RB1CC1 Protein Positively Regulates Transforming Growth Factor-β Signaling through the Modulation of Arkadia E3 Ubiquitin Ligase Activity

Daizo Koinuma,Masahiko Shinozaki,Yoshiko Nagano,Hiroaki Ikushima,Kana Horiguchi,Kouichiro Goto,Tokuhiro Chano,Masao Saitoh,Takeshi Imamura,Kohei Miyazono,Keiji Miyazawa

doi:10.1074/jbc.m111.227561

Abstract

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.

Highlights

Transforming growth factor-␤ (TGF-␤) is a multifunctional cytokine that regulates various cellular responses, including growth, motility, differentiation, and apoptosis in a wide variety of target cells
Identification of RB1-inducible coiled-coil 1 (RB1CC1) as an Arkadia-binding Protein— Arkadia is an E3 ubiquitin ligase that enhances TGF-␤ family signaling by targeting Smad7, c-Ski, and SnoN [24, 25, 28, 29]
Because Arkadia interacts with its substrates through this region [28], we examined whether RB1CC1 is a substrate of Arkadia E3 ubiquitin ligase

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screening—The yeast two-hybrid screening was performed as described previously [31]. Human RB1CC1 cDNA was described previously [37]. CDNA for a constitutively active form of TGF-␤ type I receptor (ALK-5-TD), mouse Smad, and human ubiquitin (Ub) were as previously described [24]. Radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X100, and protease inhibitors (P8340, Sigma) was used in some experiments to detect endogenous protein expression. Antibodies—Anti-RB1CC1 antibody was prepared by immunizing a rabbit with human RB1CC1 (amino acid residues 1247–1396) expressed as a fusion protein with glutathione S-transferase (GST). Anti-Arkadia antibody (#62) was raised against mouse Arkadia (amino acid residues 1–203) as described previously [24] and used for immunoblotting. Into HEK293, HEK293T, or COS7 cells using Lipofectamine2000 reagent (Invitrogen) according to the manufacturer’s instructions with 100 –120 pmol of siRNA/well of 6-well tissue culture plates. The Tukey-Kramer test of R program was used for multiple comparisons of data. p values of less than 0.01 were considered to indicate significance of the experiment

RESULTS

DISCUSSION

Miyazono and Keiji Miyazawa