E2A turnover and Notch-controlled lymphocyte development

E2A转换和Notch控制的淋巴细胞发育

• 批准号: 6675261
• 负责人: Xiao-Hong Sun
• 金额: $ 17.33万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6675261
• 关键词: B lymphocyte; T cell receptor; T lymphocyte; biological signal transduction; cell differentiation; cell line; flow cytometry; gene expression; gene targeting; genetically modified animals; immunoprecipitation; laboratory mouse; lymphocyte; mitogen activated protein kinase; phosphorylation; polymerase chain reaction; protein degradation; protein structure function; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): Notch signaling pathways are important for several crucial decisions during lymphocyte development. The basic helix-loop-helix E2A transcription factors (E12/E47) are also essential for the development for both B and T lymphocytes. Our recent findings suggest that E2A transcription factors may be downstream effectors of Notch signaling for lymphocyte development. Specifically, we have shown that signaling through Notch modulates the turnover of E2A proteins, which requires the phosphorylation of these proteins by p42/p44 MAP kinases. Expression of activated Notch l enhanced the association of E47 with the SCFSkp2 E3 ubiquitin ligase and its ubiquitination. Notch-induced degradation of E2A proteins occurred in B but not T cells studied. However, this correlated with the level of MAP kinase activity in these cells. Therefore, differences in the level of MAP kinase activity may be an effective regulatory mechanism to control E2A turnover. In this proposal, we first hypothesize that accelerated E2A degradation by Notch signals may play an important role in Notch-mediated T versus B lineage choice by blocking B cell development in the thymus. We then propose that Notch signals may be responsible for reducing E2A levels in T cells once functional pre-TCR and TCR are formed, and lower E2A levels are necessary for T cells to go through proper selections during maturation. These hypotheses will be tested by measuring E 2A degradation in progenitor B cells and in T cells upon activation of MAP kinases. Furthermore, mutations will be introduced into the E2A genome to encode E2A proteins resistant to Notch induced degradation. The knock-in mice will be used to determine if expression of the mutant protein allows B cell formation in the thymus, renders resistance to Notch-mediated inhibition of B cell development in the bone mar row, and impacts on T cell selection, as well as positive and negative selection. Finally, we propose to understand how Notch signals induce the ubiquitination and degradation of E2A proteins. Perhaps, Notch signals facilitate expression of unknown genes involved in the ubiquitination of E2A proteins, which will be identified by examination of proteins associated with the ubiquitination machinery of E2A proteins. In summary, studies outlined here expand our investigation on a novel function of Notch signaling, i.e., induction of ubiquitin-mediated E2A degradation, and evaluate the biological significance of this function in the context of lymphocyte development.

描述（由申请人提供）：Notch信号通路对于淋巴细胞发育过程中的几个关键决定很重要。碱性螺旋-环-螺旋E2 A转录因子（E12/E47）对于B和T淋巴细胞的发育也是必需的。我们最近的研究结果表明，E2 A转录因子可能是下游效应的Notch信号淋巴细胞发育。具体来说，我们已经表明，通过Notch信号调节E2 A蛋白的周转，这需要这些蛋白的磷酸化p42/p44 MAP激酶。活化的Notch 1的表达增强了E47与SCFSkp 2 E3泛素连接酶的结合及其泛素化。Notch诱导的E2 A蛋白降解发生在所研究的B细胞中，而不是T细胞中。然而，这与这些细胞中的MAP激酶活性水平相关。因此，MAP激酶活性水平的差异可能是控制E2 A周转的有效调节机制。在这个提议中，我们首先假设Notch信号加速E2 A降解可能通过阻断胸腺中B细胞发育在Notch介导的T与B谱系选择中发挥重要作用。然后，我们提出，一旦形成功能性前TCR和TCR，Notch信号可能负责降低T细胞中的E2 A水平，并且较低的E2 A水平对于T细胞在成熟过程中进行适当的选择是必要的。这些假设将通过测量MAP激酶激活后祖B细胞和T细胞中的E 2A降解来检验。此外，将突变引入E2 A基因组以编码对Notch诱导的降解具有抗性的E2 A蛋白。敲入小鼠将用于确定突变蛋白的表达是否允许胸腺中B细胞形成，是否对Notch介导的骨髓中B细胞发育抑制产生抗性，以及是否影响T细胞选择以及阳性和阴性选择。最后，我们建议了解Notch信号如何诱导E2 A蛋白的泛素化和降解。也许，Notch信号促进参与E2 A蛋白泛素化的未知基因的表达，这将通过检查与E2 A蛋白泛素化机制相关的蛋白质来鉴定。总之，这里概述的研究扩展了我们对Notch信号传导的新功能的研究，即，诱导泛素介导的E2 A降解，并评估这种功能在淋巴细胞发育中的生物学意义。