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Sumoylation of Pontin Chromatin-Remodeling Complex Reveals a Signal Integration Code in Prostate Cancer Cells

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Sumoylation of Pontin Chromatin-Remodeling Complex Reveals a Signal Integration Code in Prostate Cancer Cells SUMOylation of pontin chromatin-remodeling complex reveals a signal integration code in prostate cancer cells Jung Hwa Kim*†, Ji Min Lee*, Hye Jin Nam*, Hee June Choi*, Jung Woo Yang*, Jason S. Lee*, Mi Hyang Kim‡, Su-Il Kim‡, Chin Ha Chung*, Keun Il Kim§, and Sung Hee Baek*¶ *Department of Biological Sciences, Research Center for Functional Cellulomics and ‡School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea; §Department of Biological Sciences, Research Center for Women’s Disease, Sookmyung Women’s University, Seoul 140-742, South Korea; and †Department of Medical Sciences, Inha University, Incheon 402-751, South Korea Communicated by Michael G. Rosenfeld, University of California at San Diego, La Jolla, CA, November 6, 2007 (received for review July 20, 2007) Posttranslational modification by small ubiquitin-like modifier mammals, they constitute parts of the Tip60 coactivator complex, (SUMO) controls diverse cellular functions of transcription factors which has intrinsic histone acetyltransferase activity (8). In ze- and coregulators and participates in various cellular processes brafish embryos, the reptin/pontin ratio serves to regulate heart including signal transduction and transcriptional regulation. Here, growth during development via the ␤-catenin pathway (9). we report that pontin, a component of chromatin-remodeling Posttranslational modification of proteins plays an important role complexes, is SUMO-modified, and that SUMOylation of pontin is in the functional regulation of transcriptional coregulators. Numer- an active control mechanism for the transcriptional regulation of ous enzymatic activities have been demonstrated to be associated pontin on androgen-receptor target genes in prostate cancer cells. with coregulator complexes, including histone acetylation/ Biochemical purification of pontin-containing complexes revealed deacetylation, phosphorylation/dephosphorylation, ubiquitination, the presence of the Ubc9 SUMO-conjugating enzyme that underlies and SUMOylation (10). Small ubiquitin-like modifier (SUMO) its function as an activator. Intriguingly, 5␣-dihydroxytestosterone plays an important regulatory role in many cellular processes treatments significantly increased the SUMOylation of pontin, and including signal transduction, transcriptional regulation, chromatin SUMOylated pontin showed further activation of a subset of structure, and nuclear/cytoplasmic shuttling (11). SUMO is an nuclear receptor-dependent transcription and led to an increase in Ϸ11-kDa protein that is structurally, but not functionally, homol- proliferation and growth of prostate cancer cells. These data ogous to ubiquitin. The enzymatic machinery that adds SUMO to, clearly define a functional model and provide a link between and removes it from, target proteins is distinct from the ubiquiti- SUMO modification and prostate cancer progression. nation machinery. SUMOylation of target proteins is a multistep process involving the E1-activating molecules SAE1/SAE2, the androgen receptor ͉ SUMO ͉ Ubc9 ͉ transcription ͉ proliferation E2-conjugating enzyme Ubc9, and E3 ligases in mammalian cells (12). Several SUMO-processing enzymes have been identified that efining the signal integration pathways that involve diverse hydrolyze the SUMO moiety from target substrates in mammals Dmolecular mechanisms at the level of gene transcription and yeast (13–15). Unlike ubiquitination, SUMO modification has remains an important goal in biology. The main downstream not been found to be associated with protein degradation. Rather, effects of a signaling pathway are the modulation of the function it is similar to the nonproteolytic roles of ubiquitination including of transcription factors and coregulators in the nucleus (1). Thus, subcellular localization and the regulation of transcriptional activ- it is important to investigate the roles of transcription factors and ity. A growing number of SUMO-modified proteins have been coregulators in response to the upstream signaling pathway. The identified including RanGAP1, p53, c-Jun, and reptin (16–18). androgen receptor (AR) not only mediates prostate develop- Given that lysine is the site for a variety of modifications, it might ment but also serves as a key regulator of primary prostatic be predicted that there exists elaborate modification code for both histone and nonhistone proteins. cancer growth (2). The effects of androgens are mediated Recently, we reported the dynamic role of a chromatin- through the AR, a member of the nuclear receptor family remodeling complex in the regulation of the metastasis suppressor