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RESEARCH ARTICLE
307
Development 136, 307-316 (2009) doi:10.1242/dev.030015
PP4 and PP2A regulate Hedgehog signaling by controlling
Smo and Ci phosphorylation
Hongge Jia, Yajuan Liu, Wei Yan and Jianhang Jia*
The seven-transmembrane protein Smoothened (Smo) and Zn-finger transcription factor Ci/Gli are crucial components in Hedgehog
(Hh) signal transduction that mediates a variety of processes in animal development. In Drosophila, multiple kinases have been
identified to regulate Hh signaling by phosphorylating Smo and Ci; however, the phosphatase(s) involved remain obscured. Using
an in vivo RNAi screen, we identified PP4 and PP2A as phosphatases that influence Hh signaling by regulating Smo and Ci,
respectively. RNAi knockdown of PP4, but not of PP2A, elevates Smo phosphorylation and accumulation, leading to increased Hh
signaling activity. Deletion of a PP4-interaction domain (amino acids 626-678) in Smo promotes Smo phosphorylation and signaling
activity. We further find that PP4 regulates the Hh-induced Smo cell-surface accumulation. Mechanistically, we show that Hh
downregulates Smo-PP4 interaction that is mediated by Cos2. We also provide evidence that PP2A is a Ci phosphatase. Inactivating
PP2A regulatory subunit (Wdb) by RNAi or by loss-of-function mutation downregulates, whereas overexpressing regulatory
subunit upregulates, the level and thus signaling activity of full-length Ci. Furthermore, we find that Wdb counteracts kinases to
prevent Ci phosphorylation. Finally, we have obtained evidence that Wdb attenuates Ci processing probably by
dephosphorylating Ci. Taken together, our results suggest that PP4 and PP2A are two phosphatases that act at different positions
of the Hh signaling cascade.
INTRODUCTION
The hedgehog (hh) family members control many aspects of
development in both vertebrates and invertebrates (Jia and Jiang,
2006). Abnormal activation of Hh pathway has been observed in
several types of human cancers (Pasca di Magliano and Hebrok,
2003; Taipale and Beachy, 2001). The Hh signal is transduced
through a reception system that includes the transmembrane proteins
Patched (Ptc) and Smo. The main outcome of Hh signaling is the
modulation of transcriptional responses via the Ci/Gli family of Znfinger transcription factors. Hh signaling regulates the balance
between the transcriptional activator and repressor forms of Ci/Gli
(Lum and Beachy, 2004). In Drosophila, the absence of Hh allows
Ptc to inhibit Smo signaling activity. In the cytoplasm, the kinesinrelated protein Costal 2 (Cos2) recruits multiple kinases, including
PKA, CK1 and GSK3 to sequentially phosphorylate full-length Ci
(CiFL) (Zhang et al., 2005), which creates binding sites for the SCF
ubiquitin ligase containing the F-Box protein Slimb (Jia et al., 2005;
Smelkinson and Kalderon, 2006), leading to Ub/proteasomemediated processing into a truncated repressor form (CiREP). CiREP
functions as a repressor to block the expression of Hh responsive
genes such as decapentaplegic (dpp), as well as hh itself (Aza-Blanc
et al., 1997; Methot and Basler, 1999). The presence of Hh relieves
the inhibition of Ptc on Smo, inducing Smo cell-surface
accumulation and phosphorylation (Denef et al., 2000) by kinases
including PKA and CK1 (Apionishev et al., 2005; Jia et al., 2004;
Zhang et al., 2004). Smo phosphorylation by PKA and CK1 appears
to activate Smo by inducing a conformational switch (Zhao et al.,
2007), as well as promoting Smo cell surface accumulation(Jia et al.,
Sealy Center for Cancer Cell Biology, Department of Biochemistry and Molecular
Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
*Author for correspondence (e-mail: [email protected])
Accepted 11 November 2008
2004). In addition, peak levels of Hh promote Smo
hyperphosphorylation that is modulated by a feedback loop
involving downstream components Cos2 and Fused (Fu) (Claret
et al., 2007; Liu et al., 2007). Activated Smo blocks CiFL
phosphorylation and proteolytic processing required for generating
CiREP, and further promotes nuclear translocation and activation of
accumulated CiFL (Chen et al., 1999; Methot and Basler, 2000;
Wang et al., 2000; Wang and Holmgren, 2000).
The Drosophila wing disc has been used as an excellent model to
study the Hh signal transduction. Posterior (P) compartment cells in
the wing discs secrete Hh protein that moves into the anterior (A)
compartment and induces the expression of Hh target genes, such as
dpp, ptc and engrailed (en), which can be used to monitor the levels
of Hh signaling activity (Jia and Jiang, 2006). Ci is produced in Acompartment cells but not P-compartment cells, whereas Smo is
expressed in the whole wing but accumulated in P-compartment
cells as well as A-compartment cells near the AP boundary, where
there is Hh-mediated stimulation. Phosphorylation of Smo and Ci
has been shown to be the major post-translational event that
regulates their signaling activities, but how their phosphorylation is
regulated is still poorly understood.
Levels of cellular protein phosphorylation are often modulated
by the opposing action of protein kinases and phosphatases.
Phosphatases are typically classified into two main groups, the
Serine/Threonine (Ser/Thr) protein phosphatases (STPs) and
protein tyrosine phosphatases (PTPs). STPs can be subdivided
into the PPP and PPM families based on distinct amino acid
sequences and crystal structures (Cohen, 1997). In the Hh
signaling cascade, multiple Ser/Thr kinases are involved,
including PKA, GSK3 and CK1 family members. Even though
PP2A has been implicated as a positive regulator in Hh signaling
(Casso et al., 2008; Nybakken et al., 2005), its relevant substrates
remained undetermined. Thus, it is not clear whether
phosphatases are involved in regulating Smo and Ci
phosphorylation, and if so which phosphatases are responsible.
DEVELOPMENT
KEY WORDS: Smo, Ci, PP4, PP2A, Hh, Signal transduction, Drosophila
RESEARCH ARTICLE
In this study, we performed an in vivo RNAi screen with the
RNAi library from VDRC (Vienna Drosophila RNAi Center)
(Dietzl et al., 2007) targeting the catalytic subunits of the STPs in
the Drosophila genome (Morrison et al., 2000), in which we
identified PP4 and PP2A as phosphatases that regulate Smo
and CiFL phosphorylation, respectively. We found that Smo
phosphorylation is elevated by RNAi knockdown of PP4 or by
abolishing Smo-PP4 interaction. We also found that the signaling
activity of CiFL is positively regulated by PP2A. We provided
evidence that PP2A prevents CiFL proteolytic processing by
dephosphorylating CiFL.
MATERIALS AND METHODS
Mutants and transgenes
WdbIP is a hypomorphic allele (Hannus et al., 2002). smo3 is a null allele
(Chen and Struhl, 1998). In vivo RNAi library has been described (Dietzl et
al., 2007). MS1096, act>CD2>Gal4, ap-Gal4, UAS-P35, UAS-HA-CiFL,
UAS-mC*, UAS-CRL, dpp-lacZ, ptc-lacZ and hh-lacZ have been described
(Jia et al., 2003; Jia et al., 2005; Liu et al., 2007; Zhang et al., 2006)
(http://flybase.bio.indiana.edu). We constructed 2XHA-UAST and 2XFlagUAST backbones to generate UAS-HA- or UAS-Flag-tagged PP4
(CG18339), PP4R (PP4 regulatory subunit, CG2890), Mts (PP2A catalytic
subunit, CG7109), Wdb (PP2A regulatory subunit, CG5643) and Tws
(PP2A regulatory subunit, CG6235). We obtained full-length cDNAs either
from DGRC or by RT-PCR from fly embryonic RNA. Myc-Smo, MycSmoCT, Myc-Smo⌬CT and HA-Cos2 have been described (Jia et al., 2003;
Wang et al., 2000). We swapped each corresponding sequence of SmoCT
truncations and internal deletions (Jia et al., 2003) into the 3XFlag-UAST
backbone to raise the affinity for western blot. We constructed Flag-CiFL by
swapping the Ci coding sequence from HA-CiFL (Wang et al., 1999) into the
3XFlag-UAST. HA-PP4 transformants were generated by standard Pelement mediated transformation. Multiple independent lines were
examined. EP3559, UAS-Wdb, UAS-Tws, UAS-Mts, UAS-DN-Mts fly strains
have been described (Hannus et al., 2002; Mayer-Jaekel et al., 1993;
Sathyanarayanan et al., 2004). To construct attB-UAS-Myc-Smo and attBUAS-Myc-Smo⌬626-678, the attB sequence (Bateman et al., 2006) was
inserted in upstream of the UAS-binding sites in pUAST, then Myc-Smo or
Myc-Smo⌬626-678 sequences were inserted. The vas-phi-zh2A-VK5 flies
(gift from Dr Hugo Bellen) were used to generate UAS-Myc-Smo and UASMyc-Smo⌬626-678 transgenes at the 75B1 attP locus. Genotypes for
generating clones are as follows: wdbIP clones, yw hsp-flp/+ or Y; FRT82
wdbIP/FRT82 hs-GFP; smo clones expressing CiFL or co-expressing CiFL
with Wdb, y MS1096 hsp-flp1/yw or Y; smo3 FRT40/hs-GFP FRT40; UASHA-Ci or UAS-HA-Ci with UAS-Wdb/hh-lacZ.
Cell culture, transfection, immunoprecipitation, western blot
analysis and GST fusion protein pull-down
S2 cell culture, transfection, immunoprecipitation and immunoblot analysis
were performed with standard protocols (Liu et al., 2007). Smo cell-surface
accumulation was detected by immunostaining with anti-SmoN antibody
before cell permeabilization (Jia et al., 2004). The intensity of cell-surface
or total Smo was analyzed by Metamorph software. To target each
phosphatase gene with less than 17 nucleotide contiguous off-target
sequence, we synthesized Mts, Wdb, Tws, PP4 and PP4R dsRNA against
the cDNA regions of 301-900, 681-1236, 761-1360, 304-921 and 791-1288,
respectively (Chen et al., 2007). Cos2 and GFP dsRNA synthesis and the
method for RNAi in S2 cells have been described (Liu et al., 2007). OA
(Calbiochem) treatment was used to inhibit both PP4 and PP2A (Cohen et
al., 1990) at a final concentration of 50 nM for 3 hours before harvesting the
cells. GST-Smo557-686 fusion protein pull-down has been described (Liu
et al., 2007). His-Cos2MB and His-Cos2CT were constructed by fusing
Cos2 corresponding sequence to the pET30 vector, expressed in E. coli, and
purified with the His resins (Clontech). Antibodies used in this study were
mouse anti-Cos2 (gift from D. Robbins), anti-Flag, M2 (Sigma), anti-GFP
(Chemicon), anti-HA, F7 (Santa Cruz), anti-His, 4D11 (Upstate), anti-Myc,
9E10 (Santa Cruz), anti-SmoN (DSHB), anti-β-tubulin (DSHB); and rabbit
anti-HA, Y-11 (Santa Cruz) and anti-GST (Santa Cruz).
Development 136 (2)
Immunostaining of imaginal discs
Standard protocols for immunofluorescence staining of imaginal discs were
used with the antibodies mouse anti-Myc, 9E10 (Santa Cruz), anti-HA, F7
(Santa Cruz), anti-Flag, M2 (Sigma), anti-SmoN (DSHB), anti-Ptc (DSHB),
anti-CD2 (Serotec); rabbit anti-Flag (ABR), anti-HA, Y-11 (Santa Cruz),
anti-βGal (Cappel), anti-GFP (Clontech); and rat anti-Ci 2A (gift from R.
Holmgren). MG132 (100 μM; Calbiochem) in M3 medium (Sigma) was
used to treat wing discs for up to 6 hours before immunostaining.
RESULTS
Identification of Smo phosphatase by in vivo
RNAi screen
In response to Hh stimulation, many Hh pathway components are
phosphorylated and a number of kinases have been identified.
However, the phosphatase(s) involved have remained elusive. To
explore whether phosphatases are also involved, we obtained 45
RNAi lines from the VDRC, targeting 26 catalytic subunits of the
STPs (Morrison et al., 2000). We performed in vivo screening by
overexpressing individual RNAi lines via the wing-specific MS1096
Gal4 to determine whether they induced any adult wing phenotypes.
We found that RNAi of eight genes affected wing development
indicated by wing phenotypes or wing blisters (data not shown). All
eight genes encode members of the PPP family of phosphatase. The
fact that increased Smo phosphorylation stabilizes Smo in wing
discs (Denef et al., 2000; Jia et al., 2004) suggests that modulation
of Smo phosphatase might alter Smo levels. We thus performed the
next round of screening with a direct approach to detect Smo
changes when a specific phosphatase was knocked down by RNAi.
Each UAS-RNAi line targeting the eight candidates was expressed
via the dorsal compartment-specific ap-Gal4, and wing discs from
late third instar larvae were immunostained with anti-SmoN
antibody. Knockdown of PP4 by RNAi induced Smo accumulation
(Fig. 1B, compared with wild-type disc staining in Fig. 1A).
