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. 2011 Jun;9(6):e1001083.
doi: 10.1371/journal.pbio.1001083. Epub 2011 Jun 14.

Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened

Yongbin Chen  1 , Noriaki Sasai, Guoqiang Ma, Tao Yue, Jianhang Jia, James Briscoe, Jin Jiang
Affiliations

Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened

Yongbin Chen et al. PLoS Biol. 2011 Jun.

Erratum in

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CK1α and GRK2 regulate Smo phosphorylation and conformation.
(A) Gli-luciferase assay in NIH 3T3 cells transfected with Smo and kinase expressing constructs and treated with or without Shh-conditioned medium. (B–D) FRET analysis in NIH 3T3 cells transfected with Smo-CFPC/SmoYFPC (B), SmoCFPL2YFPC (C), or SmoCFPN/SmoYFPN (D) and treated with or without Shh-conditioned medium or cotransfected with indicated kinase expressing constructs. Filled and open circles in the cartoons denote CFP and YFP, respectively. (E–F) Cell extracts were prepared from NIH 3T3 cells transfected by indicated constructs and treated with or without indicated reagents, separated on SDS-PAGE gel containing 25 mM Phos tag-conjugated acrylamide, followed by immunoblotting with an anti-Myc antibody. The inhibitors used are: CK1 inhibitor, CKI-7 (10 µM); GRK inhibitor Heparin (HP, 1 µM). PP indicates λ-phosphatase treatment. (G) Gli-luc assay in NIH 3T3 cells stably expressing shRNA targeting CK1α, GRK2, or GRK5 and transfected with or without Smo and treated with or without Shh-conditioned medium. (H) Cell extracts were prepared from NIH 3T3 cells stably expressing shRNA targeting CK1α, GRK2, or GRK5 and transfected with Smo-Myc and treated with or without Shh-conditioned medium, separated on the Phos tag-conjugated SDS-PAGE gel and immunoblotted with an anti-Myc antibody.
Figure 2
Figure 2. CK1 and GRK phosphorylate multiple sites in Smo C-tail.
(A–B) In vitro kinase assay using a recombinant CK1δ or GRK5 and purified GST-fusion proteins carrying indicated Smo fragments. (C) A schematic drawing of a full-length Smo with the sequences of the six CK1/GRK phosphorylation sites shown underneath (S0–S5) and color coded: blue for CK1 specific sites; red for GRK specific sites; green for sites phosphorylated by both CK1 and GRK. Smo variants with the indicated substitutions are listed. Sequence alignment shows that the CK1/GRK phosphorylation sites are highly conserved among Mouse (m), Human (h), Chick (g), and Zebrafish (z) Smo proteins. (D) Cell extracts were prepared from NIH 3T3 cells transfected with Smo-Myc or SmoSA0–5-Myc and with or without cotransfection of the indicated kinase expressing constructs and treated with or without Shh-conditioned medium. The extracts were separated on Phos tag-conjugated SDS-PGAE gel and immunoblotted with an anti-Myc antibody. PP indicates λ-phosphatase treatment. (E) Cell extracts were prepared from NIH 3T3 cells transfected with Smo-Myc and with or without indicated kinase expressing constructs, followed by treating with or without Shh-conditioned medium or indicated kinase inhibitors. The extracts were subjected to SDS-PAGE, followed by immunoblotting with the PS1 antibody. The membrane was stripped and probed with Myc antibody for Smo-Myc. (F) Cell extracts prepared from shRNA lines targeting CK1α, GRK2, or GRK5 transfected by Smo-Myc and treated with or without Shh-conditioned medium were analyzed as in (E). (G) NIH 3T3 cells were transiently transfected with Smo-Myc and treated without or with increasing levels of ShhN peptides (1 nM, 2 nM, 5 nM). 8XGliBS-luc activities were normalized by control pRL-TK. Cell extracts were separated on Phos tag-conjugated (top panel) or regular SDS-PAGE gel (bottom two panels), followed by immunoblotting with Myc or PS1 antibody. (H–I) Cell extracts from NIH 3T3 cells transfected with the indicated constructs and treated with or without Shh-conditioned medium, CYC (cyclopamine; 10 µM), SAG (200 nM), or 20-OHC (20α-hydroxycholesterol; 10 µM) were separated on SDS-PAGE gel and probed with PS1 antibodies. The membranes were stripped and probed with Myc antibody to monitor Smo-Myc levels.
