
. Using antibody uptake assay in S2 cells, we have shown that
Smo reaches the cell surface but quickly internalizes in the
absence of Hh and that Hh stimulation diminishes internalized
Smo with a concomitant increase in cell surface Smo . Taken
together, these observations suggest that Hh signaling may
regulate Smo cell surface expression by blocking its endocytosis
and/or promoting its recycling back to the cell surface after
The mechanisms by which Smo endocytic trafficking and cell
surface expression are regulated have remained unknown. Smo
intracellular regions lack recognizable endosomal-lysosomal sort-
ing signals such as the NPXY and dileucine-based motifs .
However, many membrane receptors are internalized after
covalently modified by ubiquitination, as has been demonstrated
for receptor tyrosine kinases (RTKs) and G protein coupled
receptors (GPCRs) [17,18]. The close relationship between Smo
PLoS Biology | www.plosbiology.org1January 2012 | Volume 10 | Issue 1 | e1001239
and GPCRs prompted us to investigate whether Smo cell surface
expression is regulated by the ubiquitin pathway. Here we provide
both genetic and biochemical evidence that Smo trafficking and
degradation are regulated through multi-site ubiquitination of
Smo C-tail and that Hh promotes Smo cell surface expression by
inhibiting its ubiquitination. We also provide evidence that the
non-visual b-arrestin Kurtz (Krz) acts in parallel with Smo
ubiquitination to control Smo cell surface expression, and that the
deubiquitinating enzyme UBPY promotes Smo cell surface
expression by counteracting Smo ubiquitination.
Inactivation of the Ubiquitin-Activating Enzyme Uba1
Leads to Smo Accumulation
In Drosophila wing discs, Smo cell surface level is low in anterior
(A) compartment cells away from the A/P boundary but is
elevated in response to Hh in A-compartment cells near the A/P
boundary or in posterior (P) compartment cells (Figure 1A) . To
determine whether Smo is downregulated by the ubiquitin
pathway, we generated mutant clones for Uba1, which encodes
the only ubiquitin-activating enzyme (E1) in Drosophila [19,20]. We
employed a temperature-sensitive allele of Uba1, Uba1H33, which
behaves like a null allele at the restrictive temperature .
Uba1H33clones were induced at second instar larval stage (48–72 h
AEL) by FRT/FLP mediated mitotic recombination. Larva
carrying Uba1H33clones were grown at permissive temperature
(18uC) for 3 d and then shifted to non-permissive temperature
(30uC) for 24 h before dissection for immunostaining. We found
that anteriorly situated Uba1H33clones accumulated high levels of
Smo compared with neighboring wild type cells (Figure 1A–B’),
suggesting that Smo is downregulated via the ubiquitin pathway in
the absence of Hh. Immunostaining with anti-Smo antibody
before membrane permeabilization suggested that Smo was
accumulated on the cell surface in anteriorly situated Uba1H33
clones (Figure S1A–A’). A 12-h temperature shift resulted in a less
robust Smo accumulation in Uba1H33clones (Figure S1B–B’),
likely due to the perdurance of Uba1 activity. In general, Smo
elevation coincided well with Uba1 mutant clones. Intriguingly,
Uba1H33mutant cells situated in the posterior compartment also
exhibited slightly higher levels of Smo than neighboring wild type
cells (arrowhead in Figure 1B), suggesting that a fraction of Smo
still undergoes ubiquitin-mediated degradation in the presence of
Uba1 Regulates Smo Ubiquitination and Cell Surface
To examine whether Smo is directly ubiquitinated and
whether Uba1 is responsible for this activity, we carried out a
cell-based ubiquitination assay (see Materials and Methods)
. We employed RNAi and/or pharmacological inhibitor to
inactivate Uba1. S2 cells stably expressing a Myc-tagged
Smo (Myc-Smo) were treated with Uba1 or control double-
stranded RNA (dsRNA) in the absence or presence of PYR-41, a
cell permeable E1 inhibitor . The efficiency of Uba1 RNAi
was confirmed by Western blot analysis of an exogenously
expressed tagged Uba1 (Figure 1C). Myc-Smo was ubiquitinated
efficiently in the absence of Uba1 inhibition (Figure 1D);
however, ubiquitination of Smo was attenuated by Uba1 RNAi
and more significantly inhibited by PYR-41 (Figure 1D). The
incomplete blockage of Smo ubiquitination by Uba1 RNAi is
likely due to partial inactivation of Uba1 by the RNAi approach.
Indeed, a combined treatment with Uba1 RNAi and PYR-41
resulted in a more complete inhibition of Smo ubiquitination
We next applied a cell-based immunostaining assay to
determine whether Uba1 regulates Smo cell surface expression
. Myc-Smo expressing cells were treated with control or Uba1
dsRNA in the absence or presence of PYR-41. Cell surface and
total Smo were visualized by immunostaining with an anti-SmoN
antibody prior to and after cell membrane permeabilization,
respectively. As shown in Figure 1E, inhibition of Uba1 either by
RNAi or PYR-41 increased the levels of Smo cell surface
expression and combined treatment resulted in more dramatic
cell surface accumulation of Smo.
Perturbation of Endocytic Machinery Leads to Smo
Accumulation and Hh Pathway Activation
Ubiquitinated membrane proteins are internalized through the
endocytic pathway and targeted to lysosome for degradation .
