Novel function of androgen receptor-associated protein 55/Hic-5 as a negative regulator of Smad3 signaling.
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-beta-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-beta signaling through an interaction of the LIM domain of ARA55 with the MH2 domain of Smad3.
Wang
Hui
H
Ireland Cancer Center Research Laboratories and Department of Pharmacology, Case Western Reserve University/University Hospitals, Cleveland, OH 44106, USA.
Song
Kyung
K
Sponseller
Tracy L
TL
Danielpour
David
D
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497787:Smad3 protein, rat; C490351:TGFB1I1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta