LARGE-SCALE IDENTIFICATION OF C-MYC-ASSOCIATED PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 c-MYC癌基因编码转录因子，其对于诱导细胞凋亡是充分和必需的。 增殖然而，c-MYC蛋白是一种相对较弱的反式激活因子，这表明它可能具有其他功能。 为了鉴定可能揭示c-MYC新功能或代表c-MYC调节因子的蛋白质相互作用物，我们系统地 使用蛋白质组学方法在体内鉴定与c-MYC相关的蛋白质。我们将串联亲和纯化 (TAP)质谱多维蛋白质鉴定技术（MudPIT）。因此，221 c-MYC相关 蛋白质进行了鉴定。其中有17个先前已知的c-MYC相互作用因子。选定的新c-MYC相关 蛋白质（DBC-1，FBX 29，KU 70，MCM 7，Mif 2-B/CHD 4，RNA Pol II，RFC 2，RFC 3，SV 40大T抗原，TCP 1a，U 5 - 116 kD， ZNF 281）的独立确认。对于与MCM 7、SV 40大T抗原和DBC-1的结合， 重要MYCbox II区域是必需的，而FBX 29和Mi2-B通过MYC-box II和BR-HLH-LZ基序相互作用。 此外，还鉴定了c-MYC活性的调节因子：FBX 29（一种E3泛素连接酶）的异位表达降低了c-MYC活性。 MYC蛋白水平并抑制c-MYC反式激活，而FBX 29的敲低升高了c-MYC蛋白的浓度。 MYC。此外，蔗糖梯度分析表明，c-MYC存在于许多不同大小的复合物中 和组成，这可能会容纳大量的新的c-MYC在此鉴定的相关蛋白， c-MYC的多种功能。我们的研究结果表明，c-MYC，除了作为一个促有丝分裂转录因子， 细胞增殖通过与参与细胞所需的多种合成过程的蛋白质复合物直接结合而实现 分裂，例如DNA复制/修复和RNA加工。此外，这第一次全面描述 c-MYC相关的亚蛋白质组将促进旨在阐明c-MYC生物学的进一步研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The c-MYC oncogene encodes a transcription factor, which is sufficient and necessary for the induction of cellular proliferation. However, the c-MYC protein is a relatively weak transactivator suggesting that it may have other functions. To identify protein interactors which may reveal new functions or represent regulators of c-MYC we systematically identified proteins associated with c-MYC in vivo using a proteomic approach. We combined tandem affinity purification (TAP) with the mass spectral multidimensional protein identification technology (MudPIT). Thereby, 221 c-MYC associated proteins were identified. Among them were 17 previously known c-MYC interactors. Selected new c-MYC associated proteins (DBC-1, FBX29, KU70, MCM7, Mif2-b/CHD4, RNA Pol II, RFC2, RFC3, SV40 Large T Antigen, TCP1a, U5-116kD, ZNF281) were confirmed independently. For association with MCM7, SV40 Large T Antigen and DBC-1 the functionally important MYCbox II region was required, whereas FBX29 and Mi2-b interacted via MYC-box II and the BR-HLH-LZ motif. In addition, regulators of c-MYC activity were identified: ectopic expression of FBX29, an E3 ubiquitin ligase, decreased c- MYC protein levels and inhibited c-MYC transactivation, whereas knock-down of FBX29 elevated the concentration of c- MYC. Furthermore, sucrose gradient analysis demonstrated that c-MYC is present in numerous complexes with varying size and composition, which may accommodate the large number of new c-MYCassociated proteins identified here and mediate the diverse functions of c-MYC. Our results suggest that c-MYC, besides acting as a mitogenic transcription factor, regulates cellular proliferation by direct association with protein complexes involved in multiple synthetic processes required for cell division, as for example DNA-replication/repair and RNA-processing. Furthermore, this first comprehensive description of the c-MYC-associated subproteome will facilitate further studies aimed to elucidate the biology of c-MYC.