Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

Logo for

PNAS 101 (52): 17900-17907

Copyright © 2004 by the National Academy of Sciences.

Inaugural Article

BIOLOGICAL SCIENCES / BIOCHEMISTRY

Extensive phosphorylation of Smoothened in Hedgehog pathway activation

Chi Zhang{dagger},{ddagger}, Elizabeth H. Williams{dagger},{ddagger}, Yurong Guo§, Lawrence Lum{dagger},¶, and Philip A. Beachy{dagger},||

{dagger}Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and §Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224

Contributed by Philip A. Beachy, October 30, 2004

Abstract: The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in Drosophila have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes in vivo and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1α in Hh pathway activation.

Freely available online through the PNAS open access option.

|| To whom correspondence should be addressed. E-mail: pbeachy{at}jhmi.edu.

{ddagger} C.Z. and E.H.W. contributed equally to this work.

Present address: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Author contributions: C.Z., E.H.W., Y.G., L.L., and P.A.B. designed research; C.Z., E.H.W., Y.G., and L.L. performed research; C.Z., E.H.W., Y.G., and L.L. contributed new reagents/analytic tools; C.Z., E.H.W., Y.G., L.L., and P.A.B. analyzed data; and C.Z., E.H.W., Y.G., L.L., and P.A.B. wrote the paper.

Abbreviations: Hh, Hedgehog; HhN, Hh N-terminal signaling domain; Smo, Smoothened; Fu, Fused; Ci, Cubitus interruptus; CiR, repressor form of Ci; Su(fu), Suppressor of Fused; PKA, cAMP-dependent protein kinase; CK1α, casein kinase 1α; GSK3β, glycogen synthase kinase 3β; Wg, Wingless; Cos2, Costal-2.

See accompanying Biography on page 17897.

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 30, 2002.

