Smad4 Protein Stability Is Regulated by Ubiquitin Ligase SCFβ-TrCP1

Mei Wan,Yi Tang,Ewan M Tytler,Chongyuan Lu,Bingwen Jin,Selwyn M Vickers,Lei Yang,Xingming Shi,Xu Cao

doi:10.1074/jbc.c400005200

Mei Wan, Yi Tang + Show 7 more

Open Access

https://doi.org/10.1074/jbc.c400005200

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Abstract

Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 protein stability is unclear. We have identified SCF(beta-TrCP1), a ubiquitin (E3) ligase, as a critical determinant for the protein degradation of Smad4 protein. F-box protein beta-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The beta-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing beta-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous beta-TrCP1 suppression increases the expression of Smad4 protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-beta-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF(beta-TrCP1) abrogates TGF-beta function in vivo by decreasing Smad4 stability.

Highlights

From the ‡Department of Pathology, §Department of Surgery, University of Alabama at Birmingham, School of Medicine, Birmingham, Alabama 35294
To determine whether SCF complex ligase is responsible for Smad4 ubiquitination and degradation, we first tested whether F-box proteins in SCF E3 ligase interact with Smad4. ␤-TrCP1 and Skp2 are two well characterized F-box proteins in mammalian cells
To further investigate whether the ␤-TrCP1/Smad3 interaction is via Smad4, immunoprecipitation assays were performed in endogenous Smad4 silenced cells using small interfering RNA from DNA templates in vivo

Summary

Accelerated Publication

We have identified SCF␤-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the protein degradation of Smad protein. Ectopic expression of SCF complex containing ␤-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad. Small interfering RNA-triggered endogenous ␤-TrCP1 suppression increases the expression of Smad protein Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-␤-dependent transcriptional activity and an impaired cell cycle arrest function. A Smurf1-related ligase, can target Smad and sometimes Smad for degradation in a TGF-␤-dependent manner [3, 4] It remains to be determined whether and how the ubiquitinproteasome pathway regulates Smad degradation. We found an E3 ligase complex SCF␤-TrCP1 that is responsible for Smad ubiquitination and degradation. F-box protein ␤-TrCP1 in this complex associates with Smad and SCF␤-TrCP overexpressing cells display increased ubiquitination and degradation of Smad.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION