E2A turnover and Notch-controlled lymphocyte development

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。他不 碱性螺旋-环-螺旋E2 A转录因子（E12/E47）也是B和B细胞发育所必需的 和T淋巴细胞。我们最近的研究结果表明，E2 A转录因子可能是下游效应子 Notch信号对淋巴细胞发育的影响。具体来说，我们已经证明，通过Notch的信号传导 调节E2 A蛋白的周转，这需要这些蛋白通过p42/p44磷酸化 MAP激酶。活化的Notchl的表达增强了E47与SCFSkp 2 E3的结合。 泛素连接酶及其泛素化。Notch诱导的E2 A蛋白降解发生在B而不是T 细胞研究然而，这与这些细胞中的MAP激酶活性水平相关。因此，我们认为， MAP激酶活性水平的差异可能是控制E2 A的有效调节机制 周转在这个提议中，我们首先假设Notch信号加速的E2 A退化可能起作用。 通过阻断胸腺中B细胞发育，在Notch介导的T与B谱系选择中起重要作用。 然后，我们提出Notch信号可能负责降低T细胞中一旦起作用的E2 A水平。 前TCR和TCR形成，较低的E2 A水平是T细胞通过适当的免疫调节所必需的。 成熟过程中的选择这些假设将通过测量孕酮中的E2 A降解来检验 在活化MAP激酶后，在B细胞和T细胞中。此外，突变将被引入E2 A 基因组编码对Notch诱导的降解具有抗性的E2 A蛋白。基因敲入小鼠将用于 确定突变蛋白的表达是否允许胸腺中的B细胞形成，是否对 Notch介导的骨髓B细胞发育抑制，以及对T细胞选择的影响， 以及积极和消极的选择。最后，我们建议了解Notch信号如何诱导 泛素化和降解E2 A蛋白。也许，Notch信号促进了未知基因的表达， 参与E2 A蛋白泛素化的基因，其将通过蛋白质的检查来鉴定 与E2 A蛋白的泛素化机制相关。总之，这里概述的研究扩展了我们的 研究Notch信号的一种新功能，即，诱导泛素介导的E2 A降解， 并评估该功能在淋巴细胞发育背景下的生物学意义。

Notch signaling pathways are important for several crucial decisions during lymphocyte development. T he 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 N otchl 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 prog enitor B cells and in T cells upon activati on 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 selecti on. 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 i n the context of lymphocyte development.