Moreover, expressing UAS-PP4RNAi caused elevated expression
and anterior expansion of Hh target genes such as ptc-lacZ in dorsal
but not ventral compartment cells (Fig. 1B⬘), a phenotype similar to
that caused by overexpressing PKA (Jia et al., 2004; Liu et al.,
2007). These observations indicate that PP4 exerts a negative
influence on Hh signaling in responding cells, probably by reducing
Smo phosphorylation. RNAi of other phosphatases including Mts
did not elevate Smo (Fig. 1C-C⬙; data not shown).
UAS-PP4 RNAi shares a 22-nucleotide contiguous sequence with
CanA1 phosphatase (CG1455; see supplementary information).
However, CanA1RNAi had no effects on Smo accumulation in wing
disc (see Fig. S1 in the supplementary material), suggesting the
accumulation of Smo by PP4 RNAi in wing discs (Fig. 1B) was due
to the downregulation of PP4 activity. To further examine the
specificity of PP4 RNAi, we tested whether the overexpressed PP4
could rescue its RNAi phenotype. As shown in Fig. 1E, coexpressing UAS-HA-PP4 attenuated the Smo elevation caused by
PP4 RNAi. By contrast, co-expressing UAS-Mts did not alleviate
PP4 RNAi-induced Smo accumulation (Fig. 1G), indicating the
specificity of PP4 RNAi. Overexpression of UAS-HA-PP4 or UASMts alone did not downregulate Smo accumulation in wing discs
(Fig. 1D,F). Taken together, our data suggest that PP4 blocks Smo
accumulation and downregulates Hh signaling activity.
PP4 downregulates Smo phosphorylation
PP4 is a highly conserved carboxymethylated protein that belongs
to the PP2A family of STPs. Drosophila PP4 shares 91.5% identity
with human PP4. To further determine whether Smo elevation
induced by PP4 RNAi (Fig. 1B) was due to enhanced Smo
DEVELOPMENT
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RESEARCH ARTICLE
309
Fig. 1. Knockdown of PP4 elevates Smo accumulation in wing
discs. (A-A’’) A wing disc expressing GFP by ap-Gal4 was stained with
anti-SmoN, β-gal (for ptc-lacZ expression). GFP indicates the ap-Gal4
expression domain in wing disc. (B-B’’) UAS-PP4RNAi was expressed by
ap-Gal4 and immunostained for Smo and ptc-lacZ expression.
Arrowhead in B indicates the accumulation of Smo and arrowhead in B’
indicates the anterior expansion of ptc-lacZ. (C-C’’) As expressing UASMtsRNAi by the ap-Gal4 caused cell lethality, the weak act>CD2>Gal4
was used and larvae were grown at 20°C to reduce the Gal4 levels,
thus limiting the strength of Mts RNAi. (D,D’) A wing disc expressing
UAS-HA-PP4 by ap-Gal4 was immunostained for Smo and HA.
(E) A wing disc co-expressing UAS-PP4RNAi and UAS-HA-PP4 was
immunostained with anti-SmoN antibody. Arrowhead indicates that
HA-PP4 reduces the levels of Smo accumulation that are caused by PP4
RNAi. (F,F’) A wing disc expressing UAS-Mts by ap-Gal4 was
immunostained with anti-SmoN antibody. (G) A wing disc co-expressing
UAS-Mts with UAS-PP4RNAi by ap-Gal4 was stained with anti-SmoN
antibody. All wing discs shown in this study were oriented with anterior
towards the left and ventral towards the top.
accumulated by either the phosphatase inhibitor okadaic acid (OA)
(lane 3, top panel), PP4 dsRNA (lane 4, top panel) or PP4R dsRNA
(lane 5, top panel) treatment, but not by Mts, Wdb and GFP dsRNA
treatments (lanes 6, 7 and 8, respectively). This is consistent with the
observation that PP2A RNAi did not promote Smo accumulation
in wing discs (Fig. 1C). In parallel, we examined whether
overexpressing PP4 could attenuate Smo phosphorylation in S2
cells. We found that expressing HA-PP4, but not HA-Mts,
diminished the Hh-induced Smo mobility shift (Fig. 2B, lanes 3 and
4, top panel).
To test whether PP4 regulates Smo phosphorylation by interacting
with Smo, we carried out co-immunoprecipitation experiments. As
shown in Fig. 2C, both Myc-Smo and Myc-SmoCT (Smo C-tail),
but not Myc-Smo⌬CT (Smo lacking its C-tail), coimmunoprecipitated with HA-PP4. To further map the Smo domain
responsible for interacting with PP4, we tested various SmoCT
truncations or internal deletions we previously generated (Fig. 2D)
(Jia et al., 2003) for their ability to bind PP4. We found that SmoCT
deletion variants lacking amino acids 626-678, which include
Smo556-625, Smo679-1035 and SmoCT⌬626-678, did not pull
down HA-tagged PP4 when co-expressed in S2 cells (see Fig. S3A,
lanes 2, 5 and 6, top panel in the supplementary material), suggesting
that amino acids 626-678 of Smo is responsible for either direct or
indirect association between SmoCT and PP4.
We reasoned that if PP4 inhibits Smo phosphorylation through
amino acids 626-678, deletion of this Smo-PP4 interacting domain
should abolish Smo-PP4 interaction thus may lead to high basal
phosphorylation of Smo. Indeed, we found that deletion of amino
acids 626-678 in the full-length Smo background (Myc-Smo⌬626678) elevated Smo phosphorylation and stabilized Smo even in the
absence of Hh (Fig. 2E lane 3, compared to lane 1, top panel),
indicating an elevation in Smo basal phosphorylation. In addition,
we found that deletion of amino acids 626-678 potentiated Hhinduced Smo phosphorylation (Fig. 2E, lane 4; compare with lane
2, top panel) and rendered Smo resistant to dephosphorylation
induced by overexpression of PP4 (see Fig. S4 in the supplementary
material). Our data indicate that amino acids 626-678 are
responsible for PP4-mediated Smo dephosphorylation.
To determine the precise activity of the exogenously expressed
Smo, we developed an in vivo assay by taking advantage of ⌽C31
integrase-mediated transgenesis (Bischof et al., 2007) in
combination with the attP sites in the fly genome (Venken et al.,
2006). We generated UAS-VK5-Myc-Smo and UAS-VK5-MycSmo⌬626-678 transgenes at the 75B1 attP locus (Venken et al.,
2006) to ensure equal expression of Myc-Smo and Myc-Smo⌬626678. We found that the activity of Myc-Smo⌬626-678 is higher than
Myc-Smo as it induced higher level of ectopic dpp-lacZ expression
(Fig. 2G, compare with 2F), indicating that amino acids 626-678
negatively regulate Smo activity. Meanwhile, we found that MycSmo⌬626-678 is stabilized in A-compartment cells (Fig. 2G⬘,
compare with 2F⬘), suggesting that deletion of amino acids 626-678
of Smo promotes its stability probably owing to enhanced
phosphorylation.
phosphorylation, we examined the levels of Smo phosphorylation
in S2 cells with our previously established assay (Jia et al., 2004; Liu
et al., 2007). Consistent with previous findings (Apionishev et al.,
2005; Denef et al., 2000; Jia et al., 2004; Liu et al., 2007; Zhang et
al., 2004), Hh stabilized Smo and induced an electrophoretic
mobility shift of Myc-Smo, indicative of Smo phosphorylation (Fig.
2A, lane 2, top panel). The hyperphosphorylated form of Smo was
Cos2 mediates the interaction between Smo and
PP4
Although the role of Smo phosphorylation has been broadly studied,
how Smo phosphorylation is regulated remains an enigma. Hh might
promote Smo phosphorylation by regulating the phosphatase. It is
unlikely that PP4 activity per se is regulated by Hh as PP4 has been
shown to be involved in essential cellular process (Zhou et al.,
2002), and also in the nucleation of microtubules (Helps et al.,
DEVELOPMENT
Regulation of Smo and Ci by phosphatases
RESEARCH ARTICLE
1998). Our finding that Smo and PP4 exist in the same protein
complex led to the hypothesis that Hh might control the accessibility
of Smo to the phosphatase. To test this, we used a GST pull-down
assay described earlier (Liu et al., 2007; Lum et al., 2003b). We
Development 136 (2)
found that Flag-PP4 from S2 cells treated with Hh was barely
precipitated by GST-Smo557-686 (Fig. 2H, lane 3, compared with
lane 2, top panel), suggesting that Hh regulates the accessibility of
PP4 to Smo.
Fig. 2. PP4 regulates Smo phosphorylation. (A) PP4RNAi elevates Smo phosphorylation. S2 cells were co-transfected with UAS-Myc-Smo and
UAS-GFP, and treated with Hh-conditioned or control medium, or treated with OA, PP4 dsRNA, PP4R dsRNA, Mts dsRNA, Wdb dsRNA or GFP
dsRNA. Cell extracts were immunoprecipitated and blotted with anti-Myc to detect Smo phosphorylation. Arrow indicates hyperphosphorylated
forms of Smo and arrowhead indicates hypophosphorylated and unphosphorylated forms. The efficiency of knockdown of individual phosphatase
is shown in Fig. S2A in the supplementary material. (B) PP4 downregulates the Hh-induced Smo phosphorylation. S2 cells were transfected with
indicated constructs and treated with or without Hh. The levels of Smo phosphorylation were examined by immunoprecipitation. The expression
levels of HA-PP4 or HA-Mts were shown by probing cell lysates with HA antibody. (C) PP4 interacts with Smo C-tail. Extracts from S2 cells expressing
indicated constructs were immunoprecipitated with Myc antibody followed by western blot with HA antibody. The expressed proteins were shown
by probing immunoprecipitates with anti-Myc, or probing the cell lysates with anti-HA. The asterisk indicates the slow mobility of the Myc-Smo⌬CT
(Jia et al., 2003). (D) SmoCT truncations interacting with PP4. See immunoprecipitation results in Fig. S3A in the supplementary material.
(E) Deletion of the PP4-binding domain in Smo elevates its phosphorylation. Extracts from S2 cells expressing Myc-Smo or Myc-Smo⌬626-678 and
treated with or without Hh were immunoprecipitated and blotted with the anti-Myc antibody. (F-G’) Wing discs expressing UAS-Myc-Smo or UASMyc-Smo⌬626-678 by ap-Gal4 were immunostained to show Myc expression in F’ and G’, and ectopic dpp-lacZ expression in F and G. Expressing
Myc-Smo⌬626-678 induced higher level of ectopic dpp-lacZ expression (arrowhead in G), compared with expressing Myc-Smo (arrowhead in F).
(H) Hh downregulates Smo-PP4 interaction. Extracts from S2 cells expressing Flag-PP4 with or without Hh treatment were incubated with the
bacterially expressed GST or GST-Smo557-686 fusion proteins. The bound PP4 proteins were analyzed by western blot with Flag antibody. The
middle panel shows the GST and GST-Smo fusion proteins. The lower panel indicates the equal amount of input PP4. (I) Smo-PP4 interaction is
attenuated by Cos2 RNAi. S2 cells were transfected with HA-PP4 and Flag-SmoCT and treated with or without Cos2 dsRNA. The SmoCT-bound PP4
was examined by immunoprecipitation with anti-Flag and western blot with anti-HA. Equally expressed PP4 proteins were ensured by probing the
lysates with anti-HA. The efficiency of Cos2 RNAi is shown in Fig. S2B in the supplementary material. (J) PP4 directly interacts with Cos2MB and
Cos2CT. Bacterially expressed and purified GST, GST-PP4, His-Cos2MB and His-Cos2CT were used for pull-down assay. Antibodies used for western
blots are indicated.
DEVELOPMENT
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Regulation of Smo and Ci by phosphatases
RESEARCH ARTICLE
311
The Smo domain responsible for PP4 association falls into the
Cos2-binding domains (amino acids 557-686) (Liu et al., 2007; Lum
et al., 2003b). In addition, we have shown that Cos2 has a negative
role in Smo phosphorylation (Liu et al., 2007). These observations
raised the possibility that Cos2 may serve as a scaffold to bridge PP4
and Smo. If so, removing Cos2 should decrease the amount of Smobound PP4, thus attenuating Smo inhibition by PP4. In support of
this model, we have found that SmoCT-PP4 interaction is attenuated
by Cos2 RNAi (Fig. 2I, lane 2, compare with lane 1, top panel),
suggesting that Cos2 may downregulate Smo phosphorylation by
recruiting the phosphatase or facilitating the interaction between
Smo and its phosphatase. We then examined the interaction between
Cos2 and PP4 and we found that both Cos2 microtubule-binding
(MB) and cargo domain (CT) bind PP4 in our immunoprecipitation
assay (see Fig. S3B in the supplementary material, data not shown).
We verified PP4 interaction with Cos2 by using an in vitro GST pulldown assay. As shown in Fig. 2J, GST-PP4 fusion protein pulled
down both His-Cos2MB and His-Cos2CT (lanes 2 and 4, top panel),
whereas GST protein failed to pull down any His-Cos2 proteins,
suggesting that PP4 directly binds Cos2 N- and C-terminal domains.
