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. 2012 Jun 15;366(2):172-84.
doi: 10.1016/j.ydbio.2012.04.007. Epub 2012 Apr 19.

Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner

Junkai Fan  1 , Yajuan Liu, Jianhang Jia
Affiliations

Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner

Junkai Fan et al. Dev Biol. .

Abstract

The activation of Smoothened (Smo) requires phosphorylation at three clusters of Serine residues in Drosophila Hedgehog (Hh) signaling. However, the mechanism by which phosphorylation promotes Smo conformational change and subsequently activates Smo in response to Hh gradient remains unclear. Here, we show that the conformational states of Smo are determined by not only the amount but also the position of the negative charges provided by phosphorylation. By using a Smo phospho-specific antibody, we demonstrate that Smo is differentially phosphorylated at three clusters of serine residues in response to levels of Hh activity. Mutating the first cluster, compared to mutating the other clusters, impairs Smo activity more severely, whereas mutating the last cluster prohibits C-terminus dimerization. In addition, phosphorylation of the membrane proximal cluster promotes phosphorylation of the distal cluster. We propose a zipper-lock model in which the gradual phosphorylation at these clusters induces a gradual conformational change in the Smo cytoplasmic tail, which promotes the interaction between Smo and Costal2 (Cos2). Moreover, we show that Hh regulates both PKA and CK1 phosphorylation of Smo. Thus, the differential phosphorylation of Smo mediates the thresholds of Hh activity.

