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Identification of IkappaB kinase phosphorylation sites on human transcription factor REL and characterization of histone acetyltransferase P300 as a REL coactivator.

机译:鉴定人类转录因子REL上IkappaB激酶的磷酸化位点,并表征作为REL共激活因子的组蛋白乙酰转移酶P300。

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摘要

The NF-kappaB family of transcription factors is involved in the regulation of many cellular processes including inflammation, innate immunity, apoptosis, and cell growth and proliferation. One member of this family, human c-Rel (REL), is encoded by an oncogene that is amplified or otherwise altered in several types of human B-cell lymphoma. Overexpression of REL can also oncogenically transform chicken spleen cells and a human B-lymphoma cell line. This transforming activity of REL requires the activity of at least one of the two transcriptional activation subdomains in the C-terminal half of REL. The goal of this research is to understand further REL-mediated transactivation by investigating post-translational modification and protein-protein interactions involving REL transactivation domain sequences.;Although REL is primarily regulated by nuclear-cytoplasmic translocation through interaction with IkappaB inhibitors, REL undergoes post-translational modifications that have been proposed to modulate its transcriptional activation ability. Deletion analysis, site-directed mutagenesis, and immune complex kinase assays have been used to identify Ser484 and Ser494 as the primary sites of IkappaB kinase (IKK)-mediated in vitro phosphorylation in the C-terminal transactivation domain of REL. However, co-transfection studies failed to detect IKK-mediated phosphorylation of these sites in vivo, and mutation of Ser484 and Ser494 did not affect IKK's ability to enhance GAL4-REL transactivation. Taken together, these results do not support a role for IKK-mediated phosphorylation as means of regulating the activity of REL in vivo.;Results in this thesis also demonstrate that the histone acetyltransferases p300 and CREB-binding protein (CBP) can interact with the REL transactivation domain in vitro and in vivo, and can enhance REL's transactivation ability. p300 interacts with REL in both REL-transformed chicken spleen cells and in the diffuse large B-lymphoma cell line RC-K8, in which REL is constitutively active and required for proliferation. Furthermore, it is shown that only a C-terminally truncated form of p300 is expressed in RC-K8 cells. This is the first report of a p300 truncation in B-cell lymphoma. A model is presented for how mutations affect the REL/NF-kappaB pathway in the RC-K8 B-lymphoma cell line. Taken together, these results suggest a role for p300 in REL-mediated oncogenic activity.
机译:转录因子NF-κB家族参与许多细胞过程的调控,包括炎症,先天免疫,细胞凋亡以及细胞生长和增殖。该家族的一个成员,人c-Rel(REL),由致癌基因编码,该致癌基因在几种类型的人B细胞淋巴瘤中被扩增或改变。 REL的过表达还可以致癌地转化鸡脾细胞和人B淋巴瘤细胞系。 REL的这种转化活性需要REL的C末端一半中的两个转录激活亚结构域中至少一个的活性。这项研究的目的是通过研究翻译后修饰和涉及REL转激活域序列的蛋白质相互作用来进一步了解REL介导的转激活作用;尽管REL主要受与IkappaB抑制剂相互作用的核质转位调控,但REL经历了后期-已提出调节其转录激活能力的翻译修饰。缺失分析,定点诱变和免疫复合激酶测定已用于鉴定Ser484和Ser494是REL C端反式激活域中IkappaB激酶(IKK)介导的体外磷酸化的主要位点。但是,共转染研究未能在体内检测到IKK介导的这些位点的磷酸化,Ser484和Ser494的突变并不影响IKK增强GAL4-REL反式激活的能力。两者合计,这些结果不支持IKK介导的磷酸化作为调节REL体内活性的手段。;本论文的结果还证明,组蛋白乙酰转移酶p300和CREB结合蛋白(CBP)可以与内毒素相互作用。 REL的反式激活域在体内和体外均可增强REL的反式激活能力。 p300在REL转化的鸡脾细胞和弥漫性大B淋巴瘤细胞系RC-K8中均与REL相互作用,其中REL具有组成型活性,是增殖所必需的。此外,显示在RC-K8细胞中仅表达p300的C末端截短形式。这是B细胞淋巴瘤中p300截短的首次报道。提出了一个模型,说明突变如何影响RC-K8 B淋巴瘤细胞系中的REL / NF-kappaB途径。总之,这些结果表明p300在REL介导的致癌活性中起作用。

著录项

  • 作者

    Garbati, Michael R.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 260 p.
  • 总页数 260
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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