MECHANISM OF RAF-1 ACTIVATION

描述(由申请人提供)：这项提案的总体目标是继续检验我们的假设，即激活Raf-1激酶需要多种因素的有序和协同作用，包括激酶和支架蛋白。RAF家族成员是RAS下游的主要效应者，是调节细胞代谢、增殖、分化和凋亡的关键酶。Raf激酶也是肿瘤形成的重要因素。因此，在大约30%的人类癌症中发现了RAS基因的激活突变，许多非核癌基因利用Ras/Raf/MEK/Erk途径来执行它们的致癌程序。其他人和我们之前的研究已经证明，不同的输入导致S338的磷酸化，这是激活Raf-1所必需的，但不是充分的。我们的初步数据表明，S338在生长因子(如EGF或TPA)的作用下被自动磷酸化，而在微管解聚剂如诺可达唑的作用下，它被PAK磷酸化。我们已经在Raf-1上发现了几个新的磷酸化位点(例如S357)，它们似乎参与了Raf-1的调控。此外，我们还发现Raf-1在含有kiras激活突变的HCT116结肠癌细胞中被结构性激活。我们还发现，RNAi抑制KSR的表达会导致Raf/MEK相互作用和MEK激活的抑制，导致DNA片段化。最后，我们的初步结果表明，HCT116细胞提取物中含有允许体外激活Raf-1的成分(S)。基于这些发现，我们建议进行以下具体目标的研究：(1)进一步研究S338在不同条件下是如何被磷酸化的及其与其他磷酸化事件的关系；(2)研究S357磷酸化的机制(S)，并确定这种磷酸化对Raf功能和vRas介导的转化(恶性)的影响；以及(3)阐明Raf-1在HCT116细胞中受KSR调控的机制(S)，并利用这些细胞作为工具来确定Raf-1的新的上游调控因子。这些研究将提供关于Raf如何被激活以及它的激活如何影响细胞功能的新信息。它们还将作为评估Raf家族在人类恶性肿瘤中的作用的框架。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to continue to test our hypothesis that activation of Raf-1 kinase requires ordered and cooperative actions of multiple factors including kinases and scaffold proteins. Raf family members are major downstream effectors of Ras and key enzymes in the regulation of cell metabolism, proliferation, differentiation and apoptosis. The Raf kinases are also important factors in neoplasia. Thus, activating mutations of the Ras gene are found in approximately 30% of human cancers and many non-nuclear oncogenes exploit the Ras/Raf/MEK/Erk pathway to execute their oncogenic programs. Previous studies by others and us have demonstrated that different inputs lead to the phosphorylation of S338, which is necessary, but not sufficient for Raf-1 activation. Our preliminary data suggest that S338 is autophosphorylated in response to growth factors (e.g., EGF or TPA), and that it is phosphorylated by Pak in response to microtubule depolymerizing agents such as nocodazole. We have identified several novel phosphorylation sites (e.g. S357) on Raf-1 that appear to participate in its regulation. In addition, we have found that Raf-1 is constitutively activated in the HCT116 colon cancer cell that contains an activating mutation of KiRas. We have also found that suppression of KSR expression by RNAi results in inhibition of Raf/MEK interaction and MEK activation, leading to DNA fragmentation. Finally, our preliminary results indicate that the HCT116 cell extract contains component(s) that allows Raf-1 activation in vitro. Based on these findings, we are proposing studies with the following specific aims: (1) to examine further how S338 is phosphorylated under different conditions and its relationship with other phosphorylation events; (2) to examine the mechanism(s) responsible for the phosphorylation of S357 and to determine the impact of this phosphorylation on Raf function and vRas-mediated transformation (malignancy); and (3) to elucidate the mechanism(s) by which Raf-1 is regulated by KSR in HCT116 cells and to use these cells as a tool to identify novel upstream regulators of Raf-1. These studies will provide new information as to how Raf is activated and how its activation affects cellular functions. They will also serve as a framework for evaluating the role of the Raf family in human malignancy.