We therefore examined the effect of inactivation of endocytic
components on Smo accumulation in wing imaginal discs. We
found that Smo was accumulated in intracellular puncta in mutant
clones lacking the Drosophila homolog of HGF-regulated tyrosine
kinase substrate (Hrs) (Figure 2A–A’), a protein involved in sorting
ubiquitinated membrane proteins into multivesicular bodies
(MVBs) . Of note, not all hrs mutant cells exhibited Smo
puncta. This could be due to perdurance of Hrs activity and/or
disc folding so that Smo puncta are present at different focal
planes. RNAi of other endocytic components, including Tsg101
, Avalanche (Avl), a Drosophila syntaxin located in early
endosomes , and Rab5, resulted in Smo accumulation in
anterior compartment cells distant from the A/P boundary (arrows
in Figure 2B–E), as well as Hh pathway activation as indicated by
Ci accumulation and ectopic expression of a Hh target gene
decapentaplegic (dpp) (Figure 2B–E). Taken together, these observa-
tions suggest that Smo is downregulated via the endocytic pathway
in the absence of Hh.
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 ubiquiti-
nation of Smo by inducing another modification, phos-
phorylation, 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 coun-
teracts ubiquitination of Smo to promote its cell surface
accumulation. Our study demonstrates that reversible
ubiquitination plays a key role in regulating Smo traffic-
king 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.
Ubiquitin Regulation of Smoothened Trafficking
PLoS Biology | www.plosbiology.org2January 2012 | Volume 10 | Issue 1 | e1001239
Smo Is Degraded by Both Lysosome and Proteasome
Consistent with Smo being downregulated through the endocytic
pathway, treating Myc-Smo expressing S2 cells with a lysosome
inhibitor, NH4Cl, stabilized Smo (Figure 3A). Interestingly, treating
cells with a proteasome inhibitor, MG132, stabilized Smo more
dramatically than treating cells with NH4Cl (Figure 3A). Further-
more, combined treatment of cells with MG132 and NH4Cl had an
additive effect onSmostabilization(Figure 3A), suggesting that Smo
is downregulated by both lysosome- and proteasome-dependent
mechanisms. However, unlike the case of Hh stimulation or Uba1
inaction where Smo was accumulated on the cell surface,
proteasome inhibition stabilized Smo in intracellular vesicles
(Figure 3B). Double labeling with endosomal markers YFP-Rab5
(for early endosomes) or YFP-Rab7 (for late endosomes) revealed
that Smo was stabilized in Rab7 positive late endosomes after
MG132 treatment (Figure 3C). Taken together, these observations
suggest that a fraction of internalized Smo was degraded by
proteasome in the endocytic pathway before reaching to lysosome.
Hh Inhibits Smo Ubiquitination Via PKA/CK1-Mediated
Hh induces Smo cell surface accumulation both in vitro and in
vivo [7,11,13]. If Smo ubiquitination is responsible for its
internalization, Hh may increase Smo cell surface expression by
inhibiting its ubiquitination. Indeed, treating Myc-Smo stably
expressing cells with Hh-conditioned medium markedly reduced
but did not completely abolish Smo ubiquitination (Figure 4A).
Similarly, Ptc RNAi also reduced Smo ubiquitination (Figure 4B).
Our previous study demonstrated that Hh induced Smo cell
surface accumulation through PKA/CK1-mediated phosphoryla-
tion of Smo C-tail . We therefore determined whether Hh
regulates Smo ubiquitination in a manner depending on Smo
phosphorylation. We found that Hh stimulation failed to inhibit
Smo ubiquitination in the presence of a PKA inhibitor H-89
(Figure 4C). On the other hand, expressing a constitutively active
PKA catalytic domain (mC*) inhibited Smo ubiquitination in the
absence of Hh (Figure 4D). To further determine whether Smo
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 Uba1H33mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. Uba1H33mutant 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.
Ubiquitin Regulation of Smoothened Trafficking
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ubiquitination is regulated by PKA/CK1-mediated phosphoryla-
tion of its C-tail, S2 cells were transfected with Myc-tagged wild
type Smo, a phosphorylation deficient form of Smo (SmoSA) with
three PKA sites (S667, S687, and S740) mutated to Ala, or a
phospho-mimetic form of Smo (SmoSD) with three PKA/CK1
clusters mutated to Asp , treated without or with Hh
conditioned medium, and followed by the ubiquitination assay
described above. As shown in Figure 4E, Hh inhibited the
ubiquitination of Myc-Smo but did not significantly affect the
ubiquitination of Myc-SmoSA. Furthermore, the phospho-mimetic
Smo mutant, SmoSD, exhibited diminished ubiquitination and its
residual ubiquitination was further reduced by Hh treatment
(Figure 4E–F). These results support the notion that Hh-induced
phosphorylation by PKA/CK1 inhibits Smo ubiquitination,
leading to its cell surface accumulation.
The SAID Domain Promotes Smo Ubiquitination and
Our previous study revealed that the Smo autoinhibitory
domain (SAID) inhibits Smo activity in part by preventing Smo
cell surface expression because a Smo variant lacking the SAID
domain (SmoD661–818or SmoDSAID) accumulated on the cell
surface in the absence of Hh stimulation . To determine
whether the SAID domain regulates Smo ubiquitination, we
examined the ubiquitin status of a Myc-tagged SmoDSAID(Myc-
SmoDSAID). As shown in Figure 4G, deleting the SAID domain
diminished Smo ubiquitination, and the residual ubiquitination of
Myc-SmoDSAIDwas further reduced by Hh treatment.
To determine whether the SAID domain suffices to promote
ubiquitination and internalization of a heterologous membrane
protein, we fused it to the C-terminus of the Wingless (Wg)
receptor Frizzle 2 (Fz2) to construct Fz2-SAID chimeric protein
(FS). When expressed in S2 cells, CFP-tagged Fz2 (CFP-Fz2) was
largely accumulated on the cell surface with a small fraction
internalized and colocalized with the endosomal marker Rab5
(Figure 5A–A’’). In contrast, CFP-FS was barely detectable on the
cell surface but largely accumulated in Rab5-positive endosomes
(Figure 5B–B’’), suggesting that SAID can promote endocytosis of
Fz2. Introducing the phosphorylation-mimetic mutation to the
SAID domain of FS (CFP-FS-SD) reduced its endocytosis
(Figure 5C–C’’), whereas the chimeric protein carrying a
phosphorylation deficient form of SAID (CFP-FS-SA) was
internalized as efficiently as CFP-FS (Figure 5D–D’’). In addition,
we found that adding the phosphorylation-deficient form but not
the phospho-mimetic form of SAID to Fz2 promotes the
ubiquitination of the corresponding chimeric protein (Figure 5E).
Taken together, these observations suggest that the SAID domain
suffices to promote ubiquitination and internalization of a
membrane protein in a manner inhibited by phosphorylation.
Combined with our earlier work , it seems that the SAID
domain autonomously regulates ubiquitination independent of the
C-terminal negatively charged region.
Smo Is Ubiquitinated at Multiple Lysine Residues
If Smo ubiquitination is responsible for its internalization and
degradation, one would expect that ubiquitination-deficient Smo
variants should be stabilized and accumulated on the cell surface.
We therefore attempted to identify Lys residues responsible for
Smo ubiquitination. In general, ubiquitin acceptor sites lack a
strict consensus and target proteins can be ubiquitinated at
multiple Lys residues. Smo C-tail and intracellular loops contain a
total of 49 Lys residues, many of which may serve as ubiquitin
acceptor sites, making it difficult to generate Smo variants devoid
of ubiquitination. As deleting the SAID domain diminished Smo
ubiquitination (Figure 4G), we speculated that this region might
contain Lys residues critical for Smo ubiquitination. There are a
total of 13 Lys residues between aa 661 and aa 818. We therefore
constructed SmoK6Rwith K665, K695, K700, K702, K710, and
K733 mutated to Arg; SmoK7Rwith K752, K753, K762, K772,
K773, K782, and K801 mutated to Arg; and SmoK13Rwith all the
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.
Ubiquitin Regulation of Smoothened Trafficking
PLoS Biology | www.plosbiology.org 4January 2012 | Volume 10 | Issue 1 | e1001239
13 Lys residues mutated to Arg. Using the cell-based ubiquitina-
tion assay described above, we found that both Myc-SmoK6Rand
Myc-SmoK7Rexhibited reduced ubiquitination compared with
Myc-Smo (Figure 6A). The combined mutations (K13R) resulted
in a more dramatic reduction in Smo ubiquitination (Figure 6A),
suggesting that Smo is ubiquitinated at multiple Lys residues
between aa 661 and aa 818. In addition, the residual ubiquitina-
tion of Myc-SmoK13Rsuggests that Smo is also ubiquitinated at
one or more Lys residues outside the SAID domain.
SmoK13RExhibits Increased Stability and Cell Surface
We next determined whether the K13R mutation affects Smo
stability and cell surface expression. Myc-Smo and Myc-SmoK13R
expression constructs were transfected into S2 cells together with a
Myc-CFP expression construct as an internal control. The levels of
Myc-Smo and Myc-SmoK13Rwere monitored at different time
points after treatment with the protein synthesis inhibitor,
cycloheximide (CHX). As shown in Figure 6B, Myc-SmoK13R
exhibited increased half-life compared with Myc-Smo, suggesting
that inhibition of Smo ubiquitination leads to its stabilization. We
also measured the steady state levels of Myc-Smo and Myc-
SmoK13Rin the absence or presence of MG132 and/or NH4Cl.
While Myc-Smo was stabilized by both MG132 and NH4Cl, Myc-
SmoK13Rwas stabilized by NH4Cl but insensitive to MG132
treatment (Figure 6C), suggesting that inhibition of Smo
ubiquitination blocks its degradation by proteasome.
To determine whether inhibition of Smo ubiquitination leads to
its cell surface accumulation, S2 cells were transfected with Myc-
Smo or Myc-SmoK13Rexpression construct, followed by treatment
with or without Hh-conditioned medium. Cell surface and total
Smo were monitored by immunostaining with the anti-SmoN
antibody before and after cell permeabilization, respectively. As
shown in Figure 6D, Myc-SmoK13Rexhibited higher basal level of
cell surface expression than Myc-Smo; however, the level of cell
surface Myc-SmoK13Rin the absence of Hh was still lower than
that of Myc-Smo or Myc-SmoK13Rin the presence of Hh
(Figure 6D). Thus, although SmoK13Rexhibits increased stability
and cell surface expression, it is still internalized and degraded by
lysosome and can be further stabilized by Hh.