functioning as a ligand-inducible transcription factor that regu- gene KAI1 (18, 19). Reptin chromatin-remodeling complexes con- lates the expression of target genes having an androgen response tain SENP1 deSUMOylating enzyme as a component, and SUMO element. Analogous to other members of the nuclear receptor modification of reptin plays an important role for the transcrip- superfamily, the AR contains a DNA binding domain and a tional regulation of KAI1 (18). In this article, we provide evidence BIOCHEMISTRY C-terminal ligand-binding domain functioning as ligand- that pontin is modified by SUMO, but that this modification dependent nuclear receptors with AF-1 and AF-2 transcription promotes the differential functional regulation of pontin having activation domains (3, 4). The ability of many nuclear receptors different downstream target genes compared with reptin. Biochem- to bind a variety of ligands, including agonists or antagonists ical purification of pontin-containing complexes revealed a Ubc9 leading to either enhanced or diminished gene activation, raises SUMO-conjugating enzyme as a binding partner. Mutation that intriguing issues about combinatorial transcriptional mecha- affects the attachment of SUMO to pontin decreased the transcrip- nisms mediated by coactivators and corepressors. tional activation function of pontin in the regulation of AR target Gene expression is influenced by chromatin structure, and covalent modification of histones plays an important role in regu- lating transcription and chromatin dynamics (5). The transcription Author contributions: J.H.K. and S.H.B. designed research; J.H.K., J.M.L., H.J.N., H.J.C., of most genes is regulated by the coordinate action of chromatin- J.W.Y., and M.H.K. performed research; J.S.L., S.-I.K., and C.H.C. contributed new reagents/ remodeling complexes. Pontin and reptin have been reported to be analytic tools; J.H.K., K.I.K., S.H.B. analyzed data; and S.H.B. wrote the paper. components of the ATP-dependent chromatin-remodeling com- The authors declare no conflict of interest. plexes and are closely related to the bacterial DNA helicase RuvB ¶To whom correspondence should be addressed. E-mail: [email protected]. (6). Pontin has been demonstrated to bind and activate the ␤-cate- This article contains supporting information online at www.pnas.org/cgi/content/full/ nin/TCF transcription complex, whereas reptin has been demon- 0710343105/DC1. strated to repress the Wnt/␤-catenin signaling pathway (7). In © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710343105 PNAS ͉ December 26, 2007 ͉ vol. 104 ͉ no. 52 ͉ 20793–20798 Downloaded by guest on September 25, 2021 Fig. 1. Purification of pontin-containing complex. (A) The pontin-containing complex was immunoprecipitated with anti-Flag IgG-conjugated agarose beads from 293T cell extracts, and the bound proteins were eluted with Flag peptide and resolved by SDS/PAGE. (B) Peptide sequences of pontin-associated polypeptides from LC-MS/MS analysis. (C) Western blot analysis was performed by using the indicated antibodies, and reptin, ␤-catenin, p400, and Ubc9 were detected in the eluates. (D) Coimmunoprecipitation of endogenous pontin with Ubc9. Cell lysates were subjected to immunoprecipitation with either anti-Ubc9 IgG or control IgG, and the resultant precipitates were subjected to immunoblotting with anti-pontin IgG. genes, such as PSA, in prostate cancer cells. SUMOylation of pontin E2 (Ubc9) in the presence or absence of purified SUMO. Pontin is related to an active control mechanism for the transcriptional conjugates were formed after the addition of SUMO to the activation function of pontin through its nuclear retention and the SUMOylation mixture (Fig. 2A). These data revealed that pontin enhancement of coactivator binding, whereas SUMOylation of undergoes SUMO modification along with the identity of the reptin is involved in the potentiation of transcriptional repression. SUMO protein from LC-MS/MS analysis [supporting information Further, SUMOylation of pontin increased proliferation and (SI) Fig. 5]. To find the functional lysine residue(s) of pontin that growth of prostate cancer cells. Taken together, we address a serve as the SUMO acceptor sites, we searched the consensus molecular mechanism in which SUMO modification of pontin SUMOylation sequence ␺KxE (␺ represents a large hydrophobic mediates and elaborates the transcriptional regulation, thereby amino acid) (Fig. 2B) (22). Each of the six lysines of pontin was strongly activating a subset of AR target genes involved in tumor changed independently to arginine, and the resultant mutants were progression. tested for the ability of in vitro or in vivo SUMO modification (Fig. 2 C and D). Mutation of K2R, K108R, K171R, K231R, and K268R Results revealed little or no alteration in SUMO modification of pontin, Pontin Chromatin-Remodeling Complexes Contain a
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