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Research Article
Developmental Biology

Hedgehog induces formation of PKA-Smoothened complexes to promote Smoothened phosphorylation and pathway activation

Science Signaling
1 Jul 2014
Vol 7, Issue 332
p. ra62

PKA Switches Partners to Promote Hedgehog Signaling

Hedgehog is a secreted protein that controls developmental patterning and cell fate specification. Hedgehog binds to the transmembrane receptor Patched to relieve inhibition of the seven-transmembrane protein Smoothened (Smo), which in turn activates the transcription factor Ci by inhibiting its phosphorylation by the kinase PKA. Li et al. found that, in fruit flies, activated Smo directly bound to and was phosphorylated by the catalytic subunit of PKA. Phosphorylated Smo recruited more of the catalytic subunit of PKA, which prevented the interaction between PKA and Ci, enabling Ci to activate Hedgehog-regulated genes. Thus, this switching of PKA-substrate interactions controls activation of Hedgehog signaling.


Hedgehog (Hh) is a secreted glycoprotein that binds its receptor Patched to activate the G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor–like protein Smoothened (Smo). In Drosophila, protein kinase A (PKA) phosphorylates and activates Smo in cells stimulated with Hh. In unstimulated cells, PKA phosphorylates and inhibits the transcription factor Cubitus interruptus (Ci). We found that in cells exposed to Hh, the catalytic subunit of PKA (PKAc) bound to the juxtamembrane region of the carboxyl terminus of Smo. PKA-mediated phosphorylation of Smo further enhanced its association with PKAc to form stable kinase-substrate complexes that promoted the PKA-mediated transphosphorylation of Smo dimers. We identified multiple basic residues in the carboxyl terminus of Smo that were required for interaction with PKAc, Smo phosphorylation, and Hh pathway activation. Hh induced a switch from the association of PKAc with a cytosolic complex of Ci and the kinesin-like protein Costal2 (Cos2) to a membrane-bound Smo-Cos2 complex. Thus, our study uncovers a previously uncharacterized mechanism for regulation of PKA activity and demonstrates that the signal-regulated formation of kinase-substrate complexes plays a central role in Hh signal transduction.

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Supplementary Material


Fig. S1. Hh signaling stabilizes PKAc depending on Smo C-tail.
Fig. S2. Characterization of AKAR3 and Myr-AKAR3 in S2 cells.
Fig. S3. Hh increases Myr-AKAR3 FRET in S2 cells.
Fig. S4. Hh signaling increases Myr-AKAR3 FRET in wing discs.
Fig. S5. Characterization of PKAc-Smo interaction by coimmunoprecipitation assay.
Fig. S6. Multiple basic clusters mediate binding of PKAc to the SAID domain.
Fig. S7. Hh induces colocalization between mC* and truncated Smo independent of endogenous Smo.
Fig. S8. Hh induces FRET between CFP-tagged Smo variants and mC*-YFP.
Fig. S9. Characterization of Smo expression driven by different Gal4 drivers.
Fig. S10. Smo-PKAc complex formation stabilizes PKAc.
Fig. S11. Hh switches the binding of PKAc from Cos2 to Smo.


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Published In

Science Signaling
Volume 7 | Issue 332
July 2014

Submission history

Received: 24 April 2014
Accepted: 11 June 2014


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We thank J. Jia, J. Zhang, Y. Zhao, R. Holmgren, G. Struhl, DSHB, and Bloomington Stock Centers for fly stocks and reagents. Funding: This work is supported by grants from the NIH (GM061269 and GM067045), National Natural Science Foundation of China (31328017), and Welch Foundation (I-1603). J.J. is a Eugene McDermott Endowed Scholar in Biomedical Science at University of Texas Southwestern Medical Center. Author contributions: S.L. and J.J. designed the experiments; S.L., G.M., and B.W. performed the experiments; S.L., G.M., and J.J. analyzed the data; and S.L. and J.J. wrote the manuscript. Competing interests: The authors declare that they have no competing interests.



Shuang Li*
Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Guoqiang Ma*
Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Bing Wang
Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.


These authors contributed equally to this work.
Corresponding author. E-mail: [email protected]

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