To determine whether the K13R mutation affects Smo stability
in vivo, we generated transgenic flies expressing either UAS-Myc-
Smo or UAS-Myc-SmoK13Rfrom the same genetic locus using the
phiC31 integration system to ensure similar expression level from
different constructs . We used the wing specific Gal4 driver
MS1096 coupled with tub-Gal80tsto drive a pulse of UAS-Myc-Smo
or UAS-Myc-SmoK13Rexpression by shifting late third instar larvae
to the non-permissive temperature for 12 h. After chasing for
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).
Ubiquitin Regulation of Smoothened Trafficking
PLoS Biology | www.plosbiology.org5 January 2012 | Volume 10 | Issue 1 | e1001239
different periods of time, wing discs were immunostained with an
anti-Myc antibody. As shown in Figure S2, after a 10 h chase,
Myc-Smo was barely detectable in A-compartment cells distant
from the A/P boundary, whereas Myc-SmoK13Rpersisted in these
cells, suggesting that Myc-SmoK13Rhas a longer half-life than
Krz Promotes Smo Internalization by Binding to Its C-Tail
Internalization of SmoK13R
ubiquitination at a Lys residue(s) outside the SAID domain. In
addition, SmoK13Rcould also be internalized by Smo interacting
proteins,ashave beenshownforotherreceptors[27,28].Ithas been
shown that the non-visual arrestin, b-arrestin 2, can bind and
internalize mammalian Smo . The Drosophila non-visual arrestin
is encoded by krz . We therefore carried out both gain- and loss-
of-function studies to determine whether Krz regulates Smo cell
surface expression. We found that overexpression of Krz in wing
imaginal discs using a dorsal compartment specific Gal4 driver, ap-
Gal4, blocked Smo accumulation in posterior-dorsal compartment
cells (compare Figure 7B with Figure 7A). However, we found that
Smo was not accumulated in krz mutant clones located in the
anterior compartment of wing discs (Figure 7C). Similar observa-
tions were obtained by a recent study .
is likely due to its residual
Using a coimmunoprecipitation assay, we found that Smo
interacted with Krz through its C-tail as both Myc-Smo and Myc-
SmoCT(a Smo variant only containing its C-tail) but not Myc-
SmoDCT(a Smo variant with its C-tail deleted) pulled down a C-
terminally YFP-tagged Krz (Krz-YFP) when expressed in S2 cells
(Figure 7D). Furthermore, Krz-YFP could internalize SmoSDbut
not SmoDCTin S2 cells (Figure 7F), suggesting that Krz
internalizes Smo by binding to its C-tail. The association between
Smo and Krz was attenuated by Hh stimulation because Myc-
Smo pulled down less Krz-YFP in the presence of Hh conditioned
medium (Figure 7E). In addition, Myc-SmoSDpulled down less
Krz-YFP than Myc-SmoSA(Figure 7E), suggesting that Smo/Krz
interaction is inhibited by Hh and PKA/CK1-mediated phos-
The observations that overexpression of Krz promoted Smo
internalization but its loss of function did not lead to Smo cell
surface accumulation suggest that a redundant mechanism(s) may
act in parallel with Krz to internalize Smo. For example, in the
absence of Krz, ubiquitination of Smo might be sufficient to
promote its internalization and degradation. On the other hand,
Krz could internalize Smo when Smo ubiquitination is compro-
mised. This may explain, at least in part, why SmoK13Ris still
internalized and degraded by lysosome. To test this model, we
Figure 4. Smo ubiquitination is inhibited by Hh and PKA/CK1-mediated phosphorylation. (A–C) Cell extracts from control or Myc-Smo
expressing S2 cells treated with or without Hh-conditioned medium (A) in the presence or absence of a PKA inhibitor H-89 (C), or treated with control
(Luc) or Ptc dsRNA (B), were immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub to visualize ubiquitinated
Smo (top) or anti-Myc antibody to visualize Myc-Smo (bottom). (D) Cell extracts from control or Myc-Smo transfected cells with or without
cotransfection of mC* were immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub or anti-Myc antibody. (E–G)
S2 cells were transfected with the indicated Myc-tagged Smo constructs and treated with or without Hh-conditioned medium. Cell extracts were
immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub or anti-Myc antibody. Of note, in all the panels, cells
were treated with MG132 for 4 h before harvest and loading was normalized by the amount of Myc-Smo monomer.
Ubiquitin Regulation of Smoothened Trafficking
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examined the effect of Krz inactivation on the cell surface
expression of Myc-Smo and Myc-SmoK13Rin S2 cells. Consistent
with the finding that loss-of-Krz has no effect on the cell surface
expression of endogenous Smo in wing discs (Figure 7C), Krz
RNAi did not significantly affect the cell surface expression of
Myc-Smo in S2 cells (Figure 7G). In contrast, Krz RNAi increased
the cell surface expression of Myc-SmoK13R(Figure 7G), suggest-
ing that SmoK13Ris, at least in part, internalized by Krz. Similarly,
Krz RNAi enhanced the cell surface accumulation of Myc-Smo
induced by Uba1 RNAi or PYR41 (Figure S3), suggesting that Krz
acts in parallel with ubiquitination to internalize Smo. On the
other hand, overexpression of Krz-YFP blocked the cell surface
accumulation of Myc-SmoK13Rand this blockage was alleviated by
Hh treatment (Figure 7I), suggesting that Hh inhibits Krz-
mediated Smo internalization.
