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A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction

Abstract

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

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Fig. 1: SMO binds PKA-C as a pseudosubstrate.
Fig. 2: SMO induces changes in the amide fingerprint of PKA-C.
Fig. 3: SMO inhibits PKA-C enzymatic activity.
Fig. 4: The SMO PKI motif is required for Hh signal transduction.
Fig. 5: SMO colocalizes with endogenous PKA-C in primary cilia.
Fig. 6: The SMO PKI motif is required for Hh signal transduction.
Fig. 7: An avidity-based mechanism for SMO inhibition of PKA-C.

Data availability

Source data, including uncropped gels and western blots, are provided with this paper. All unique biological materials are available on request from the authors.

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Acknowledgements

We thank J. Zalatan for making us aware of the parallels between SMO/PKA-C regulation in the Hh pathway and LRP/GSK-3β regulation in the Wnt pathway. We thank S. Lusk and K. Kwan (University of Utah) for providing smo-null zebrafish (smohi1640), and D. Klatt Shaw and D. Grunwald (University of Utah) for sharing advice and reagents regarding zebrafish immunohistochemistry. We thank J. Müller and S. Kasten (University of Kassel) for excellent technical assistance. We thank the Johnson Foundation Structural Biology and Biophysics Core at the Perelman School of Medicine (University of Pennsylvania, Philadelphia, PA, USA) for performing multi-angle light scattering coupled with size-exclusion chromatography analyses. We thank D. Julius, S. Nakielny, A. Manglik, K. Basham and M. He for providing feedback on the manuscript. B.R.M. acknowledges support from the 5 for the Fight Foundation (award no. 6000-32705) and a Cancer Center Support Grant Pilot Project Fund from the Huntsman Cancer Institute (award no. 200206). This work was supported by the DFG (the German Research Foundation) grant no. GRK2749/1 (F.W.H.) and National Institutes of Health grant nos. R01GM100310-08 (G.V.), 1R35GM130389 (S.S.T.), 1R03TR002947 (S.S.T.) and 1R35GM133672 (B.R.M.).

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Authors

Contributions

J.T.H. designed, executed and interpreted CREB and GLI reporter assays. C.D.A. designed, executed and interpreted HEK293 BRET assays. J.B. designed, executed and interpreted fluorescence polarization studies and peptide array studies. D.B., J.W.B. and F.W.H. designed, executed and interpreted in vitro PKA-C activity assays and SPR studies. I.B.N. developed SMO pCT purification approaches and purified this domain for in vitro PKA-C activity assays. C.O. designed, executed and interpreted NMR studies. J.Z. designed, executed and interpreted all NIH3T3 imaging studies, under supervision from X.G. D.S.H. designed, executed and interpreted zebrafish embryology studies. J.-F.Z. designed, executed and interpreted coimmunoprecipitation and IMCD3 BRET assays. J.L.C. designed, executed and interpreted HEK293 confocal imaging studies. L.V. performed initial fluorescence polarization studies. C.C.K. collaborated with J.B. to develop SMO peptide arrays. V.L.R.-P. provided advice and guidance on mutagenesis experiments to disrupt SMO-PKA-C interactions. S.S.T. and B.R.M. conceived the project. G.V., F.W.H., S.S.T. and B.R.M. interpreted data and provided overall project supervision. B.R.M. performed IMCD3 ciliary imaging studies and wrote the manuscript with assistance from J.T.H., C.D.A. and I.B.N.

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Correspondence to Susan S. Taylor or Benjamin R. Myers.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Philip Ingham and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Carolina Perdigoto, Beth Moorefield and Anke Sparmann in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Sequence alignment of SMO PKI motif.

Extended alignment of a portion of the pCT from the indicated SMO orthologs, with key PKI motif residues colored as in Fig. 1a.

Extended Data Fig. 2 Additional binding and peptide array studies, and SPR sensorgrams.

a, Fluorescence polarization assays using mouse PKA-Cɑ, performed as in Fig. 1b. Triplicate points from representative experiments are shown. b, Peptide array, performed as in Fig. 1c, but with individual residues in the human SMO PKI motif mutated to alanine. c, SPR sensorgram for 625 nM PKA-Cɑ binding to GST-tagged wild-type (blue) or WRR mutant (purple) SMO pCT, or a PKIɑ positive control (red), in the presence of ATP and MgCl2. d, Exemplary steady-state analysis of binding interactions between human PKA-Cɑ and a recombinant wild-type SMO pCT, with a KD of 703 + /− 0.003 nM (dotted line) as assessed by SPR. This measurement was made three times, resulting in a mean KD value of 752 + /− 34 nM. e, SPR sensorgram, performed as in c, but with ATP and MgCl2 omitted from the buffer. PKA-Cɑ was present at 2.5 µM. Note that although removal of ATP and MgCl2 does not completely eliminate steady-state binding to the PKIɑ positive control, it dramatically accelerates the dissociation rate, as expected.

