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Modulation of Androgen Receptor Activity by Reversible NEDD8 Modification

by
Kai-Hsiung Chang
M.S., 2004
National Taiwan University

Thesis Advisor: J. Don Chen, Ph.D.
Graduate Program in Cellular and Molecular Pharmacology

RWJMS - Research Tower - 4th floor
Pharmacology Conference Room

Wednesday, October 15, 2008
3:30 pm


Abstract

Androgen receptor (AR) is a crucial ligand-dependent transcription factor in the progression of PCa and the androgen-AR axis is the principle therapeutic target for this disease. While post-translational modification on both histones and transcriptional factors has significant impacts on gene expression, how it affects ARs activity is largely unknown. In searching of post-translation modifications of AR, we show that NEDD8, an ubiquitin-like small protein modifier, is a novel covalent modifier of AR and functionally regulated its transcriptional activity. We found that PIASy acts as the E3 ligase to catalyze neddylation of AR and inhibit ARs transcriptional activity. The integrity of RING domain of PIASy is prerequisite for both NEDD8-E3 ligase activity and inhibitory effect on AR. Mutagenesis studies suggested NEDD8 acceptor sites located in a consensus lysine residues in the hinge region. Conversely, we found Jab1 interacts with AR and enhances ARs transcriptional activity dependent on its JAMM domain, suggesting the deneddylation activity was involved. In agreement, knockdown of Jab1 increases neddylation level of endogenous AR in prostate cancer cells and hinders androgen-induced gene activation. Finally, we show that Jab1 knockdown also impedes prostate cancer cell growth.

Importantly, PIASy-dependent neddylation and transcriptional inhibition are applicable to other nuclear receptors such as PR, ER and hPXR. PIASy had been shown to function as a RING-type SUMO-E3 ligase for many transcription factors. Intriguingly, studies also implicated PIASy serves as a strong corepressor in other transcription regulations through mechanisms independent of sumoylation pathway. Using hPXR as another model substrate, we also demonstrated that PIASy specifically interacts with hPXR in vitro and in vivo and acts as a RING-dependent NEDD8-E3 ligase for hPXR in cells. Mutagenesis studies mapped a NEDD8 acceptor site at K108 in the hinge region. Moreover, the interacting domain located in the SAP domain of PIASy and DNA binding domain of hPXR. In particular, this interaction prevents PXR-responsive DNA elements recognition and results in the transcriptional repression. Altogether, these results suggested that PIASy contributes to stringent modulation of hPXRs activity through either stimulating neddylation or blocking DNA binding ability.

Taken together, these two studies suggest a novel NEDD8 modification strongly repress transcriptional activities among nuclear receptors. Deciphering more regulation pathways of this modification would provide important therapeutic implications for diseases resulted from aberrant regulation of nuclear receptors.


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