Fig. 3. PP4 RNAi elevates the Hh-induced Smo cell-surface
accumulation. (A-P) CFP-tagged Smo constructs were transfected into
S2 cells and treated with phosphatase inhibitor (OA), phosphatase
dsRNA or Hh-conditioned medium. Cell-surface-localized Smo was
visualized by immunostaining with anti-SmoN antibody. The total
amount of expressed Smo was indicated by the CFP signal. (Q) Ratio of
cell surface level to total level of Smo. Each data set was based on 15
individual cells. WT, CFP-Smo; SD123, CFP-SmoSD123; ⌬626-678, CFPSmo ⌬626-678.
accumulation by controlling the phosphorylation of other sites in
addition to the three PKA-CK1 phosphorylation clusters. By
contrast, neither RNAi of the PP2A catalytic nor of its regulatory
subunit enhanced the cell-surface accumulation of SmoSD123 (see
Fig. S5K-L in the supplementary material). Consistent with the
above observations, we found that, upon Hh stimulation, deletion of
the PP4-binding region in Smo (Smo⌬626-678) elevated Hhinduced Smo cell-surface accumulation (Fig. 3P, compared with
3B). We also found that Smo⌬626-678 did not accumulate on the
cell surface in the absence of Hh (Fig. 3O), which was consistent
with the observation that PP4 RNAi alone was not sufficient to
promote Smo cell-surface expression (Fig. 3E). The above finding,
that PP4 regulates Smo cell-surface accumulation, was further
supported by quantification analysis of the cell-surface localized
Smo (Fig. 3Q and Fig. S5N in the supplementary material).
PP2A is essential for Hh signaling
An RNAi screen in cultured cells implicated PP2A as a potential
phosphatase involved in Hh signaling (Nybakken et al., 2005). We
found that Mts RNAi affected neither Smo accumulation in wing
discs (Fig. 1C-C⬙) nor Smo phosphorylation in S2 cells (Fig. 2A,
lane 6, top panel). However, when UAS-MtsRNAi was expressed in
DEVELOPMENT
PP4 regulates Smo cell-surface accumulation
Hh-induced Smo phosphorylation promotes its cell-surface
accumulation (Jia et al., 2004); we therefore investigated whether
PP4 regulates Smo cell-surface localization using a cell-based assay
(Jia et al., 2004; Liu et al., 2007). Consistent with our previous
observations (Jia et al., 2004), Smo accumulated on the cell surface
upon Hh stimulation (Fig. 3B, compare with Fig. 3A without Hh
treatment). The Hh-induced Smo cell-surface accumulation was
enhanced by OA treatment (Fig. 3D), even though OA treatment
alone did not promote Smo cell-surface expression (Fig. 3C),
suggesting that the blockade of phosphatase activity elevates the Hhinduced Smo cell-surface accumulation. Knockdown of PP4 but not
of Mts elevated the Hh-induced Smo cell-surface accumulation (Fig.
3F,H). Furthermore, RNAi of PP4 regulatory subunit promoted Hhinduced Smo cell-surface accumulation (see Fig. S5D in the
supplementary material) but RNAi of PP2A regulatory subunit did
not (see Fig. S5F in the supplementary material). Without Hh
treatment, neither PP4 RNAi (Fig. 3E) nor Mts RNAi (Fig. 3G)
promoted Smo cell-surface accumulation, similar to the effect of OA
treatment in the absence of Hh (Fig. 3C). However, overexpression
of PP4 but not Mts blocked the Hh-induced Smo cell-surface
accumulation (see Fig. S5G,H in the supplementary material). These
data suggest that PP4 attenuates the Hh-induced Smo cell-surface
accumulation. In the absence of PP4, Smo is not fully
phosphorylated and its activation still depends on Hh, which may
explain why the accumulation of Smo by PP4 RNAi in anterior-most
regions of wing disc is unable to induce ectopic ptc-lacZ expression
(Fig. 1B).
Smo intracellular domain has a total of 26 Ser/Thr sites that are
phosphorylated upon Hh stimulation (Zhang et al., 2004). We have
previously shown that SmoSD12, or SmoSD123, in which the two
or three clusters of PKA and CK1 phosphorylation sites are replaced
by Asp to mimic phosphorylation, has elevated cell-surface
expression and signaling activity (Jia et al., 2004). We wondered
whether PP4 regulates the cell-surface accumulation of SmoSD12
or SmoSD123. Consistent with our previous findings (Jia et al.,
2004), Hh induced further cell-surface accumulation of both
SmoSD12 and SmoSD123 (Fig. 3J,M, respectively). Importantly,
the cell-surface accumulation of SmoSD12 or SmoSD123 was
elevated by PP4 RNAi (Fig. 3K,N) in a manner similar to Hh
treatment, suggesting that PP4 may regulate Smo cell-surface
RESEARCH ARTICLE
wing discs by the wing-specific MS1096 Gal4, CiFL levels were
significantly reduced (Fig. 4B) and dpp-lacZ expression diminished
(compare Fig. 4B,B⬘ with 4A,A⬘). Similar results were obtained
when a dominant-negative form of Mts (DN-Mts) was
overexpressed (see Fig. S6B-B⬘ in the supplementary material). In
these experiments, P35 was co-expressed to prevent cell death
owing to loss of PP2A activity (Zhang et al., 2006).
PP2A functions are mediated largely by the regulatory B subunit
that directs the phosphatase to distinct substrate (Virshup, 2000).
Thus, we examined the roles of PP2A regulatory subunits in
regulating Hh signaling. We found that Wdb RNAi inhibited the
activity of Hh signaling, indicated by the attenuated CiFL levels (Fig.
4C) and reduced dpp-lacZ expression (Fig. 4C⬘). Knockdown of
Tws by RNAi caused early lethality (not shown). We did not observe
Hh-related defects when knocking down other PP2A regulatory
subunits, including PP2A-B⬘ (CG7913) and PP2A-B⬙ (CG4733)
(not shown).
We also used wdb mutants to examine its physiological function
in regulating CiFL. As null mutation of wdb causes cell lethality
(Hannus et al., 2002), we used a wdb hypomorphic allele, WdbIP,
that harbors a stop codon at amino acid 332, resulting a significant
reduction of Wdb activity (Hannus et al., 2002). We examined Smo
and CiFL distribution in wing discs carrying clones homozygous for
WdbIP and found that CiFL levels were reduced (Fig. 4F) with Smo
accumulation unaffected (Fig. 4F⬘) in wdb mutant cells. These
results suggest that Wdb has a positive role in Hh signaling by
regulating Ci and Hh target genes, which is consistent with the
findings in a genetic screen where mts was identified as a positive
regulator in Hh pathway (Casso et al., 2008).
