REGULATION OF C-MYB EXPRESSION BY PHOSPHORYLATION

c-Myb的截短与其致癌激活相关， Myb的不同截短形式导致 表型不同的造血细胞类型。 因此，结构 修饰改变了c-Myb的功能。 在正常分化期间 结构修饰可以通过磷酸化或 RNA差异剪接 很明显，c-Myb是一种磷蛋白， 我们最近发现pp 42 mapk在体外磷酸化c-Myb， 多个站点。 有趣的是，这些网站似乎位于 已显示影响c-Myb序列的负调控结构域 特异性DNA结合、转录反式激活、转化和 可能参与蛋白质之间的相互作用。 的 通过磷酸化调节转录因子的功能是一个重要的机制。 这是一个有吸引力的假设，因为它将允许一个数字的整合 通过蛋白激酶和磷酸酶的信号通路， 核水平。 本提案的目的是探讨 通过磷酸化调节c-Myb功能。 因此，该提案 三个具体目标。 首先，我们将使用二维磷酸肽 定位以表征体内c-Myb磷酸化， 重点是确定pp 42 mapk位点被 磷酸化。 第二，我们将使用四种c-myb活性测量方法， 确定磷酸化在调节c-myb功能中的作用 包括：1）序列特异性DNA结合，2）转录 反式激活，3）转化和4）阻断末端的能力 在小鼠红白血病细胞中的分化。 第三，我们将确定 并在功能上表征c-Myb磷酸化的新位点。 在 我们的第一个具体目标，我们希望确定的网站的变化 除了PP 42 MAPK磷酸化位点之外， 这将 让我们超越pp 42 mapk网站，并开始了解 其他激酶对c-Myb功能的影响。 这个广泛的目标 我们的建议是了解磷酸化在 调节c-Myb功能。

Truncation of c-Myb is associated with its oncogenic activation and differentially truncated forms of Myb result in the transformation of phenotypically distinct hematopoietic cell types. Thus, structural modifications alter the function of c-Myb. During normal differentiation structural modifications could be mediated by phosphorylation or differential RNA splicing. It is clear the c-Myb is a phosphoprotein and we have recently found that pp42mapk phosphorylates c-Myb in vitro at multiple sites. Interestingly, these sites appear to be located in the negative regulatory domain which has been shown to affect c-Myb sequence specific DNA binding, transcription transactivation, transformation and to potentially be involved in protein/protein interactions. The regulation of transcription factor function by phosphorylation is an attractive hypothesis as it would allow for the integration of a number of signalling pathways via protein kinases and phosphatases at the nuclear level. It is the purpose of this proposal to explore the regulation of c-Myb function by phosphorylation. Thus, this proposal has three specific aims. First, we will use two dimensional phosphopeptide mapping to characterize c-Myb phosphorylation in vivo with particular emphasis on determining conditions under which the pp42mapk sites are phosphorylated. Second, we will use four measures of c-myb activity to determine the role of phosphorylation in regulating c-myb function including: 1) sequence specific DNA binding, 2) transcription transactivation, 3) transformation and 4) the ability to block terminal differentiation in murine erythroleukemia cells. Third, we will identify and functionally characterize novel sites of phosphorylation c-Myb. In our first specific aim we expect to identify changes in sites of phosphorylation aside from the pp42mapk phosphorylation sites. This will allow us to move beyond the pp42mapk sites and begin to understand the impact of other kinases on c-Myb function. The broad goal of this proposal is to understand the role played by phosphorylation in regulating c-Myb function.