Identification of a Suppressive Mechanism for Hedgehog Signaling through a Novel Interaction of Gli with 14-3-3

Yoshinari Asaoka,Fumihiko Kanai,Tohru Ichimura,Keisuke Tateishi,Yasuo Tanaka,Miki Ohta,Motoko Seto,Motohisa Tada,Hideaki Ijichi,Tsuneo Ikenoue,Takao Kawabe,Toshiaki Isobe,Michael B Yaffe,Masao Omata

doi:10.1074/jbc.m109.038232

Abstract

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.

Highlights

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 14-3-3⑀, and that Gli2 and Gli3 bind to 14-3-3⑀ through homologous sites
Identification of 14-3-3⑀ as a Novel Interacting Protein with Gli1—The MEF (Myc-tobacco etch virus (TEV)-FLAG) technique uses two different affinity modules (Myc and FLAG) separated by a cleavage site for the TEV protease to reduce nonspecific protein binding to the complex (Fig. 1A)

Summary

Introduction

Gli transcription factors are central effectors of Hedgehog signaling in development and tumorigenesis. To confirm the interaction between Gli1 and 14-3-3⑀ in a physiological condition, the lysates from PLC/PRF/5 cells, where Hh signaling is constitutively active [19], were applied to immunoprecipitation using anti-Gli1 and anti-14-3-3⑀ Abs. As a result, Gli1 and 14-3-3⑀ co-precipitated each other (Fig. 1, C and D), which suggests the in vivo interaction of endogenous Gli1 and 14-3-3⑀.

Results

Conclusion