© 2004 by The National Academy of Sciences of the USA

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Zebrafish Cxcr4a determines the proliferative response to Hedgehog signalling.
T. Stuckemann, T. Wegleiter, E. Stefan, O. Nagele, K. Tarbashevich, G. Bock, E. Raz, and P. Aanstad (2012)
Development 139, 2711-2720
   Abstract »    Full Text »    PDF »
RACK1 Promotes Non-small-cell Lung Cancer Tumorigenicity through Activating Sonic Hedgehog Signaling Pathway.
S. Shi, Y.-Z. Deng, J.-S. Zhao, X.-D. Ji, J. Shi, Y.-X. Feng, G. Li, J.-J. Li, D. Zhu, H. P. Koeffler, et al. (2012)
J. Biol. Chem. 287, 7845-7858
   Abstract »    Full Text »    PDF »
Drosophila G-protein-coupled receptor kinase 2 regulates cAMP-dependent Hedgehog signaling.
S. Cheng, D. Maier, and D. R. Hipfner (2012)
Development 139, 85-94
   Abstract »    Full Text »    PDF »
Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube.
M. Tuson, M. He, and K. V. Anderson (2011)
Development 138, 4921-4930
   Abstract »    Full Text »    PDF »
The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation.
Q. Shi, S. Li, J. Jia, and J. Jiang (2011)
Development 138, 4219-4231
   Abstract »    Full Text »    PDF »
Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling.
Y. Su, J. K. Ospina, J. Zhang, A. P. Michelson, A. M. Schoen, and A. J. Zhu (2011)
Science Signaling 4, ra43
   Abstract »    Full Text »    PDF »
International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors.
G. Schulte (2010)
Pharmacol. Rev. 62, 632-667
   Abstract »    Full Text »    PDF »
Casein Kinase 2 Promotes Hedgehog Signaling by Regulating both Smoothened and Cubitus Interruptus.
H. Jia, Y. Liu, R. Xia, C. Tong, T. Yue, J. Jiang, and J. Jia (2010)
J. Biol. Chem. 285, 37218-37226
   Abstract »    Full Text »    PDF »
Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function.
S. M. Dudek, E. T. Chiang, S. M. Camp, Y. Guo, J. Zhao, M. E. Brown, P. A. Singleton, L. Wang, A. Desai, F. T. Arce, et al. (2010)
Mol. Biol. Cell 21, 4042-4056
   Abstract »    Full Text »    PDF »
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.
Y. Chen, S. Li, C. Tong, Y. Zhao, B. Wang, Y. Liu, J. Jia, and J. Jiang (2010)
Genes & Dev. 24, 2054-2067
   Abstract »    Full Text »    PDF »
Mechanism and evolution of cytosolic Hedgehog signal transduction.
C. W. Wilson and P.-T. Chuang (2010)
Development 137, 2079-2094
   Abstract »    Full Text »    PDF »
Neuroprotective Effect of Upregulated Sonic Hedgehog in Retinal Ganglion Cells Following Chronic Ocular Hypertension.
J. Wu, S. Zhang, and X. Sun (2010)
Invest. Ophthalmol. Vis. Sci. 51, 2986-2992
   Abstract »    Full Text »    PDF »
Activation of Hedgehog Signaling in Human Cancer: Basic Mechanisms and Clinical Implications.
J. Xie (2009)
Am. Assoc. Cancer Res. Educ. Book 2009, 25-41
   Full Text »    PDF »
PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation.
H. Jia, Y. Liu, W. Yan, and J. Jia (2009)
Development 136, 307-316
   Abstract »    Full Text »    PDF »
Smoothened Signaling in Vertebrates Is Facilitated by a G Protein-coupled Receptor Kinase.
M. Philipp, G. B. Fralish, A. R. Meloni, W. Chen, A. W. MacInnes, L. S. Barak, and M. G. Caron (2008)
Mol. Biol. Cell 19, 5478-5489
   Abstract »    Full Text »    PDF »
Kinome siRNA Screen Identifies Regulators of Ciliogenesis and Hedgehog Signal Transduction.
M. Evangelista, T. Y. Lim, J. Lee, L. Parker, A. Ashique, A. S. Peterson, W. Ye, D. P. Davis, and F. J. de Sauvage (2008)
Science Signaling 1, ra7
   Abstract »    Full Text »    PDF »
Hedgehog: functions and mechanisms.
M. Varjosalo and J. Taipale (2008)
Genes & Dev. 22, 2454-2472
   Abstract »    Full Text »    PDF »
A Screen for Modifiers of Hedgehog Signaling in Drosophila melanogaster Identifies swm and mts.
D. J. Casso, S. Liu, D. D. Iwaki, S. K. Ogden, and T. B. Kornberg (2008)
Genetics 178, 1399-1413
   Abstract »    Full Text »    PDF »
Fused Costal2 protein complex regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation.
Y. Liu, X. Cao, J. Jiang, and J. Jia (2007)
Genes & Dev. 21, 1949-1963
   Abstract »    Full Text »    PDF »
The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila.
C. Molnar, H. Holguin, F. Mayor Jr., A. Ruiz-Gomez, and J. F. de Celis (2007)
PNAS 104, 7963-7968
   Abstract »    Full Text »    PDF »
A large complex containing Patched and Smoothened initiates Hedgehog signaling in Drosophila.
S. L. Walthall, M. Moses, and J. I. Horabin (2007)
J. Cell Sci. 120, 826-837
   Abstract »    Full Text »    PDF »
Hedgehog signaling.
M. Varjosalo and J. Taipale (2007)
J. Cell Sci. 120, 3-6
   Full Text »    PDF »
Smoothened Signal Transduction Is Promoted by G Protein-Coupled Receptor Kinase 2.
A. R. Meloni, G. B. Fralish, P. Kelly, A. Salahpour, J. K. Chen, R. J. Wechsler-Reya, R. J. Lefkowitz, and M. G. Caron (2006)
Mol. Cell. Biol. 26, 7550-7560
   Abstract »    Full Text »    PDF »
Human Receptors Patched and Smoothened Partially Transduce Hedgehog Signal When Expressed in Drosophila Cells.
M. De Rivoyre, L. Ruel, M. Varjosalo, A. Loubat, M. Bidet, P. Therond, and I. Mus-Veteau (2006)
J. Biol. Chem. 281, 28584-28595
   Abstract »    Full Text »    PDF »
Proteomic Analysis of Pharmacological Preconditioning: Novel Protein Targets Converge to Mitochondrial Metabolism Pathways.
D. K. Arrell, S. T. Elliott, L. A. Kane, Y. Guo, Y. H. Ko, P. L. Pedersen, J. Robinson, M. Murata, A. M. Murphy, E. Marban, et al. (2006)
Circ. Res. 99, 706-714
   Abstract »    Full Text »    PDF »
Activation of heterotrimeric G proteins by Smoothened.
N. A. Riobo, B. Saucy, C. DiLizio, and D. R. Manning (2006)
PNAS 103, 12607-12612
   Abstract »    Full Text »    PDF »
The Contributions of Protein Kinase A and Smoothened Phosphorylation to Hedgehog Signal Transduction in Drosophila melanogaster.
Q. Zhou, S. Apionishev, and D. Kalderon (2006)
Genetics 173, 2049-2062
   Abstract »    Full Text »    PDF »
Phosphoinositide 3-kinase and Akt are essential for Sonic Hedgehog signaling.
N. A. Riobo, K. Lu, X. Ai, G. M. Haines, and C. P. Emerson Jr. (2006)
PNAS 103, 4505-4510
   Abstract »    Full Text »    PDF »
Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling via Two Distinct Mechanisms.
S. K. Ogden, D. J. Casso, M. Ascano Jr., M. M. Yore, T. B. Kornberg, and D. J. Robbins (2006)
J. Biol. Chem. 281, 7237-7243
   Abstract »    Full Text »    PDF »
CKI, there's more than one: casein kinase I family members in Wnt and Hedgehog signaling..
M. A. Price (2006)
Genes & Dev. 20, 399-410
   Abstract »    Full Text »    PDF »
Regulation of Gli1 Localization by the cAMP/Protein Kinase A Signaling Axis through a Site Near the Nuclear Localization Signal.
T. Sheng, S. Chi, X. Zhang, and J. Xie (2006)
J. Biol. Chem. 281, 9-12
   Abstract »    Full Text »    PDF »
Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates.
D. Huangfu and K. V. Anderson (2006)
Development 133, 3-14
   Abstract »    Full Text »    PDF »
Robust Enrichment of Phosphorylated Species in Complex Mixtures by Sequential Protein and Peptide Metal-Affinity Chromatography and Analysis by Tandem Mass Spectrometry.
M. O. Collins, L. Yu, H. Husi, W. P. Blackstock, J. S. Choudhary, and S. G. N. Grant (2005)
Sci. STKE 2005, pl6
   Abstract »    Full Text »    PDF »
Loss of the Serine/Threonine Kinase Fused Results in Postnatal Growth Defects and Lethality Due to Progressive Hydrocephalus.
M. Merchant, M. Evangelista, S.-M. Luoh, G. D. Frantz, S. Chalasani, R. A. D. Carano, M. van Hoy, J. Ramirez, A. K. Ogasawara, L. M. McFarland, et al. (2005)
Mol. Cell. Biol. 25, 7054-7068
   Abstract »    Full Text »    PDF »
Biography of Philip A. Beachy.
M. Marino (2004)
PNAS 101, 17897-17899
   Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882