Source data

Extended Data Fig. 3 Raw NMR spectra for wild-type SMO peptide binding to PKA-Cα at varying kinase:peptide ratios.

2D NMR spectra from an experiment in which SMO peptide was titrated into ATPγN-bound PKA-Cα at the indicated kinase:peptide ratios. Upper left panel represents an overlay of all three spectra to highlight the concentration dependence of the SMO peptide-induced changes observed in each spectrum. Black spectrum represents ATPγN-bound PKA-Cα without SMO peptide (see also Extended Data Figs. 4, 5). In the overlay plot, a box denotes one example of a peak (likely corresponding to an unassigned tryptophan residue) that changes linearly according to SMO peptide concentration (magnified in the inset at left).

Extended Data Fig. 4 Raw NMR spectra for WRR mutant peptide binding to PKA-Cα, as shown in Fig. 2a.

a, The WRR mutant SMO peptide was titrated into ATPγN-bound PKA-Cα at the indicated kinase:peptide ratios. ATPγN-bound PKA-Cα without peptide (dark green spectrum, upper left) is shown for reference. See Extended Data Fig. 5 for raw NMR spectrum corresponding to PKA-Cα:wild-type SMO peptide at 1:6 ratio. b, Overlay of the spectra in a.

Extended Data Fig. 5 Raw NMR spectra for wild-type SMO peptide binding to PKA-Cα, as shown in Fig. 2a, and PKIα(5-24)-induced displacement of SMO peptide from PKA-Cα, as shown in Fig. 2c.

a, 2D NMR spectra for ATPγN-bound PKA-Cα, either alone (left) or with SMO peptide added at a 1:6 kinase:peptide ratio (right). b, 2D NMR spectra for titration of PKIα(5-24) peptide into the SMO peptide:ATPγN:PKA-Cα complex at the indicated kinase: PKIα(5-24) ratios. Upper left panel represents an overlay of the individual spectra to highlight the concentration dependence of the PKIα(5-24) peptide-induced effects.

Extended Data Fig. 6 Coimmunoprecipitation studies and ciliary colocalization studies to assess SMO / PKA-C interactions.

a, Coimmunoprecipitation of PKA-Cɑ-YFP with the indicated FLAG-tagged wild-type or mutant SMO constructs was assessed using FLAG chromatography from lysates of transfected HEK293 cells. Data shown are representative of two independent experiments. b, Left, Colocalization of FLAG-tagged wild-type or mutant SMO674 (magenta) with mNeonGreen-tagged PKA-Cɑ (green) in ciliated IMCD3 cells stably expressing both constructs and treated with the SMO agonist SAG21k. Cilia are marked by the SMO (FLAG-647) stain. mNeonGreen-tagged Nbβ2AR80 (which does not bind SMO29) serves as a negative control. 3D reconstructions from Z-stacks of confocal live-cell images are shown. Right, quantification of microscopy studies with the median represented by a dashed line and the upper and lower quartiles indicated by dotted lines (n = 142–244 cilia per condition). P < 0.0001 (****). See Supplementary Table 1 for full statistical analysis.

Source data

Extended Data Fig. 7 Characterization of NIH3T3 cell line expressing epitope-tagged SMO.

The NIH3T3 cell line used in Fig. 5 exhibited trace amounts of SMO in cilia under vehicle (‘Ctrl’) -treated conditions, and a dramatic accumulation of SMO in cilia under SAG-treated conditions. SMO (magenta) is visualized using an anti-V5 antibody, and cilia are visualized using an anti-Arl13b antibody (green). Scale bar = 10 µm. Data shown are representative of two independent experiments.

Extended Data Fig. 8 Controls for SMO / PKA-C binding, colocalization, and signaling studies.

a, Expression levels of SMO constructs in Fig. 3a, assessed by whole-cell nanoluc measurements. Data represent the mean + /− standard deviation, n = three biologically independent samples. NS = not significant. b, Surface levels of N-terminally FLAG-tagged wild-type or mutant SMO674 constructs were quantified via expression in HEK293 cells followed by FLAG staining and flow cytometry. Mock-infected cells stained with FLAG antibody (red) serve as a negative control. A representative histogram is shown. The % of FLAG-positive cells (that is, those to the right of the vertical dashed line) are: 0.3 + /− 0.3% (Ctrl); 95.9 + /− 0.9% (wt), 96.0 + /− 2.5% (WRR); 93.4 + /− 4.2% (A635S); values represent the mean + /− standard deviation from two biologically independent samples. See Supplementary Table 1 for full statistical analysis, and Supplementary Figure 2 for gating strategy. c, Ciliary localization in IMCD3 cells of myc-tagged wild-type or mutant SMO proteins (magenta). Cilia were visualized with Arl13b antibody (green). Scale bar = 5 µm. Data shown are representative of two independent experiments. d, GRK2/3-dependent phosphorylation of FLAG-tagged wild-type or mutant SMO674 constructs was determined via expression in HEK293 cells treated with or without the GRK2/3 inhibitor cmpd101, followed by FLAG purification. Levels of total and phosphorylated SMO were assessed by Stain Free imaging and ProQ Diamond fluorescence, respectively. SMO566, which is not phosphorylated by GRK2/3 (as it does not contain the C-tail and therefore lacks all previously mapped physiological GRK2/3 phosphorylation sites), serves as a negative control. Data shown are representative of two independent experiments.

Source data

Extended Data Fig. 9 Complete data set from SMO C-tail peptide array studies.

a, The same SMO tiled peptide array from Fig. 7c, but including the sequences of all positive hits in each array cluster. b, Complete human SMO C-tail sequence used to create the peptide array. In a,b, the SMO PKI motif identified in the pCT is indicated in red. Key residues in this PKI motif, along with ones in the candidate PKI motif in the dCT, are colored as in Fig. 1a.

Source data

Extended Data Fig. 10 Similarity between signal transduction mechanisms in the Hh and Wnt pathways.

Schematic diagram of transmembrane signal transduction in the Hh (left) and Wnt (right) pathways. During Hh signal transduction, active SMO is phosphorylated on its cytoplasmic tail by GRK2/3, triggering membrane sequestration and inhibition of PKA-C, and ultimately stabilization and activation of GLI. During Wnt signal transduction, active LRP5/6 is phosphorylated on its cytoplasmic tail by glycogen synthase kinase (GSK)-3β and casein kinase (CK)-1ɑ, triggering membrane sequestration and inhibition of GSK-3β, and ultimately stabilization and activation of β-catenin. Note that this is a simplified and highly schematized diagram and is not intended to be comprehensive; many other components of both pathways (for example, the destruction complex in which GSK-3β and β-catenin reside) are omitted in order to highlight mechanistic similarities between the underlying transmembrane signaling mechanisms.

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1 and 2, Discussion and Source Data for Supplementary Fig. 1.

Reporting Summary.

Source data

Source Data Fig. 1

Uncropped peptide array blots and raw data from fluorescence polarization and SPR studies.

Source Data Fig. 2

Raw data (list + CSP files) from NMR studies (also applies to Extended Data Figs. 3–5).

Source Data Fig. 3

Raw data from PKA-C in vitro enzymatic activity assays.

Source Data Fig. 4

Raw data from BRET studies and SMO-PKA-C colocalization studies in HEK293 cells

Source Data Fig. 5

Raw data from BRET studies and SMO-PKA-C colocalization studies in NIH3T3 cells.

Source Data Fig. 6

Raw data from CREB and GLI transcriptional reporter assays.

Source Data Fig. 7

Uncropped peptide array blots and raw data from BRET studies.

Source Data Extended Data Fig. 2

Uncropped peptide array blots and raw data from fluorescence polarization and SPR studies.

Source Data Extended Data Fig. 6

Uncropped protein gels, western blots and raw data from SMO-PKA-C colocalization studies in IMCD3 cells.

Source Data Extended Data Fig. 8

Uncropped protein gels and raw data from BRET studies.

Source Data Extended Data Fig. 9

Uncropped peptide array blots.

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Happ, J.T., Arveseth, C.D., Bruystens, J. et al. A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction. Nat Struct Mol Biol 29, 990–999 (2022). https://doi.org/10.1038/s41594-022-00838-z

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