MECHANISM OF RAF1 ACTIVATION

描述：RAF-1是MEK1/2上游的一种生理性激酶，其 下游靶点MAPK/ERK首先被鉴定为胰岛素反应性 激活剂。Raf-1是原癌基因c-Ras的主要下游效应因子 而它的激活对于调控增殖至关重要， 分化和其他细胞功能。尽管如此，知道 Raf激活的详细机制尚不完全。研究来自 许多实验室已经表明，细胞外信号，如 作为胰岛素，IGF和EGF最初与其氨基末端相互作用 监管领域。其他人和这些调查人员的发现表明 这种绑定打开了不活动的Raf的紧密结构-并使其 可由尚不确定的激活器访问，从而将Raf锁定在活动的 通过磷酸化和可能的其他机制来维持状态。我们最近做了 显示该激活过程需要14-3-3，或者通过 调节Raf-1的二聚化或使其与其他 帮助组装可激活的Raf的蛋白质因子 激活。拟议的研究将使用生物化学来检验这一模型。 和分子方法。具体目标如下：1. 阐明齐聚/二聚作用的作用机理 与激活Raf-1有关。2.探讨14-3-3在细胞周期调控中的作用。 Raf激酶活性的调节。3.鉴定Raf上的序列 对它的激活是至关重要的。重点将放在确定一个新的 EGF激活时出现在Raf-1肽图上的磷酸肽 它。4.鉴定使所述序列磷酸化的激酶 在目标3中。这些研究应该通过以下方式为机制提供新的见解 哪个Raf-1被激活以响应其细胞外的变化和 胞内环境。他们还应该扩大我们对 14-3-3和二聚体在细胞信号转导中的作用。

DESCRIPTION: Raf-1 is a physiological kinase upstream of MEK 1/2, whose downstream target, MAPK/Erk was first identified as an insulin-responsive kinase. Raf-1 is a major downstream effector of the proto-oncogene c-Ras and its activation is crucial for the regulation of proliferation, differentiation and other cellular functions. Despite this, knowledge of the detailed mechanism of Raf activation is still incomplete. Studies from a number of laboratories have suggested that extracellular signals, such as insulin, IGF and EGF initially interacting with its amino-terminal regulatory domain. Findings by others and these investigators suggest that this binding opens the tight structure of inactive Raf- and makes it accessible to as yet unidentified activators, which lock Raf in an active state by phosphorylation and possibly other mechanisms. We have recently shown that 14-3-3 is required for this activation process, either by mediating dimerization of Raf-1 or causing it to interact with other protein factors that assist in the assembly of an activatable Raf activation. The proposed studies will examine this model using biochemical and molecular approaches. The specific aims are as follows: 1. To elucidate the mechanism by which oligomerization/dimerization contributes to the activation of Raf-1. 2. To explore the role of 14-3-3 in the regulation of Raf kinase activity. 3. To identify sequences on Raf that are critical for its activation. The focus will be on identifying a new phospho-peptide that appear on the peptide map of Raf-1 when EGF activates it. 4. To identify the kinase that phosphorylates the sequences described in aim 3. These studies should provide new insights into the mechanism by which Raf-1 is activated in responses to changes in its extracellular and intracellular milieu. They should also expend our understanding of the role 14-3-3 and of dimerization in cellular signal transduction.