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. 2010 Sep 15;24(18):2054-67.
doi: 10.1101/gad.1948710.

G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila

Yongbin Chen  1 , Shuang Li, Chao Tong, Yun Zhao, Bing Wang, Yajuan Liu, Jianhang Jia, Jin Jiang
Affiliations

G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila

Yongbin Chen et al. Genes Dev. .

Abstract

G protein-coupled receptor kinase 2 (Gprk2/GRK2) plays a conserved role in modulating Hedgehog (Hh) pathway activity, but its mechanism of action remains unknown. Here we provide evidence that Gprk2 promotes high-level Hh signaling by regulating Smoothened (Smo) conformation through both kinase-dependent and kinase-independent mechanisms. Gprk2 promotes Smo activation by phosphorylating Smo C-terminal tail (C-tail) at Ser741/Thr742, which is facilitated by PKA and CK1 phosphorylation at adjacent Ser residues. In addition, Gprk2 forms a dimer/oligomer and binds Smo C-tail in a kinase activity-independent manner to stabilize the active Smo conformation, and promotes dimerization/oligomerization of Smo C-tail. Gprk2 expression is induced by Hh signaling, and Gprk2/Smo interaction is facilitated by PKA/CK1-mediated phosphorylation of Smo C-tail. Thus, Gprk2 forms a positive feedback loop and acts downstream from PKA and CK1 to facilitate high-level Hh signaling by promoting the active state of Smo through direct phosphorylation and molecular scaffolding.

