Proteomics Analysis of the Interactome of N-myc Downstream Regulated Gene 1 and Its Interactions with the Androgen Response Program in Prostate Cancer Cells*□S

Abstract

NDRG1 is known to play important roles in both androgen-induced cell differentiation and inhibition of prostate cancer metastasis. However, the proteins associated with NDRG1 function are not fully enumerated. Using coimmunoprecipitation and mass spectrometry analysis, we identified 58 proteins that interact with NDRG1 in prostate cancer cells. These proteins include nuclear proteins, adhesion molecules, endoplasmic reticulum (ER) chaperons, proteasome subunits, and signaling proteins. Integration of our data with protein-protein interaction data from the Human Proteome Reference Database allowed us to build a comprehensive interactome map of NDRG1. This interactome map consists of several modules such as a nuclear module and a cell membrane module; these modules explain the reported versatile functions of NDRG1. We also determined that serine 330 and threonine 366 of NDRG1 were phosphorylated and demonstrated that the phosphorylation of NDRG1 was prominently mediated by protein kinase A (PKA). Further, we showed that NDRG1 directly binds to -catenin and E-cadherin. However, the phosphorylation of NDRG1 did not interrupt the binding of NDRG1 to E-cadherin and -catenin. Finally, we showed that the inhibition of NDRG1 expression by RNA interference decreased the ER inducible chaperon GRP94 expression, directly proving that NDRG1 is involved in the ER stress response. Intriguingly, we observed that many members of the NDRG1 interactome are androgen-regulated and that the NDRG1 interactome links to the androgen response network through common interactions with -catenin and heat shock protein 90. Therefore we overlaid the transcriptomic expression changes in the NDRG1 interactome in response to androgen treatment and built a dual dynamic picture of the NDRG1 interactome in response to androgen. This interactome map provides the first road map for understanding the functions of NDRG1 in cells and its roles in human diseases, such as prostate cancer, which can progress from androgen-dependent curable stages to androgen-independent incurable stages. Molecular & Cellular Proteomics 6:575–588, 2007. N-myc downstream regulated gene 1 (NDRG1) is known to be involved in cell differentiation, carcinogenesis, atherosclerosis, survival, and metastasis (1). The induction of NDRG1 expression is associated with the processes of cell differentiation such as trophoblast recovery from hypoxia-induced injury (2) and mast cell maturation (3). Therefore, it has been characterized as a differentiation-regulated gene (4, 5). The expression levels of NDRG1 in normal and cancer tissue remain controversial. Some studies showed that NDRG1 is down-regulated in cancer such as prostate, breast, and colon (6–8). Other studies showed that NDRG1 is overexpressed in a variety of cancers including lung, brain, melanoma, liver, prostate, breast, and renal cancers (9). Nonetheless, overexpression of NDRG1 is known to inhibit tumor growth and metastasis by inducing cell differentiation (10, 11). Thus it is postulated that NDRG1 can be a tumor suppressor and a potential marker for cancer diagnosis (9–11). Wang et al. (12) showed that increased expression of NDRG1 was correlated with tumor progression during colorectal carcinogenesis. In contrast, others demonstrated that NDRG1 was induced in primary colon carcinoma but reduced in metastatic colon cancer (11). Overexpression of NDRG1 in highly metastatic colon cancer cells resulted in the suppression of cancer cell invasion and metastasis both in vitro and in vivo (8). Moreover, NDRG1 expression was significantly reduced in patients with lymph node or bone metastasis as compared with those with localized prostate cancer (6). These evidences indicate that NDRG1 can be a metastasis suppressor and may be a predictor of cancer metastasis (12). However, how NDRG1 mediates its multiple functions remains largely unknown. NDRG1 belongs to a new family of proteins that contains no protein motifs of known function (13). It is a cytoplasmic protein that contains three 10-amino acid tandem repeats at the C terminus (4) and is also a phosphoprotein that can be phosphorylated by several protein kinases (14). NDRG1 was categorized into the / hydrolase superfamily based on protein structure, but it does not have a catalytic site (15). The

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@inproceedings{Tu2007ProteomicsAO, title={Proteomics Analysis of the Interactome of N-myc Downstream Regulated Gene 1 and Its Interactions with the Androgen Response Program in Prostate Cancer Cells*□S}, author={Lan Chun Tu and Xiaowei Yan and Leroy Hood and Biaoyang Lin}, year={2007} }