Smo Ubiquitination Is Counteracted by the
Deubiquitinating Enzyme UBPY
Ubiquitination is a reversible process and ubiquitin attached to
target proteins can be removed by deubiquitinating enzymes/
DUBs . Compared with the large number of E3 ubiquitin
ligases that catalyze ubiquitination of targeted proteins, each
genome encodes a much smaller number of DUBs. For example,
the Drosophila genome encodes over 200 annotated E3s but less
than 30 annotated DUBs (Flybase; Table S1). To determine
whether Smo ubiquitination is regulated by DUBs, we systemat-
ically knocked down individual DUBs by RNAi and examined the
effect on Smo ubiquitination in S2 cells stably expressing Myc-
Smo. From this screen, we found that RNAi of the Drosophila
UBPY/USP8 significantly increased the basal levels of Smo
ubiquitination (Figure S4). The effect of UBPY RNAi on Smo
ubiquitination was confirmed by an independent dsRNA for
UBPY (Figure 8A). We also found that inactivation of UBPY by
RNAi increased Smo ubiquitination in the presence of Hh
(Figure 8A), suggesting that UBPY counteracts Smo ubiquitination
in both Hh signaling ‘‘off’’ and ‘‘on’’ states. Consistent with UBPY
being able to counteract Smo ubiquitination independent of Hh
signaling states, overexpression of UBPY reduced Smo ubiquitina-
tion in S2 cells both in the absence and presence of Hh (Figure 8B).
We then carried out coimmunoprecipitation assays to determine
whether UBPY physically interacts with Smo. As shown in
Figure 8C, Myc-Smo and Myc-SmoCTbut not Myc-SmoDCT
pulled down a flag-tagged UBPY (Fg-UBPY) when expressed in S2
cells, suggesting that UBPY interacts with Smo through its C-tail.
The association between UBPY and Myc-Smo was not signif-
icantly affected by Hh stimulation (Figure 8D). Furthermore,
UBPY appears to interact equally well with Myc-Smo, Myc-
SmoSA, and Myc-SmoSD, suggesting that the bulk of Smo/UBPY
association is not regulated by Hh signaling.
We next examined the effect of loss- or gain-of-UBPY on Smo
cell surface expression. In wing discs carrying UBPY mutant
clones, Smo cell surface accumulation was attenuated in P-
compartment situated UBPY mutant cells (Figure 8E–E’’). On the
contrary, expression of UAS-UBPY using the wing specific Gal4
driver MS1096 resulted in Smo accumulation in anterior
compartment cells away from the A/P boundary (Figure 8G,J).
Similarly, overexpression of UBPY in S2 cells markedly increased
the cell surface expression of Myc-Smo (Figure 8K). Overexpres-
sion of UBPY in wing discs stabilized full-length Ci (Figure 8G’,J’)
and induced ectopic expression of dpp-lacZ in anterior dorsal
compartment cells where MS1096 was expressed at high levels
(Figure 8G’’). Smo RNAi suppressed the ectopic dpp-lacZ
expression induced by UBPY overexpression as well as the
(Figure 8H–H’’). However, overexpression of UBPY induced little
if any ectopic expression of ptc-lacZ (Figure 8J’’), which is normally
induced by higher levels of Hh signaling than dpp-lacZ. Taken
together, these results suggest that UBPY can reverse Smo
ubiquitination to promote its cell surface accumulation and induce
low but not high levels of Hh pathway activation. This is in line
with our previous finding that overexpression of wild type Smo
only induced low levels of Hh pathway activation and full
activation of Smo requires additional steps, including a phosphor-
ylation-mediated conformational switch in Smo C-tail [7–10,13].
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.
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Smo Is Regulated by Both Mono- and Polyubiquitination
It is generally thought that monoubiquitination or multi-
ubiquitination (monoubiquitination at multiple sites) is responsible
for receptor internalization and degradation by lysosome, whereas
Lys 48-linked polyubiquitination targets proteins for proteasome-
mediated degradation. The observation that Smo is degraded by
both lysosome and proteasome dependent mechanisms implied
that Smo might undergo both types of modification. To determine
if Smo could be monoubiquitinated, Myc-Smo or its KR variants
was coexpressed with a HA-tagged mutant form of Ub with all Lys
residues mutated to Arg (HA-UbK0) in S2 cells. In this case,
addition of HA-UbK0prevents the formation of polyubiquitination
chains, generating modified proteins with one or more sites
monoubiquitinated. We found that Myc-Smo was effectively
modified by HA-UbK0(Figure 9A). HA-UbK0was also incorpo-
rated into Myc-SmoK6R, Myc-SmoK7R, and Myc-SmoK13R, albeit
with reduced efficiency compared with Myc-Smo (Figure 9A),
suggesting that Smo can be monoubiquitinated at multiple sites.
In the absence of proteasome inhibitor, HA-UbK0and wild type
HA-Ub were incorporated into Myc-Smo at similar levels
(Figure 9B), suggesting that the ubiquitinated Smo species
modified by HA-UbK0or HA-Ub detected under these conditions
were mostly mono- or multi-ubiquitinated. Furthermore, Hh
stimulation inhibited Smo ubiquitination under these conditions
(Figure 9B). However, after MG132 treatment, more HA-Ub
conjugated Smo was detected than HA-UbK0modified Smo
(Figure 9B), suggesting that a fraction of Myc-Smo underwent
polyubiquitination that was normally degraded by proteasome.
The proteasome inhibitor also increased the level of HA-UbK0
conjugated Smo (Figure 9B), suggesting that a fraction of HA-
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-SmoK13Rwere 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-SmoK13Rtogether 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-SmoK13Rtogether 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-SmoK13Rand 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.
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UbK0conjugated Smo might undergo polyubiquitination via
To confirm that Smo could be modified by Lys 48-linked
polyubiquitination, we probed Smo immunopurified from S2 cells
stably expressing Myc-Smo with a Lys 48-linkage specific
polyubiquitin antibody (K48, Cell Signaling). As shown in
Figure 9C, immunoprecipitated Myc-Smo was recognized by the
K48 antibody and the signal was markedly increased by MG132
treatment, suggesting that Smo can also be modified by Lys 48-
linked polyubiquitination that targets it for proteasome-mediated
Regulation of Smo cell surface expression is a key step in Hh
signal transduction [7,11,13], but the underlying mechanism has
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-SmoDCT. (F) Confocal images of S2 cells
transfected with CFP-SmoSD, CFP-SmoDCT, or CFP-SmoWTeither alone (left) or together with Krz-YFP (right). Overexpression of Krz-YFP internalized
CFP-SmoSDbut not CFP-SmoDCT. (G) S2 cells transfected with Myc-Smo or Myc-SmoK13Rin 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-SmoK13Ralone or together with Krz-YFP with or
without Hh treatment, followed by immunostaining to visualize cell surface Myc-SmoK13R(green) and Krz-YFP (red).
Ubiquitin Regulation of Smoothened Trafficking
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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-SmoDCT. (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.
Ubiquitin Regulation of Smoothened Trafficking
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remained unknown. In this study, we provide the first evidence
that Smo is ubiquitinated in a manner regulated by Hh signaling
and PKA/CK1-mediated Smo phosphorylation. We provide both
genetic and biochemical evidence that Smo ubiquitination
regulates its endocytic trafficking and cell surface expression. In
addition, we provide evidence that the non-visual b-arrestin Krz
acts in parallel with Smo ubiquitination to promote its internal-
ization and that Smo ubiquitination is antagonized by the
deubiquitinating enzyme UBPY.
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 ubiquiti-
nation 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.
Early studies with yeast membrane receptors provided evidence
that monoubiquitination of GPCRs mediates their agonist-
induced internalization [33,34]. Later studies with mammalian
GPCRs and other receptors suggested that both mono- and
polyubiquitination could be involved in receptor endocytosis and
degradation . However, it has been shown that ‘‘polyubiqui-
tination’’ of some receptors is due to monoubiquitination at
multiple sites (multiubiquitination) instead of forming a poly-
ubiquitination chain at a single site [35,36]. Here we provide
evidence that Smo is both mono- and polyubiquitinated. It is
possible that mono- or multiubiquitination may lead to Smo
internalization and that internalized Smo could be further
ubiquitinated in the endocytic pathway, leading to the formation
of Lys 48-linked polyubiquitin chain that targets Smo for
proteasome-mediated degradation (Figure 10). Thus, multiple
ubiquitination events provide a robust mechanism for Smo
downregulation to prevent aberrant Smo activity in the absence
Regulation of Smo trafficking and cell surface expression
provides a new paradigm for how the ubiquitin pathway controls
the activity of a membrane receptor. Unlike all the other cases
whereby receptor ubiquitination is triggered by ligand or agonist
stimulation and serves as a mechanism to control the duration of
cell signaling, Smo ubiquitination occurs in the absence of ligand
stimulation and serves as a mechanism to keep the basal pathway
Figure 9. Smo is regulated by both multi- and polyubiquitination. (A) S2 cells were transfected with HA-UbK0and 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-UbK0or 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.
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activity in check. Smo ubiquitination is inhibited upon ligand
stimulation; as a consequence, Smo is accumulated on the cell
surface where it becomes activated. Thus, the regulation of Smo
ubiquitination by the upstream signal is in the opposite direction
compared with other receptors.
How does Hh block Smo ubiquitination? Smo intracellular
regions such as SAID could recruit one or more E3 ubiquitin
ligases to catalyze Smo ubiquitination and E3 recruitment could
be inhibited by Hh stimulation and PKA/CK1-mediated Smo
phosphorylation. An alternative but not mutually exclusive
mechanism is that Hh and Smo phosphorylation could promote
Smo deubiquitination by regulating the binding and/or activity of
one or more DUBs. In a systematic RNAi-based screen, we
identified UBPY as a Smo DUB. UBPY binds Smo C-tail and
antagonizes Smo ubiquitination. UBPY may modulate Smo cell
surface expression by attenuating Smo endocytosis and/or
promoting Smo recycling (Figure 10). 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 phosphor-
ylation, suggesting that Smo deubiquitination by UBPY is unlikely
to be a major mechanism by which Hh inhibits Smo ubiquitina-
tion, 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. The mechanism underlying
the regulation of Smo ubiquitination might be analogous to those
regulating the phosphorylation of many proteins in which kinases
instead of phosphatases are usually regulated by upstream signals.
Thus, identifying the E3 ligase(s) involved in Smo ubiquitination
may shed important light on the mechanism by which Smo
ubiquitination is regulated.
We have also obtained evidence that the non-visual b-arrestin
Krz can promote Smo internalization by binding to its C-tail and
this activity is inhibited by Hh. However, while Krz overexpres-
sion effectively internalized Smo, loss-of-Krz-function did not lead
to a significant change in Smo cell surface expression (Figure 7C,G)
. Our results suggest that Smo ubiquitination can act
independently of Krz to internalize Smo, leading to its
degradation by both proteasome and lysosome so that the
requirement of Krz in internalizing Smo can only be revealed
when Smo ubiquitination is compromised (Figure 7G–I). It is
possible that Smo ubiquitination plays a major role while Krz only
plays a minor role in the regulation of Smo trafficking and cell
The mechanisms that regulate Smo trafficking and cell surface
expression exhibit interesting similarities to as well as important
differences from those regulating GPCRs. For example, it has
been shown that agonist-induced downregulation of b2-Adrener-
gic Receptor (b2AR) is mediated by both b-arrestin and receptor
ubiquitination . In addition, b2AR internalization and
degradation is regulated by both proteasome- and lysosome-
dependent mechanisms [27,37]. However, b2AR ubiquitination is
induced by agonist and serves as a mechanism for desensitization
[27,37], whereas Smo ubiquitination is inhibited by Hh and serves
as a mechanism for keeping pathway activity off in the absence of
the ligand. b-arrestin binding to b2AR is induced by agonists and
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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requires GRK2-mediated phosphorylation of the activated
receptor , whereas Krz binding to Smo is attenuated by Hh
and Smo phosphorylation (Figure 7). Although GPRK2/GRK2
also regulates Smo in Drosophila, its function appears to be
uncoupled from that of Krz because loss of GPRK2 exhibits a
phenotype distinct from that exhibited by loss of Krz [31,38–40].
Furthermore, Krz can internalize Smo in the absence of GPRK2
. b-arrestin is required for b2AR ubiquitination [27,37],
whereas Krz inactivation does not significantly affect Smo
ubiquitination (unpublished observations). Finally, while the
proteasome inhibitor MG132 blocks agonist-induced b2AR
internalization , it does not prevent Smo internalization but
instead inhibits Smo degradation after internalization (Figure 3).
It is also interesting to note that b-arrestin has been implicated
in the regulation of Smo trafficking and Shh signaling in
vertebrates [29,41,42]. Furthermore, b-arrestin binds to mamma-
lian Smo (mSmo) in a manner promoted by Shh and GRK2-
mediated phosphorylation of mSmo C-tail [42,43], which is
analogous to agonist-induced b-arrestin binding to GPCRs.
However, instead of internalizing mSmo for degradation, b-
arrestin appears to promote mSmo ciliary accumulation ,
which correlates with its positive role in Shh signaling. Both
Drosophila and vertebrate Smo proteins can activate trimeric G-
proteins [44–46], suggesting that they are not only structurally but
also functionally related to GPCRs. It is conceivable that Smo
proteins may employ multiple mechanisms utilized by GPCRs to
control their intracellular trafficking and activity. Thus, it will be
interesting to determine whether vertebrate Smo is also regulated
by the ubiquitin pathway.
Materials and Methods
Mutations and Transgenes
Mutations used in this study are Uba1H33, l(2)23AdD28/hrs
, krz1, and UBPYKO. Mutant clones were generated
by FLP/FRT-mediated mitotic recombination as previously
described . The genotypes for making clones are as follows:
Uba1 clones: yw 122; FRT42 Uba1H33/FRT42 hs-Myc-GFP; hrs
clones: yw 122; l(2)23AdD28FRT40/ hs-Myc-GFP FRT40; krz or
UBPY clones: yw 122; FRT82 krz1or UBPYKO/FRT82 hs-Myc-GFP.
Transgenic RNAi lines used are UAS-Tsg101-RNAi (VDRC#
23944), UAS-Avl-RNAi (VDRC# 5413), and UAS-Rab5-RNAi
(VDRC# 34096). UAS-Krz and UAS-UBPY are previously
described [47,48]. Constructs for various tagged forms of wild
type Smo, SmoDCT, SmoCT, SmoD661–818, SmoSA, and SmoSDare
previously described [8,13,50]. CFP-tagged Fz2 is described .
To construct Fz2/Smo chimeric proteins, the coding sequence for
the wild type and mutant forms of SAID (aa 661–818) was
amplified by PCR and inserted at a Kpn I site between the coding
sequence for Fz2 and CFP. To construct Krz-YFP, the coding
sequence of Krz was amplified by PCR and inserted between Not
I/ Kpn I digestion sites of pUAST vector, and YFP was inserted in
frame to the C-terminus of Krz between Kpn I/ XbaI digestion
sites. SmoK6R, SmoK7R, and SmoK13Rwere generated using PCR-
based site-directed mutagenesis to introduce K to R mutations in
corresponding Lys residues.
Cell Culture, Transfection, Immunoprecipitation, Western
Blot, and Immunostaining
Drosophila S2 cells were cultured in Drosophila SFM (Invitrogen)
with 10% fetal bovine serum, 100 U/ml of penicillin, and
100 mg/ml of streptomycin at 23uC. Transfection was carried
out by Calcium Phosphate Transfection Kit (Specialty Media)
according to the manufacturer’s instructions. Hh-conditioned
medium treatment was carried out as described . Cells were
treated with 50 mM MG132 (Calbiochem) for 4 h to inhibit
proteasome or 20 mM NH4Cl (Sigma) for 18 h to inhibit
lysosome. Immunoprecipitation and Western blot analysis were
carried out using standard protocols as previously described .