We next performed gain-of-function study and found that
expressing UAS-Mts or UAS-Wdb by ap-Gal4 elevated CiFL levels
(Fig. 4D,E) and induced ectopic dpp-lacZ expression (Fig. 4D⬘,E⬘,
respectively). By contrast, overexpressing HA-PP4 neither elevated
CiFL levels nor upregulated Hh target genes (see Fig. S7 in the
supplementary material; not shown). Although expressing UAS-Mts
Development 136 (2)
or UAS-Wdb alone induced little, if any, ectopic Ptc expression that
is indicative of high level of Hh signaling activity (see Fig. S8E,F in
the supplementary material), co-expressing UAS-Mts with UASWdb induced ectopic Ptc expression (see Fig. S8G in the
supplementary material). Expressing HA-CiFL induced ectopic ptclacZ in the P-compartment but rarely in the A-compartment (Fig.
4G); co-expressing Wdb with CiFL induced ectopic ptc-lacZ
expression in A-compartment cells away from the AP boundary
(Fig. 4H). These results strengthen the view that PP2A-Wdb
positively regulates Hh signaling activity.
Overexpression of Wdb and Mts elevated CiFL levels (Fig. 4D,E;
see Fig. S8A,B in the supplementary material) but did not influence
the Hh-induced Smo accumulation (see Fig. S8A⬘,B⬘ in the
supplementary material). Knockdown of endogenous Wdb by RNAi
decreased CiFL levels (see Fig. S8C in the supplementary material),
which was in line with the result of Mts RNAi (see Fig. S8D in the
supplementary material). However, we did not observe changes in
Smo accumulation when Wdb or Mts was knocked down (see Fig.
S8C⬘,D⬘ in the supplementary material). Consistently, expression of
DN-Mts did not result in Smo accumulation in wing discs (see Fig.
S6B⬙ in the supplementary material). Our data indicate that PP2A is
involved in Hh signaling by specifically regulating CiFL.
Genetic interaction between PP2A and Ci kinases
In the absence of Hh, CiFL is phosphorylated by multiple kinases
including PKA, GSK3 and CK1 (Jia et al., 2005; Smelkinson and
Kalderon, 2006), leading to its processing to generate CiREP that
blocks the expression of Hh target genes. To determine whether
PP2A positively regulates Hh signaling by counteracting Ci kinases,
thus downregulating CiFL phosphorylation, we examined the genetic
interaction between PP2A and kinases in wing discs. Consistent with
previous findings (Jia et al., 2005; Wang et al., 1999),
overexpressing the constitutively active form of PKA (mC*) by
MS1096 Gal4 attenuated the levels of CiFL (Fig. 5B, compare with
wild-type CiFL staining in Fig. 5A) and downregulated the
Fig. 4. PP2A is essential for Hh
signaling. (A,A’) A wild-type wing disc
was stained to show the expression of
CiFL and dpp-lacZ. The Ci antibody only
recognizes CiFL in wing discs. (B,B’) A
wing disc co-expressing UAS-MtsRNAi
with UAS-P35 by MS1096 Gal4 was
immunostained with Ci and β-gal
antibodies. Arrowhead in B indicates
the decreased CiFL levels. Arrowhead in
B’ indicates the downregulated dpplacZ expression. MS1096 Gal4 is
expressed at lower level in the ventral
region than in the dorsal region of the
wing disc (Jia et al., 2003). (C-C’) A
wing disc expressing UAS-WdbRNAi by
MS1096 Gal4. Arrowhead in C
indicates the decreased CiFL and
arrowhead in C⬘ indicates the
downregulated dpp-lacZ. (D-E’) Wing
discs expressing UAS-Mts or UAS-Wdb
by ap-Gal4. Arrowheads in D and E
show the elevation of CiFL and
arrowheads in D’ and E’ show the induced ectopic dpp-LacZ expression. (F-F’’) A wing disc bearing wdbIP homozygous clones that were marked by
the lack of GFP expression was immunostained with anti-Ci and anti-SmoN antibodies. Arrowhead in F shows the reduction of CiFL. Arrowhead in F’
shows the unaffected Smo accumulation in wdb mutant cells. (G,H) Wing discs expressing UAS-HA-CiFL alone, or along with UAS-Wdb by C765
Gal4. Arrowhead in H indicates the ectopic Ptc-lacZ expression in A-compartment cells.
DEVELOPMENT
312
Regulation of Smo and Ci by phosphatases
RESEARCH ARTICLE
313
Fig. 5. PP2A counteracts kinases to regulate Ci and Hh target gene expression. (A,A’) A wild-type wing disc was immunostained to show
CiFL and ptc-lacZ expression. (B,B’) A wing disc overexpressing UAS-mC* by MS1096 Gal4. Arrowhead in B shows the downregulated CiFL level and
arrowhead in B’ shows the attenuated ptc-lacZ expression. (C,C’) A wing disc overexpressing UAS-Wdb by MS1096 Gal4. Arrowhead in C shows
the upregulated CiFL. (D,D’) A wing disc co-expressing UAS-mC* with UAS-Wdb by MS1096 Gal4. Arrowhead in D shows reinstated CiFL and
arrowhead in D’ shows the partially restored ptc-lacZ expression. (E,F) Wing discs expressing UAS-CRL or co-expressing UAS-CRL with UAS-MtsRNAi
by MS1096 Gal4 and stained to show the levels of CiFL.
PP2A dephosphorylates Ci and attenuates Ci
processing
To determine whether PP2A regulates Ci phosphorylation, we then
examined CiFL phosphorylation status when PP2A was knocked
down by RNAi in S2 cells. As shown in Fig. 6, upon OA treatment,
CiFL underwent rapid changes in phosphorylation indicated by an
electrophoretic mobility shift (Fig. 6A, lane 2, compare with lane 1,
top panel). We found that Flag-CiFL showed low mobility when
endogenous Mts or Wdb was knocked down by RNAi (lanes 3 and
4, top panel). By contrast, Flag-CiFL did not exhibit a mobility shift
by PP4 or GFP RNAi (lanes 5 and 6, top panel). These data suggest
that CiFL is specifically regulated by PP2A. To further determine
whether the elevated levels of CiFL produced by overexpressing Mts
or Wdb in wing discs (Fig 4D,E; see Fig. S8A,B in the
supplementary material) were due to changes in CiFL
phosphorylation, we examined whether overexpressing
phosphatases could attenuate CiFL phosphorylation in S2 cells. OA
treatment consistently induced the mobility shift of CiFL (Fig. 6B,
lane 2; compare with lane 1, top panel). Expressing HA-Mts (Fig.
6B, lane3, top panel) or HA-Wdb (lane 4), but not HA-PP4 (lane 5),
alleviated the OA-induced CiFL mobility shift, suggesting that
PP2A, but not PP4, prevents CiFL phosphorylation and, thus, the
accumulation in wing discs.
We next asked whether downregulation of Ci phosphorylation
affects its processing. The proteolytic processing of CiFL requires
the activity of the proteasome. The levels of CiFL in wing discs were
elevated by treatment with the proteasome inhibitor MG132 (Fig.
6D, compare with 6C). We found that overexpressing UASWdbRNAi by ap-Gal4 destabilized CiFL in dorsal compartment cells
(Fig. 6E). However, downregulation of CiFL by Wdb RNAi was
prevented by the treatment with MG132 (Fig. 6F), suggesting that
PP2A acts upstream of the proteasome to dephosphorylate CiFL and
promote CiFL accumulation. Similarly, MG132 treatment prevented
the downregulation of CiFL by Mts RNAi (not shown).
To determine whether the accumulation of CiFL was due to the
blockade of CiFL processing, we assessed CiFL processing using an
in vivo function assay, in which UAS-HA-CiFL or UAS-HA-CiFL plus
UAS-Wdb were misexpressed in the P-compartment wing discs
carrying smo mutant clones and hh-lacZ reporter gene. Consistent
with previous findings (Jia et al., 2005), P-compartment smo mutant
cells expressing HA-CiFL blocked hh-lacZ expression (Fig. 6G-G⬘),
indicating that CiFL was processed to generate CiREP. By contrast, Pcompartment smo mutant cells co-expressing HA-CiFL with Wdb
partially suppressed hh-lacZ expression (Fig. 6H-H⬘), suggesting
that HA-CiFL is partially blocked to produce CiREP in the presence
of Wdb. We also assessed CiFL processing by immunoprecipitation
and western blot analysis. UAS-HA-CiFL was expressed either alone
or along with UAS-Wdb by MS1096 Gal4 in wing discs. HA-CiFL
did not give rise to detectable CiREP when Wdb was co-expressed,
whereas HA-CiFL alone was partially processed into CiREP (Fig. 6I).
Taken together, our data suggest that PP2A is a positive regulator in
Hh signaling by inhibiting the phosphorylation and processing of
CiFL.
DISCUSSION
Regulated phosphorylation of Smo and Ci are crucial events in
mediating Hh signal transduction. Previous studies have identified
multiple Ser/Thr kinases that regulate Hh signaling by
phosphorylating Smo and Ci; however, the Smo and Ci
phosphatases remain unknown. We are able to identify the
phosphatase that regulates Smo phosphorylation by using an in vivo
screen. Our screen differs from the previous screens because we use
an in vivo assay to examine Smo expression levels, which is a more
direct readout, and because knockdown of specific phosphatase
DEVELOPMENT
expression of Hh target genes such as ptc-lacZ (Fig. 5B⬘, compare
with wild-type ptc-lacZ expression in Fig. 5A⬘). Expressing UASWdb by MS1096 Gal4 elevated CiFL levels (Fig. 5C) but did not
affect ptc-lacZ expression (Fig. 5C⬘). Strikingly, co-expressing UASWdb with UAS-mC* attenuated the effect of mC*, as evident by the
rescue of CiFL levels (Fig. 5D) and ptc-lacZ expression near the AP
boundary (Fig. 5D⬘), suggesting that Wdb counteracts PKA to
regulate CiFL and Hh target genes. We previously generated CK1
RNAi transformant, UAS-CRL, which produces dsRNA that
efficiently interferes with the activity of endogenous CK1 (Jia et al.,
2005). Expressing UAS-CRL in wing discs caused accumulation of
CiFL (Fig. 5E) (Jia et al., 2005). To determine whether PP2A also
counteracts CK1 to regulate Ci, we co-expressed UAS-CRL with
UAS-MtsRNAi by MS1096 Gal4. We found that the elevation of CiFL
caused by CRL expression was severely restricted by co-expressing
UAS-MtsRNAi (Fig. 5F), suggesting that PP2A also counteracts CK1
activity in regulating CiFL.
314
RESEARCH ARTICLE
Development 136 (2)
Fig. 6. PP2A downregulates Ci phosphorylation and blocks Ci proteolytic processing. (A) CiFL phosphorylation is upregulated by PP2A RNAi.
S2 cells were transfected with Flag-Ci and treated with OA or indicated dsRNA. Cell extracts were subjected to direct western blot with anti-Flag
antibody. Arrow indicates hyperphosphorylated forms of Ci and arrowhead indicates the hypophosphorylated or unphosphorylated forms. βTubulin serves as loading control. The knockdown efficiency of individual phosphatase was estimated by the method used for Fig. 2A. (B) PP2A
downregulates CiFL phosphorylation. S2 cells were transfected with Flag-Ci alone or along with indicated HA-tagged phosphatase and treated with
or without OA. Cell lysates were probed with anti-Flag or anti-HA antibodies. (C) A disc shows the wild-type CiFL staining. (D) A wing disc shows the
CiFL stabilization by the treatment of proteasome inhibitor MG132. (E,F) Wing discs expressing UAS-WdbRNAi by ap-Gal4 were treated with or
without MG132 and stained to show CiFL. Arrowhead in E indicates the destabilized CiFL by Wdb RNAi. Arrowhead in F indicates that the
destabilized CiFL by Wdb RNAi was restored by MG132 treatment. (G-H’) Wing discs bearing smo3 clones and expressing UAS-HA-CiFL alone or
along with UAS-Wdb by MS1096 Gal4 were stained to show the expression of GFP (green) and hh-lacZ (red). Arrowheads in G and H indicate smo3
clones that are marked by the lack of GFP expression. Arrowheads in G’ and H’ indicate the hh-lacZ expression in smo3 cells. (I) Western blot
analysis of protein extracts from wing discs expressing UAS-HA-CiFL or co-expressing UAS-HA-CiFL with UAS-Wdb using the MS1096 Gal4. Protein
extracts were prepared from 400 wing discs, immunoprecipitated and blotted with HA antibody. (J) A model for the involvement of PP4 and PP2A
in Hh signaling. PP4 negatively regulates Hh signal transduction by antagonizing the phosphorylation of Smo. PP2A positively regulates Hh pathway
by counteracting kinases to downregulate CiFL phosphorylation and attenuate its proteasome-mediated processing.
PP4 and PP2A play distinct roles in the Hh
pathway
In this study, we have identified PP4 and PP2A to be negative and
positive regulators in the Hh pathway, and we showed that they exert
their roles through Smo and Ci, respectively. Are PP4 and PP2A the
only phosphatases in the Hh pathway? Although our data suggest that
PP4 is a phosphatase for Smo, we cannot exclude the possibility of
the involvement of other phosphatase(s). Hh induces extensive Smo
phosphorylation at numerous Ser/Thr sites, and multiple kinases are
involved in these phosphorylation events (Apionishev et al., 2005;
Jia et al., 2004; Zhang et al., 2004). It might be possible that multiple
phosphatases could be involved. In addition, our loss-of-function
studies on PP2A regulating Ci are not based on null mutations. This
was due to the fact that genetic null mutations of the catalytic and
regulatory subunits cause cell lethality (Sathyanarayanan et al.,
2004). Thus, the results might not be exclusive.
