Novel Function of Androgen Receptor-associated Protein 55/Hic-5 as a Negative Regulator of Smad3 Signaling*
- Ireland Cancer Center Research Laboratories and Department of Pharmacology, Case Western Reserve University/University Hospitals, Cleveland, Ohio 44106
- ‡ To whom correspondence should be addressed: Case Comprehensive Cancer Center, Case Western Reserve University, Wolstein Research Bldg., Rm. 3532, 2103 Cornell Rd., Cleveland, OH 44106. Tel.: 216-368-5670; Fax: 216-368-8919; E-mail: david.danielpour{at}case.edu.
Abstract
Androgen receptor-associated protein 55 (ARA55/Hic-5) belongs to the LIM protein superfamily and is featured by three or four N-terminal LD motifs and four C-terminal zinc finger-like LIM domains. Both LD motifs and LIM domains can serve as protein-protein interaction interfaces. Recently, we found that enforced expression of ARA55 inhibits transforming growth factor-β-mediated up-regulation of Smad binding element-luciferase reporter activity in NRP-154 and NRP-152 rat prostate and LNCaP human prostate cell lines. Moreover, ARA55 also inhibits the induction of Smad-binding element 4-luciferase and 3TP-luciferase (a plasminogen activator inhibitor-1 (PAI-1) promoter construct) reporters by constitutively active (CA)-Smad3 in these cell lines. Co-immunoprecipitation studies suggest an interaction between ARA55 and either CA-Smad3 or wild-type Smad3 in HEK293 cells that occurs through the MH2 domain of Smad3 and the C terminus of ARA55 with wild-type Smad3 having stronger affinity than CA-Smad3 to ARA55. Glutathione S-transferase pull-down assays demonstrate that this interaction can occur in a cell-free system. These results are consistent with the luciferase data showing that the C terminus of ARA55 is critical for suppression of Smad3 activity. Furthermore, using a mammalian two-hybrid system, we confirmed that ARA55 interacts with the MH2 domain of Smad3 and suppresses CA-Smad3-induced transcriptional responses. In conclusion, these results support that ARA55 selectively intercepts transforming growth factor-β signaling through an interaction of the LIM domain of ARA55 with the MH2 domain of Smad3.
Footnotes
-
↵1 The abbreviations used are: TGF-β, transforming growth factor-β; AR, androgen receptor; GST, glutathione S-transferase; ARA55, AR-associated protein 55; CA, constitutively active; WT, wild-type; SBE, Smad-binding element; co-IP, co-immunoprecipitation; MH1, Mad homology 1; MH2, Mad homology 2; ML, middle linker; FBS, fetal bovine serum; HEK293, human embryonic kidney cell line 293; TβRII, TGF-β receptor type II; TβRI, TGF-β receptor type I; FAK, focal adhesion kinase; PYK2, proline-rich tyrosine kinase 2; BD, Gal4 DNA-binding domain; AD, NF-κB transcription activation domain; DMEM/F-12, Dulbecco's modified Eagle's medium/Ham's F-12; RT, reverse transcription; DHT, dihydrotestosterone; RIPA, radioimmunoprecipitation assay; RL, renilla luciferase.
-
↵2 H. Wang, K. Song, T. L. Sponseller, and D. Danielpour, unpublished data.
-
↵* This work was supported by NCI Grants 1R01CA102074 and 1R01CA092102 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
- Received October 12, 2004.
- The American Society for Biochemistry and Molecular Biology, Inc.