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Figures

Fig. 1
Fig. 1. Characterization of a Smo phospho-specific antibody (SmoP)
(A) Smo sequence indicating the three phosphorylation clusters located in the C-tail, with PKA sites marked in red and CK1 sites in green. (B) Smo phosphorylation by PKA and CK1 is detected by the anti-SmoP antibody. An in vitro kinase assay is shown here using the purified GST-Smo proteins and the commercial PKA and CK1 kinases. GST is used as a control. (C) S2 cells were transfected with Myc-SmoWT or Myc-SmoSAS and treated with HhN-conditioned medium or control medium, in combination with H-89 and/or CK1–7 inhibitors. To normalize the levels of Smo, 50 μM MG132 and 15 mM NH4Cl was used to block Smo degradation, and samples were normalized for loading. All figures in this study showing the levels of Myc-Smo were normalized by this method. Cell extracts were immunoprecipitated with the anti-Myc antibody and blotted with either anti-Myc to detect Smo phosphorylation, indicated by its mobility shift on the SDS gel, or anti-SmoP, to directly detect Smo phosphorylation. GFP served as a transfection and loading control. (D) S2 cells were transfected with Myc-SmoWT and treated with OA or HhN-conditioned medium followed by immunoprecipitation with the anti-Myc antibody and western blot with anti-Myc or anti-SmoP antibodies. GFP served as a transfection and loading control. Myc-Smo was normalized by the method described above. (E–E′) A wing disc expressing Flag-SmoWT under MS1096 Gal4 control were immunostained with anti-SmoP and anti-Flag antibodies. (F–F″) A large magnification of a wing disc expressing Flag-Smo under MS1096 Gal4. The arrow indicates SmoP staining in A compartment cells near the A/P boundary. The dash line indicates the A/P boundary that is marked by Ci staining. (G–H′) Wing discs expressing Flag-SmoWT with Hh or Flag-SmoSAS by MS1096-Gal4 were stained for SmoP and Flag. All wing imaginal discs shown in this study were oriented with anterior on the left and ventral on the top.
Fig. 2
Fig. 2. Gradient of Hh signaling activity induces differential phosphorylation of Smo
(A–B) A ptc-luc reporter assay in S2 cells. S2 cells in 6-well plates were co-transfected with 40 ng (Hh+), 80 ng (Hh++), or 160 ng (Hh+++) of UAST-HhN and the indicated amount of either UAST-Ci or tub-Ci. The y-axis represents normalized ptc-luc activity. (C–C’) A time course experiment using S2 cells co-transfected with Myc-SmoWT and tub-Ci and treated with 60% of Hh-medium in 6-well plates. Cells were then split into two halves, one half for detection of Smo phosphorylation by western blot analysis using the Myc-IP followed by western blot with the anti-Myc or anti-SmoP antibodies, and the second half for the luciferase assay. Myc-Smo was normalized by the method described above. (D) A time course experiment to examine Smo cell surface accumulation by using the same treatments of S2 cells as used for C–C’. Cells were immunostained with the anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panel). Quantification of cell surface and total Smo levels is shown in the bottom panel (mean ± SD; n ≥ 20). The numbers below the bars indicate the percentage of Smo on the cell surface. The intensity of whole cell signal from 24 h treatment was set as 100. (E–E’) S2 cells in 6-well plates were co-transfected with the indicated construct and treated with different amounts of Hh-medium. The cells were then split into two halves, one for detection of Smo phosphorylation, and the second for the luciferase assay. Myc-Smo was normalized by the method described above. (F) A parallel experiment similar to that used in E–E’ to examine Smo cell surface accumulation under different conditions. Quantification is shown in the bottom panel (mean ± SD; n ≥ 20). The numbers below the bars indicate the percentage of Smo on the cell surface. The intensity of whole cell signal from the highest Hh treatment was set as 100.
Fig. 3
Fig. 3. Changing the sequence of the phosphorylation clusters has no effect on Smo activity and responsiveness to Hh
(A) Sequence alignment of SmoWT and Smo222 in which the 1st and 3rd clusters were substituted with the 2nd cluster. (B and C) Wing discs carrying smo mutant clones immunostained for Ptc (red), En (red), or GFP (green). The smo mutant clones were recognized by the lack of GFP expression and the A/P boundaries were determined by Ci staining (not shown). (D–G) Wing discs containing smo mutant clones and expressing either Myc-Smo222 by MS1096 Gal4 or Smo222 by the tubulinα promoter were immunostained to show Ptc, En, and GFP. The arrows indicate the rescued ptc or en expression by expressing Smo222 in smo cells. (H and I) Wing discs expressing Myc-SmoWT or Myc-Smo222 by MS1096 Gal4 were stained for dpp-lacZ expression. The arrows indicate the comparable dpp-lacZ expression when either forms of Smo are expressed. (J) A wing discs expressing Smo222 by the tubulinα promoter is stained for SmoP and SmoN. (K) A large magnification image of the P compartment in the wing disc from Figure J. The arrowheads indicate the co-localization of SmoP and SmoN on the cell membrane. (L and M) Large magnification images of the cells in P compartment of the wing discs expressing either Myc-SmoWT or Myc-Smo222. The arrowheads indicate the co-localization of Myc and SmoP signals. (N) S2 cells were transfected with Myc-Smo222 and treated with Hh-medium in combination with H-89 or CK1–7. Cell lysates were subjected to immunoprecipitation with anti-Myc followed by a western blot with anti-Myc or anti-SmoP antibodies. GFP served as a transfection and loading control. Myc-Smo was normalized by the method described above. (O and P) Wing discs expressing SmoWT or Smo222 by the tubulinα promoter, or co-expressing SmoWT or Smo222 by the tubulinα promoter along with UAS-Hh by MS1096 Gal4 were collected from flies cultured at either 25 °C or 19 °C, and then subjected to direct western blot with the anti-SmoP antibody to detect the phosphorylated forms of SmoWT or Smo222, or with anti-Flag antibody to detect the total Smo. The ratio of phosphorylated Smo to total Smo is shown in the right panel (each data set was from three repeats).
