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Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription
Journal article   Open access   Peer reviewed

Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription

Jordy J Hsiao, Melinda M Smits, Brandon H Ng, Jinhee Lee and Michael E Wright
The Journal of biological chemistry, Vol.291(36), pp.18818-18842
09/02/2016
DOI: 10.1074/jbc.M116.732313
PMCID: PMC5009256
PMID: 27365400
url
https://doi.org/10.1074/jbc.M116.732313View
Published (Version of record) Open Access

Abstract

Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the "AR-interactome." Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope (streptavidin-binding peptide-tagged wild-type androgen receptor; SBP-AR). A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex in vitro Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers.
 Proteomics Prostatic Neoplasms - metabolism Humans Receptors, Androgen - metabolism Male Transcription Factors - genetics Coat Protein Complex I - genetics Coat Protein Complex I - metabolism DNA-Binding Proteins - genetics Neoplasm Proteins - metabolism DNA-Binding Proteins - metabolism Transcription Factors - metabolism Cell Nucleus - metabolism Prostatic Neoplasms - genetics Receptors, Androgen - genetics Cell Nucleus - genetics Cell Line, Tumor Golgi Apparatus - metabolism Transcription, Genetic Active Transport, Cell Nucleus Neoplasm Proteins - genetics Golgi Apparatus - genetics

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