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PNAS 101 (52): 17900-17907

Copyright © 2004 by the National Academy of Sciences.

Inaugural Article


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}

{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

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