Fig. 4
Fig. 4. Intra-molecular mutations contribute differently to Smo activity
(A) A schematic drawing of Smo variants and their in vivo activity. In the sequence, enlarged black Ser residues are the PKA sites and enlarged grey Ser residues are CK1 sites. Individual constructs were assayed for Hh signaling activity by overexpression under the MS1096 Gal4 driver. Ectopic activity was scored by activated dpp-lacZ expression. Rescue experiments were carried out to examine whether expressing the indicated constructs by MS1096-Gal4 can restore Hh target gene expression in smo mutant cells. (B–O) Examples of the ability of each individual construct to induce ectopic dpp-lacZ expression in wing discs, when driven by MS1096 Gal4. Of note, the activity of SmoDDA is dramatically reduced (L) compared to SmoDDS (G), and the activity of SmoAPKADDCK1 is comparable to SmoDDD by inducing similar ectopic dpp-lacZ expression (compare M with C). (P–Q′) Wing discs expressing SmoDDD or SmoAPKADDCK1 were stained for Ptc-lacZ (P and Q) and En (P′ and Q′). (R) The activity of each individual construct was assayed by the ptc-luc reporter in S2 cells using the method described above. (S) The cell surface accumulation of each individual construct was measured with anti-SmoN antibody staining by using the method described above. Please note that the cell surface accumulation of SmoDDA is dramatically reduced compared to SmoDDS. (T) FRET efficiency from the indicated WT or mutant Smo with CFP or YFP tagged to their C-termini. SmoDDA, SmoDAD, and SmoADD have significantly reduced FRET values, and are much less responsive to Hh.
Fig. 5
Fig. 5. Dominant-negative activity of Smo with phospho-deficient mutations at different combination of clusters
(A) A schematic drawing of Smo mutants tested in this figure. (B–O) Examples of dominant-negative activity of the indicated Smo constructs when driven by ap-Gal4 in wing discs. Arrowheads in B, H, L, and N indicate the constrained ptc-lacZ expression in A compartment cells near the A/P boundary. Arrows in C, I, M, and O indicate the attenuated/blockade of en expression in A compartment cells near the A/P boundary. The impaired ptc-pacZ and en expression suggest the dominant-negative activity of each individual Smo construct. (P–S) Wing discs containing smo mutant clones and expressing SmoAAS or SmoSAA from the tubulinα promoter were immunostained to show Ptc, En, and GFP. The smo mutant clones are recognized by the lack of GFP expression. The arrows indicate that SmoAAS and SmoSAA do not rescue ptc or en expression in cells lacking endogenous smo. (T) The ptc-luc reporter activity of the indicated Smo constructs that were expressed in S2 cells. (U) FRET efficiency from S2 cells transfected with the indicated Smo constructs and treated with Hh-medium or control medium. Mutations at any of the two clusters interfere with their responsiveness to Hh.
Fig. 6
Fig. 6. Phosphorylation at one cluster promotes the phosphorylation at the nearby distal cluster
(A) S2 cells were transfected with the indicated Smo constructs and treated with Hh-medium. Cell extracts were immunoprecipitated with anti-Myc antibody followed by western blot with either anti-SmoP or anti-Myc antibodies. GFP served as a transfection and loading control. Myc-Smo was normalized by the method described above. The bottom panel shows that the same set of Smo constructs were also subjected to luciferase assay in S2 cells with co-transfection of tub-Ci and the treatment of Hh-medium. The luciferase value of SmoASS was set as 1. (B–E) Wing discs expressing the indicated Smo constructs by MS1096 Gal4 were stained for SmoP. (F) S2 cells were transfected with the indicated Smo constructs and treated with HhN-conditioned medium or control medium. Immunoprecipitation was carried out with the anti-Myc antibody. To detect phosphorylation at a specific position, the anti-SmoP antibody was used to detect phosphorylation of the 2nd cluster sequence. GFP served as a transfection and loading control. Myc-Smo was normalized by the method described above. (G) S2 cells transfected with Myc-Smo222DSS or Myc-Smo222ASS were treated with Hh-medium or control medium. Cell extracts were immunoprecipitated and a western blot was performed with the indicated antibodies. GFP served as a transfection and loading control. Myc-Smo was normalized by the method described above.
Fig. 7
Fig. 7. Mechanisms of Smo phosphorylation and dimerization at the C-terminus
(A) S2 cells were co-transfected with HA-Cos2 and Smo variants. Immunoprecipitation was carried out with anti-Myc antibody followed by western blot with anti-Myc to detect the expression of Smo and anti-HA antibody to detect the bound Cos2. Myc-Smo was normalized by the method described above. Cell lysates were also subjected to direct western blot with the anti-HA antibody to examine the expression of Cos2. (B–D) A model of Smo differential phosphorylation that mediates thresholds of Hh signaling activity: Gene expression pattern in response to Hh activity gradient (B); Increasing Hh activity elevates the levels of Smo phosphorylation (C); Gradient Hh activity signal transduction by differential Smo phosphorylation (D). (E) A zipper-lock model demonstrates the intra-molecular mechanism of the Smo conformational change that is regulated by Hh. Dimerization of the Smo C-terminal tail promotes Cos2 binding.
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