REGULATION OF C-MYB EXPRESSION BY PHOSPHORYLATION

C-Myb的截短与其致癌活性和 Myb的差值截断形式导致了 表型不同的造血细胞类型。因此，结构性的 修饰改变了c-Myb的功能。在正常分化过程中 结构修饰可以通过磷酸化或 差异RNA剪接。很明显，c-Myb是一种磷酸蛋白， 我们最近发现pp42mapk在体外使c-Myb磷酸化。 多个站点。有趣的是，这些站点似乎位于 已被证明影响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 潜在地参与蛋白质/蛋白质的相互作用。这个 通过磷酸化调节转录因子的功能是一种 有吸引力的假设，因为它允许一个数的积分 通过蛋白激酶和磷酸酶介导的信号通路 核能级。这项建议的目的是探讨 C-Myb功能的磷酸化调节。因此，这项建议具有 三个具体目标。首先，我们将使用二维磷酸肽 定位c-Myb在体内的磷酸化 重点是确定pp42mapk站点在什么条件下 磷酸化。第二，我们将使用c-myb活动的四个衡量标准来 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 激活，3)转换和4)阻止终端的能力 小鼠红白血病细胞的分化。第三，我们将确定 并从功能上表征了c-Myb磷酸化的新位点。在……里面 我们的第一个具体目标是确定以下地点的变化 除pp42mapk的磷酸化位点外，还有其他的磷酸化。这将是 允许我们超越pp42mapk站点，并开始理解 其他激酶对c-Myb功能的影响。这样做的总体目标是 建议理解磷酸化在蛋白合成中的作用。 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.