PP4 and regulation of Smo phosphorylation
We showed that removal of PP4 by RNAi in wing discs induced
Smo accumulation in A-compartment cells both near and away from
the AP boundary (Fig. 1B). In addition, PP4 RNAi induced the
elevation and anterior expansion of Hh target gene expression (Fig.
1B⬘). However, the accumulated Smo caused by PP4 RNAi did not
ectopically activate Hh target genes in cells away from the AP
boundary (Fig. 1B⬘, data not shown). In addition, although Smo
phosphorylation was potentiated by knocking down PP4 or
abolishing Smo-PP4 interaction (Fig. 2A,E), the elevated
DEVELOPMENT
gene(s) involved in Smo dephosphorylation might not affect the
pathway activity in a significant way and such gene(s) could have
been missed in the previous RNAi screens with cultured cells (Lum
et al., 2003a; Nybakken et al., 2005). In this study, we have
identified PP4 as a novel Hh signaling component that regulates
Smo phosphorylation. Our study provides the first evidence for the
physiological Smo and Ci phosphatases, and uncovers the
underlying mechanism of Smo regulation by phosphatase (Fig. 6J).
phosphorylation did not suffice to promote Smo cell-surface
accumulation (Fig. 3E,O). These data suggest that the basal
phosphorylation of Smo regulated by PP4 is not sufficient to activate
Smo, and that de novo Smo activation still depends on Hh.
Previous studies have shown that PKA and CK1 are required for
Hh-induced Smo accumulation and signaling activity.
Phosphorylation-deficient forms of Smo (with PKA or CK1 sites
mutated to Ala) are defective in Hh signaling, whereas SmoSD123,
the phosphorylation-mimicking Smo, has potent signaling activity
and high level of cell-surface accumulation (Jia et al., 2004; Zhang
et al., 2004). Thus, the PKA and CK1 sites are apparently crucial in
mediating Smo phosphorylation and activation. Hh treatment may
cause increased phosphorylation at these sites. In addition to PKA
and CK1 sites, there are many other Ser/Thr residues that are
phosphorylated upon Hh stimulation (Zhang et al., 2004). Although
phosphorylation-mimicking mutations at these sites alone did not
have discernible effect on Smo, their phosphorylation could
modulate the cell-surface accumulation and activity of Smo
phosphorylated at the three PKA/CK1 sites, which may at least in
part explain why cell-surface accumulation and activity of
SmoSD123 is still regulated by Hh (Jia et al., 2004) (Fig. 3M). Here,
we found that removing PP4 alone promoted Smo phosphorylation
but did not elevate the cell-surface accumulation of Smo. It is
possible that high levels of basal Smo phosphorylation in the
absence of PP4 do not reach the threshold for promoting Smo cellsurface accumulation. It is also possible that basal Smo
phosphorylation mainly occurs at sites other than the crucial
PKA/CK1 phosphorylation clusters. In support of this notion, we
found that knockdown PP4 by RNAi promoted SmoSD123 to
further accumulate on the cell surface in the absence of Hh (Fig. 3N).
How is Smo phosphorylation regulated? Hh may regulate Smo
phosphorylation by regulating the accessibility of its kinase and/or
phosphatase. In this study, we found that Smo interacts with PP4
through amino acids 626-678, a region we previously mapped to be
a Cos2-interacting domain (Liu et al., 2007). We further found that
Smo-PP4 association diminished when Cos2 was knocked down by
RNAi (Fig. 2I). Our previous study revealed that Cos2 impedes Hhinduced Smo phosphorylation by interacting with amino acids 626678 of Smo and Hh-induced phosphorylation of Cos2 at Ser572
dissociates Cos2 from amino acids 626-678 of Smo, thereby
alleviating its inhibition on Smo phosphorylation (Liu et al., 2007).
In this study, we found that Cos2 inhibits Smo phosphorylation by
recruiting PP4 and Hh promotes Smo phosphorylation by preventing
Cos2-PP4 complex from binding to amino acids 626-678 of Smo.
Smo⌬626-678, when not interacting with PP4, could still interact
with Cos2 via a Cos2-interaction domain near the Smo C terminus
(Jia et al., 2003). The Cos2-binding Smo C terminus might not
recruit PP4. Taken together, our findings suggest that Hh may
promote Smo phosphorylation at least in part by reducing the
accessibility of a phosphatase.
PP2A and regulation of Ci phosphorylation
As stated in the Introduction, phosphorylation of Ci/Gli controls the
balance of its activator and repressor activity. Here, we demonstrate
a role of PP2A in dephosphorylating Ci and attenuating Ci
processing. However, it is not known whether Hh regulates PP2A to
dephosphorylate Ci. Previous studies have shown that Hh interferes
with the Cos2-Ci-kinase protein complex (Zhang et al., 2005). It is
possibly that Hh also regulates Ci phosphatase, or the accessibility
of the phosphatase. Future studies should determine whether PP2A
interacts with Cos2-Ci and whether such interaction is regulated by
Hh.
RESEARCH ARTICLE
315
Many aspects of Smo and Ci/Gli regulation are conserved across
species. For example, both Drosophila and mammalian Smo
proteins undergo a conformational switch in response to Hh
stimulation (Zhao et al., 2007). Ci/Gli proteolysis is mediated by
the same set of kinases and E3 ubiquitin ligases (Jiang, 2006). In
addition, it has been shown that PP2A is involved in vertebrate Hh
signaling, probably by regulating Gli nuclear localization and
activity (Krauss et al., 2008; Rorick et al., 2007). Therefore, it
would be interesting to determine whether PP4 and PP2A play
similar roles in regulating phosphorylation of vertebrate Smo and
Gli.
We thank Drs Suzanne Eaton, David Glover, Amita Sehgal, David Robbins,
Robert Holmgren, C.-ting Wu and Jin Jiang for providing valuable reagents;
VDRC for fly strains; DGRC for cDNA clones; and DSHB for antibodies. We
thank Dr Hugo Bellen and Koen Venken for vas-phi-zh2A-VK5 fly stock. We
are grateful to Drs Jin Jiang for comments on the manuscript, Leoncio Vergara
for assistance with cell-surface Smo quantification and Tianyan Gao for helpful
discussions. This work was supported by grants from the National Institute of
Health (R01 GM 079684), the American Heart Association and the American
Cancer Society Institutional Research Award to J.J. Deposited in PMC for
release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/307/DC1
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