Figure 3
Figure 3. Phosphorylation at multiple CK1 and GRK sites regulates Smo activity both in vitro and in vivo.
(A–C) Gli-luc activity assays for smo−/− MEFs transfected with the indicated Smo expressing constructs together with the 8XGliBS-luc reporter gene and a control pRL-TK. The cells were treated with or without Shh-conditioned medium 2 d after transfection. (D–E) HH st11–12 chick neural tubes were electroporated with the indicated expression constructs and assayed by immunohistochemistry 48 h after transfection. (D) The expression of Pax7, Islet1/2, Olig2, and Nkx2.2 in anterior thoracic regions of embryos transfected with wild-type Smo (SmoWT), SmoSD123, SmoA1, or SmoA1SA1–5. Cells expressing the constructs were identified by CFP (green). Arrows indicate the expanded expression of Islet1/2, Olig2, and Nkx2.2 and the reduction of Pax7 expression. SmoA1 exhibited potent constitutive activity, leading to the repression of Pax7 and induction of ectopic Islet1/2, Olig2, and Nkx2.2. Mutating multiple CK1/GRK sites in SmoA1 (A1SA1–5) diminished its constitutive activity. SmoSD123 also exhibited constitutive activity, resulting in a reduction of Pax7 expression and expansion of Islet1/2, Olig2, and Nkx2.2, albeit less dramatic than SmoA1. Note that there was also a subtle expansion of Olig2 in some embryos transfected with SmoWT. (E) Embryos co-transfected at a ratio of 2∶1 with Ptc1Δloop2 (PtcΔ2) and either Smo-WT, SmoSD0–5, or SmoA1 and assayed for Pax7, Islet1/2, Olig2, and Nkx2.2 expression. Ptc1Δloop2 inhibits Shh signaling resulting in the repression of Islet1/2, Olig2, and Nkx2.2, and a ventral expansion of Pax7. SmoSD123 and SmoSD0–5 but not SmoWT overcame the dominant inhibitory effect of Ptc1Δloop2.
Figure 4
Figure 4. Regulation of Smo ciliary localization by CKI/GRK-mediated phosphorylation.
(A–B) Wild-type MEFs infected with retrovirus encoding CFP-tagged wild-type Smo or indicated Smo variants and treated with or without Shh-conditioned medium were immunostained to show the expression of Acetylated (Ac)-tubulin (Red) that labels the primary cilium, GFP (green) that labels the CFP-tagged Smo proteins, and DRAQ5 (blue) that labels the nucleus (A). The insets show enlarged views of the selected regions with shifted overlays. Quantification of ciliary localization of infected Smo variants as indicated by the percentage of GFP+ cilia is shown in (B). Over 100 ciliated cells were counted for each Smo construct. (C) Shh promotes the interaction between Smo with β-arr2. NIH 3T3 cells were transfected with the indicated Myc-tagged Smo variants and YFP tagged β-arr2. Cells lysates were subjected to western blot analysis with Myc and GFP antibodies or immunoprecipitated with the Myc antibody, followed by western blot analysis with the GFP antibody. Asterisk indicates IgG heavy chain. (D) FRET analysis of NIH 3T3 cells transfected with indicated C-terminally CFP-tagged Smo variants and C-terminally YFP-tagged β-arr2 and treated with or without Shh-conditioned medium.
Figure 5
Figure 5. Shh, A1, SAG, and cyclopamine regulate Smo conformation through CK1/GRK-mediated phosphorylation.
(A–C) FRET analyses of NIH 3T3 cells transfected with C-terminally CFP/YFP-tagged wild type Smo or Smo variants with the indicated mutations and treated with or without Shh-conditioned medium (mean ± s.d., n≥10). (D) FRET analysis in NIH 3T3 cells transfected with Smo-CFPC/YFPC, SmoSA0–5-CFPC/YFPC, or SmoSD0–5-CFPC/YFPC, and treated without or with Shh-conditioned medium, 200 nM SAG, or a combination of Shh-conditioned medium and 10 µM cyclopamine (mean ± s.d., n≥10).
Figure 6
Figure 6. Smo phosphorylation at the primary cilium.
(A) NIH 3T3 cells stably expressing Smo-CFP were either untreated (control) or treated with Shh-conditioned medium (Shh), SAG (200 nM), 20-OHC (10 µM), CYC (10 µM), or a combination of Shh-conditioned medium and CYC (10 µM). Cells were immunostained to show the expression of acetylated tubulin (red; primary cilium), GFP (green; Smo), and PS1 (phosphorylated Smo; blue). Images in the insets are shifted overlays of the selected fields. (B–C) The percentage of Smo-CFP (GFP) or phosphorylated Smo (PS1) positive primary cilia at different time after cells were treated with or without Shh or SAG (200 nM). Over 100 ciliated cells were counted for each time point, n = 3. (D) NIH 3T3Smo-CFP cells were treated with 200 nM SAG for 1, 2, 4, or 24 h, followed by immunostaining to show the expression of acetylated tubulin (red), GFP (green), and PS1 (blue). Representative images with shifted overlay were shown for each time point. Histograms underneath show the relative intensities of PS1 or GFP fluorescence signals and their ratios (PS1/CFP) at each time point. The ratios are normalized to that of 24 h time point, which is set at 100%. (E) NIH 3T3Smo-CFP cells were treated with Shh-conditioned medium for 0, 1, 2, 4, or 24 h. Cell extracts at each time point were separated on SDS-PAGE and probed with the indicated antibodies. Histograms show the relative intensities of PS1 and GFP bands quantified by the ImageJ software and their ratio (PS1/CFP) normalized to that at 24 h. (F) Cell extracts prepared from NIH 3T3 cells transfected with the indicated constructs and treated with or without Shh-conditioned medium were subjected to western blot analysis with the indicated antibodies.
Figure 7
Figure 7. Shh promotes CK1α/GRK2 binding to Smo.
(A) Schematic drawings of full-length and truncated Smo with point mutations indicated by the asterisks. Black boxes denote the transmembrane domains. L430A is located in the third intracellular loop; A1 in the seventh transmembrane domain; M1, S570A, and I573A are located in the membrane proximal region of the Smo C-tail. (B–C, E–H) Coimmunoprecipitation assays to determine the interaction between CK1α/GRK2 with different forms of Smo. NIH 3T3 cells were transfected with the indicated Myc-tagged or HA-tagged Smo constructs, followed by immunoprecipitation and western blot analysis with indicated antibodies. Cell lysates were also directly immunoblotted by the indicated antibodies. Histogram in (C) shows the quantification of (B) with CK1α binding normalized by the Smo input and compared with lane 1. *p<0.05, **p<0.01, ***p<0.005. Western blots were quantified using the ImageJ software followed by Prism analysis, n = 3. Quantifications of (G) and (H) are shown in Figure S5. (D) NIH 3T3 cells untreated (control) or treated with Shh-conditioned medium were immunostained to show the expression of acetylated tubulin (red), CK1α (green), and DRAQ5 (blue). Images in the insets are shifted overlays of the selected fields. (I) NIH 3T3 cells were transfected with the indicated Myc-tagged Smo constructs either alone or together with a Flag-tagged CK1α construct and treated with or without Shh-conditioned medium, followed by immunoprecipitation and western blot analysis with the indicated antibodies. Cell lysates were also directly immunoblotted by the indicated antibodies. (J) A model for how Smo phosphorylation is regulated by Shh. See text for details.
Figure 8
Figure 8. A unified mechanism for Smo activation in different species.
Multi-site phosphorylation by distinct but overlapping sets of kinases activates mSmo and dSmo by regulating their subcellular localization and conformation. See text for details.
See this image and copyright information in PMC

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