For Smo cell surface staining assay, S2 cells were harvested and
washed with PBS, fixed with 4% formaldehyde at room
temperature for 20 min, and incubated with the mouse anti-
SmoN antibody in PBS at room temperature for 90 min. Cells
were washed 3 times by PBS followed by secondary antibody
staining. Immunostaining of imaginal discs was carried out as
described [13,49]. Quantification of immunostaining and autora-
diography densitometric analysis was performed using ImageJ
software. Antibodies used in this study were: mouse anti-SmoN
(DSHB), rat anti-Ci 2A1 , rabbit and mouse anti-Flag (Sigma),
mouse anti-Myc (Santa Cruz), mouse anti-HA (Santa Cruz),
mouse anti-GFP (Millipore), rabbit anti-GFP (Santa Cruz), rabbit
anti-LacZ (ICN Pharmaceuticals, Inc.), anti-Ub (P4D1) (Santa
Cruz), and anti-Poly-UbK48(Cell signaling).
Ubiquitination assays were carried out based on the protocol
described previously . Briefly, Myc-Smo stably expressing S2
cells or S2 cells transfected with Smo variants with or without HA-
Ub (wild type or mutants) were treated with MG132 or NH4Cl
before harvesting. Cells were lysed in 100 ml of denaturing buffer
(1% SDS/50 mM Tris, pH 7.5/0.5 mM EDTA/1 mM DTT).
After incubation for 5 min at 100uC, the lysates were diluted 10-
fold with lysis buffer and then subjected to immunoprecipitation
and Western blot analysis.
RNAi in Drosophila S2 Cells
dsRNA was generated by MEGAscript High Yield Transcrip-
tion Kit (Ambion: #AM1334) according to the manufacturer’s
instruction. DNA templates targeting Uba1(aa 1–172), Krz(aa
191–365), UBPY(aa 25–191 and aa 124–290) or other DBUs
(Table S1) were generated by PCR and used for generating
dsRNA. Ptc RNAi was carried out as previously described .
dsRNA targeting the Fire Fly Luciferase coding sequence was used
as a control. For RNAi knockdown experiments, S2 cells were
cultured in serum free medium containing indicated dsRNA at
23uC for 8 h. After adding fetal bovine serum to a final
concentration of 10%, dsRNA treated cells were cultured
overnight before transfection. 48 h after transfection, cells were
harvested for further analysis.
mutant clones. Low (A, B) and high (A’, B’) magnification view of
wing imaginal discs carrying Uba1H33
immunostained with anti-SmoN (red) and anti-GFP (green)
antibodies. Larvae were grown at 18uC after clone induction
and shifted to 30uC for 24 (A, A’) or 12 (B, B’) h, followed by
immunostaining with anti-SmoN antibody prior to membrane
permeabilization. Uba1H33mutant clones are marked by the lack
of GFP staining. Arrows indicate anterior clones that accumulate
Smo on the cell surface.
Smo is accumulated on the cell surface in Uba1
mutant clones and
Wing discs expressing UAS-Myc-Smo (left) or UAS-Myc-SmoK13R
under the control of MS1096 in conjunction with Gal80ts. Larvae
were grown at 18uC until late third instar, shifted to 30uC for 12 h,
and then put back to 18uC for the indicated hours before
SmoK13Ris more stable than wild type Smo in vivo.
Ubiquitin Regulation of Smoothened Trafficking
PLoS Biology | www.plosbiology.org13January 2012 | Volume 10 | Issue 1 | e1001239
immunostaining with anti-Myc antibody. Arrows indicate Myc-
Smo or Myc-SmoK13Raccumulation in anterior compartment
cells distant from the A/P boundary.
Smo. Myc-Smo expressing S2 cells were treated with Luc, Uba1,
or Uba1 plus Krz dsRNA in the absence or presence of PYR41,
followed by immunostaining to visualize cell surface Smo or total
Smo. 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.
Krz acts in parallel with ubiquitination to internalize
cells stably expressing Myc-Smo were treated with control dsRNA
or dsRNA targeting the indicated DUB. After treatment with
MG132, cell extracts were immunoprecipitated with anti-Myc
antibody, followed by immunoblotting with anti-Myc or anti-Ub
antibody. Loading was normalized by the amount of Myc-Smo
monomer. IP, immunoprecipitation; IB, immunoblot.
An RNAi screen identified UBPY as a Smo DUB. S2
annotated Drosophila DUBs with gene names, CG numbers, and
primer sequences for making dsRNA are indicated. The dsRNAs
against individual DUBs are designed based on the sequence and
primer information through the Gene and Reagent Lookup tool
on the DRSC website: http://www.flyrnai.org/cgi-bin/RNAi_
Annotated DUBs in the Drosophila genome. A list of
We thank Drs. Andrea Bergmann, Hugo Bellen, Satoshi Goto, Spyros
Artavanis-Tsakonas, Robert Holmgren, and Jianhang Jia for reagents;
Bloomington stock center for fly stocks; and DSHB for antibodies.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: JJ SL YC.
Performed the experiments: SL YC QS BW. Analyzed the data: SL YC
QS JJ. Wrote the paper: JJ.
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