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Figures

Figure 1.
Figure 1.
Reduction of Gprk2 modifies the phenotypes caused by a dominant-negative Smo. (A,B) A wild-type wing (A) or a wing expressing UAS-Smo−PKA12 with the C765 Gal4 driver (C765-Smo−PKA12) (B). (Arrow in B) Overexpression of Smo−PKA12 causes a partial fusion between vein 3 and vein 4. (C–F) Heterozygote for a Gprk2 deficiency (C), a P-element insertion allele of Gprk2 (Gprk206936) (D), an excision allele (Gprk2Δ15) (E), or Gprk2 RNAi (F) enhanced the fused wing phenotype caused by C765-Smo−PKA12. (G–J) C765-Smo−PKA12 reduces ptc expression (H), which is enhanced by the Gprk2Δ15 heterozygote (I) or Gprk2 RNAi (J).
Figure 2.
Figure 2.
Genetic characterization of Gprk2. (A–C) A Gprk2-lacZ enhancer trap is expressed along the A/P border (A), and is induced ectopically by misexpression of either Hh (B) or an activated form of Ci (C) using MS1096 Gal4 driver. (D–F″) A-compartment Gprk2Δ15 mutant cells near the A/P boundary (marked by the lack of GFP) (arrows in D–D″,F–F″) exhibited reduced expression of ptc (D′) and en (F′). Insets in D′ and D″ show enlarged images of the region indicated by the arrows. (E–E″) P-compartment Gprk2Δ15 mutant cells abutting the A/P boundary did not affect ptc expression. (G–J) Wing discs expressing SmoΔSAID (G,H) or SmoSD123 (I,J) with (H,J) or without (G,I) multiple copies of a Gprk2 RNAi transgene using MS1096 were immunostained to show en expression. SmoΔSAID but not SmoSD123 induced ectopic expression of en when Gprk2 was knocked down. (K–N) Gprk2Δ15 mutant discs expressing a wild-type Gprk2 (L), a kinase-dead Gprk2 (Gprk2KM) (M), or a mammalian GRK5 (mGRK5) (N) with MS1096 were immunostained to show en expression. Expression of the wild-type Gprk2 or mGRK5 but not Gprk2KM rescued anterior en expression in Gprk2Δ15 mutant discs. The red lines demarcate the A/P border based on Ci expression (not shown).
Figure 3.
Figure 3.
GRK phosphorylates multiple sites in Smo C-tail. (A) A schematic drawing of Smo with the sequence surrounding the PKA/CK1 sites and GRK sites (GPS1 and GPS2) shown below. PKA, CK1, and GRK sites are indicated by red, blue, and green residues, respectively. The transmembrane domains are indicated by the black boxes, and the SAID is indicated by the gray bar. Amino acid substitutions for SmoSD123 and its derivatives are listed. (B) In vitro kinase assay using a recombinant GRK5 and GST fusion proteins carrying indicated fragments from the Smo C-tail. (Lanes 12,18) Two minimal fragments, amino acids 700–748 and amino acids 997–1035, were phosphorylated by GRK5. (Lanes 14,19) Mutating the S741/T742 or S1013/S1015 abolished phosphorylation of the corresponding fragments. (C) In vitro kinase assay using recombinant GRK5 (shown in lanes 1–8) or immunoprecipitated Fg-Gprk2 with GST-Smo700–748 bearing the wild-type sequence or indicated point mutations. PKA/CK1 pretreatment was carried out in the presence of cold ATP. (D–G′) Wing discs expressing CFP-tagged SmoSD123 (D,D′), SmoSDGPSA1 (E,E′), SmoSDGPSA2 (F,F′), or SmoSDGPSA12 (G,G′) were immunostained to show the expression of CFP (green) and en (blue). SmoSD123 and SmoSDGPSA2 but not SmoSDGPSA1 or SmoSDGPSA12 induced ectopic en expression.
Figure 4.
Figure 4.
Gprk2 promotes Smo activation through phosphorylation-dependent and phosphorylation-independent mechanisms. (A–D′) Wing discs expressing UAS-SmoSD123-CFP (A,A′,C,C′) or UAS-SmoSDGPSD-CFP (B,B′,D,D′) with the C765 Gal4 driver were immunostained to show the expression of ptc (A,B), en (C,D), and CFP (A′,B′,C′,D′). (E,F). en expression (visualized by anti-En antibody) in Gprk2Δ15 heterozygous (E) or homozygous (F) wing discs expressing UAS-SmoSDGPSD-CFP with the MS1096 Gal4 driver. Loss of Gprk2 diminished the ectopic en expression induced by SmoSDGPSD. (G,G′) A Gprk2Δ15 homozygous wing disc expressing SmoSDGPSD and Flag-tagged Gprk2KM with MS1096 was immunostained with En and Flag antibodies. The kinase-dead Gprk2 rescued SmoSDGPSD-induced ectopic en expression in Gprk2Δ15 mutant discs. (H–J′) Gprk2Δ15 mutant discs expressing SmoSD123 alone (I), or together with wild-type Gprk2 (H) or Gprk2KM (J,J′) under the control of MS1096 were immunostained with anti-En and anti-Flag antibodies. Wild-type Gprk2 but not Gprk2KM rescued SmoSD123-induced ectopic en expression in Gprk2Δ15 mutant discs.
Figure 5.
Figure 5.
Gprk2 regulates Smo levels. (A) Wing discs carrying Gprk2Δ15 mutant clones were immunostained to show the expression of Smo (red channel) and GFP (green channel). Gprk2 mutant cells are marked by the lack of GFP expression. Smo levels were elevated in anterior Gprk2 mutant clones located near (big arrows) or away from (small arrows) the A/P boundary, but not in anterior Gprk2 mutant cells immediately abutting the A/P boundary or in P-compartment cells (arrowheads). The A/P boundary is marked by red lines based on Ci costaining (not shown). (B) S2 cells stably expressing a Myc-tagged Smo under the control of metallothionein promoter were treated with Gprk2 dsRNA or control (Luciferase) dsRNA in the absence of Hh, or in the presence of low (one-tenth of high) or high levels of Hh. 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 are shown (mean ± SD; n ≥ 20). The numbers below the bars indicate the percentage of Smo on the cell surface.
Figure 6.
Figure 6.
Gprk2 regulates Smo conformation. (A–C) FRET efficiency from indicated wild-type or mutant Smo-CFPC/YFPC (A), Smo-CFPL3YFPC (B), or Smo-CFPN/YFPN (C) expressed in S2 treated with Gprk2 dsRNA or control (luciferase) dsRNA in the absence or presence of Hh treatment. Gprk2 RNAi reduced Hh-induced FRET from Smo-CFPC/YFPC (A) and attenuated Hh-induced reduction in the FRET from Smo-CFPL3YFPC (B), but did not affect the FRET from Smo-CFPN/YFPN (C). (A) Gprk2 RNAi reduced both the basal and Hh-induced FRET from SmoSD123-CFPC/YFPC or SmoSDGPSD-CFPC/YFPC. Mean ± SD; n ≥ 15. The cartoons above the graphics indicate Smo biosensors, with filled and open circles representing CFP and YFP, respectively. The inset in A shows that Gprk2 RNAi (G-RNAi) but not the control RNAi (C-RNAi) effectively knocked down transfected Myc-Gprk2 in S2 cells. Myc-GFP was cotransfected as a control for transgene expression and RNAi specificity. (D) S2 cells treated with Gprk2 or control dsRNA were transfected with SmoSD123-CFPC/YFPC or SmoSDGPSD-CFPC/YFPC with or without cotransfection of Fg-Gprk2 or Fg-Gprk2KM in the presence of Hh-conditioned medium, followed by FRET analysis. Both the wild-type and kinase-dead Gprk2 restored high FRET from SmoSDGPSD-CFPC/YFPC after endogenous Gprk2 was depleted by Gprk2 RNAi. In contrast, only the wild-type but not the kinase-dead Gprk2 rescued the FRET from SmoSD123-CFPC/YFPC after Gprk2 RNAi. Mean ± SD; n ≥ 15.
Figure 7.
Figure 7.
Gprk2 interacts with the SAID and promotes Smo C-tail dimerization. (A) Schematic drawing of full-length Smo and its deletion mutants, Fz2, and Fz2-Smo chimeric protein (Fz2-SAID), in which the SAID is fused to the C terminus of Fz2. The ability of individual constructs to interact with Gprk2 is indicated on the right. (B–D) Coimmunoprecipitation assays to determine Smo/Gprk2 interaction. S2 cells were transfected with indicated Myc-tagged or Flag (Fg)-tagged Smo or Fz2 and Gprk2 constructs, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. Cell lysates were also directly immunoblotted by the indicated antibodies. Asterisks in C indicate the positions of full-length or truncated Smo. The arrow in C indicates IgG. (E) GST pull-down assay to determine Smo/Gprk2 interaction and its regulation by PKA/CK1-mediated phosphorylation. GST-Smo fusion protein containing Smo656–755 with wild-type sequence (WT); PKA sites mutated to Ala (SA) or PKA/CK1 sites mutated to Asp (SD) were purified from bacteria, treated with (+) or without (−) PKA and CK1, and incubated with cell lysates derived from S2 cells expressing a Flag-tagged Gprk2 (Fg-Gprk2). Fg-Gprk2 bound to GST-Smo fusion proteins were pulled down by glutathione beads and detected by Western blot with anti-Flag antibody. (F) Self-association of Gprk2. S2 cells were transfected with Myc-tagged or Flag-tagged Gprk2 individually or in combination. Cell lysates were subjected to immunoprecipitation and Western blot analysis with the indicated antibodies. (G) FRET analysis to determine Smo/Gprk2 interaction and Gprk2 self-association in intact cells. Numbers indicate the FRET efficiency from indicated CFP- and YFP-tagged constructs expressed in S2 cells. Mean ± SD; n ≥ 20. (H) Gprk2 promotes dimerization of Smo C-tail. Flag- and HA-tagged wild-type Smo C-tail (SmoC) or its phosphomimetic form (SmoSD-C) were transfected into S2 cells with or without Myc-Gprk2, or into S2 cells treated with Gprk2 dsRNA (Gprk2 RNAi). Cell lysates were subjected to immunoprecipitation and Western blot analysis with the indicated antibodies. Arrows indicate IgG.
Figure 8.
Figure 8.
Model for regulating Smo activation state by Gprk2. Hh-induced phosphorylation by PKA and CK1 leads to unfolding of Smo C-tail and promotes its association with and phosphorylation by Gprk2. Both Gprk2 binding and phosphorylation stabilize Smo in the active conformation by preventing refolding. In addition, Gprk2 forms a dimer/oligomer and promotes the active state of Smo by cross-linking Smo C-tails.
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