MECHANISM OF RAF-1 ACTIVATION

描述（由申请人提供）：本提案的总体目标是继续验证我们的假设，即Raf-1激酶的激活需要多种因子（包括激酶和支架蛋白）的有序和协同作用。Raf家族成员是Ras的主要下游效应子，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Ras基因的激活突变，并且许多非核癌基因利用Ras/Raf/MEK/Erk途径来执行其致癌程序。其他人和我们以前的研究表明，不同的输入导致S338的磷酸化，这是Raf-1激活所必需的，但不是充分的。我们的初步数据表明，S338是响应于生长因子（例如，EGF或TPA），并且其响应于微管解聚剂如诺考达唑而被Pak磷酸化。我们已经确定了几个新的磷酸化位点（如S357）Raf-1似乎参与其调节。此外，我们发现Raf-1在包含KiRas激活突变的HCT 116结肠癌细胞中被组成性激活。我们还发现，通过RNAi抑制KSR表达导致Raf/MEK相互作用和MEK活化的抑制，导致DNA片段化。最后，我们的初步结果表明，HCT 116细胞提取物含有允许Raf-1体外活化的组分。基于这些发现，我们提出了以下具体目标的研究：（1）进一步研究S338在不同条件下如何磷酸化及其与其他磷酸化事件的关系;（2）研究S357磷酸化的机制，并确定这种磷酸化对Raf功能和vRas介导的转化的影响（恶性肿瘤）;和（3）阐明HCT 116细胞中Raf-1受KSR调节的机制，并使用这些细胞作为鉴定Raf-1的新型上游调节因子的工具。这些研究将为Raf如何被激活以及其激活如何影响细胞功能提供新的信息。它们也将作为评估Raf家族在人类恶性肿瘤中作用的框架。

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.