Identification of a suppressive mechanism for Hedgehog signaling through a novel interaction of Gli with 14-3-3.
Gli transcription factors are central effectors of Hedgehog signaling in development and tumorigenesis. Using a tandem affinity purification (TAP) strategy and mass spectrometry, we have found that Gli1 interacts with 14-3-3epsilon, and that Gli2 and Gli3 also bind to 14-3-3epsilon through homologous sites. This interaction depends on their phosphorylation, and cAMP-dependent protein kinase (PKA), a known negative regulator of Hedgehog signaling serves as a responsible kinase. A Gli2 mutant engineered to eliminate this interaction exhibited increased transcriptional activity (2 approximately 3x). Transcriptional repression by 14-3-3 binding was also observed with Gli3, when its N-terminal repressor domain was deleted. The phosphorylation sites responsible for the binding to 14-3-3 are distinct from those required for proteolysis, the known mechanism for PKA-induced repression of Hh signaling. Our data propose a novel mechanism in which PKA down-regulates Hedgehog signaling by promoting the interaction between Gli and 14-3-3 as well as proteolysis. Given the certain neuronal or malignant disorders in human caused by the abnormality of 17p13 encompassing 14-3-3epsilon overlap with increased Hh signaling, the Gli-14-3-3 interaction may have pathological significance for those human diseases.
Asaoka
Yoshinari
Y
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan. Electronic address: yasa-tky@umin.ac.jp.
Kanai
Fumihiko
F
the Departments of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
Ichimura
Tohru
T
the Department of Applied Chemistry, National Defense Academy, Kanagawa 239-8686, Japan.
Tateishi
Keisuke
K
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
Tanaka
Yasuo
Y
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
Ohta
Miki
M
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
Seto
Motoko
M
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
Tada
Motohisa
M
the Departments of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan.
Ijichi
Hideaki
H
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
Ikenoue
Tsuneo
T
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
Kawabe
Takao
T
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan; Departments of Endoscopy and Endoscopic Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
Isobe
Toshiaki
T
the Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan, and.
Yaffe
Michael B
MB
the Center for Cancer Research and Departments of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Omata
Masao
M
From the Departments of Gastroenterology, The University of Tokyo, Tokyo 113-8655, Japan.
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006367:HeLa Cells; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D034622:RNA Interference; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3
D048948:14-3-3 Proteins; C400551:GLI1 protein, human; C082593:GLI2 protein, human; C065683:GLI3 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C482983:YWHAE protein, human; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases