The mechanism(s) of Ras deactivation in growth factor signalling: role and regulation of the tumor suppressor gene product neurofibromin

小G蛋白Ras是基本上所有有核细胞中细胞增殖的中心调节因子。响应于生长因子的Ras激活在本质上是短暂的，并且大量数据已经提供了Ras信号传导的时间动态在细胞命运决策中的关键作用的证据。例如，Ras/Erk信号传导的持续时间在诱导PC 12神经内分泌细胞的增殖（瞬时Ras/Erk信号）与分化（延长信号）中是决定性的。类似地，由致癌Ras引起的活性Ras的延长和连续发射被假设为驱动Ras转化的细胞衰老的关键参数。因此，不仅要了解细胞如何协调Ras-GTP的形成，而且要了解Ras信号如何终止以限制Ras信号的持续时间，这在概念上是非常重要的。它是通过生长因子诱导的核苷酸交换因子（GEF）活性的增强而上调Ras上的核苷酸交换的结果。相比之下，Ras失活所涉及的机制，即随后的Ras-GTP水平的逆转，仍然不清楚。在对RU 860/4-1进行的工作中，我们已经公开了在负Ras/MEK/Erk反馈环的背景下GTP水解酶活化蛋白（GAP）神经纤维蛋白的活化作为Ras失活的基础机制。这是一个了不起的发现，因为：第一，它首次以生物化学方式记录了生长因子信号传导中GAP的刺激，第二，它揭示了肿瘤抑制蛋白神经纤维蛋白作为反馈信号转导器的作用，一个GAP物种的信号网络中的功能不明。在这里申请的项目中，我们计划调查和详细描述的生化过程连接MEK/Erk对Ras失活背景下的神经纤维蛋白刺激的影响。我们设想这种连接的三种潜在机制：第一直接磷酸化的神经纤维蛋白的Erk;第二Erk依赖磷酸化的中间蛋白，和第三神经纤维蛋白独立的变化，影响神经纤维蛋白的行动Ras的Ras微环境。实验计划，包括一个公正的神经纤维蛋白相互作用蛋白质组学屏幕和修改的上下文中的几个新开始的合作，已被设计为所有这些情况。总之，我们从RU 860/4-1的发现揭示了一个根本性的新的图片促有丝分裂瞬时Ras激活的特征是连续激活的GEF，Ras，和神经纤维蛋白GAP。我们在这里申请资源，继续这项工作一年，以获得一个缺乏的信息，这将使我们能够关闭Erk和神经纤维蛋白之间的机制差距，并将帮助我们获得有丝分裂Ras激活/失活过程的全貌。

The small G-protein Ras is a central regulator of cellular proliferation in essentially all nucleated cells. Ras activation in response to growth factors is transient in nature, and a large body of data has provided evidence for a critical role of the temporal dynamics of Ras signalling in cell-fate decision making. For example the duration of Ras/Erk signalling is decisive in inducing proliferation (transient Ras/Erk signal) versus differentiation (prolonged signal) in PC12 neuroendocrine cells. Similarly, prolonged and continuous firing from active Ras, as elicited by oncogenic Ras, is hypothesized to be a critical parameter driving Ras-transformed cells into senescence. It is hence of conceptual importance to understand not only how cells orchestrate Ras-GTP formation but also how the Ras signal is terminated to limit Ras signal duration.The process of Ras activation, i.e. the surge in Ras-GTP levels following mitogen stimulation, is understood in detail. It results from the up-regulation of nucleotide exchange on Ras via the growth factor induced enhancement of nucleotide exchange factor (GEF) activity. By contrast the mechanisms involved in Ras deactivation, i.e. the ensuing reversion of Ras-GTP levels, have remained obscure. During the ongoing work to RU860/4-1 we have disclosed activation of the GTP hydrolase activating protein (GAP) neurofibromin in the context of a negative Ras/MEK/Erk feedback loop as the mechanism underlying Ras deactivation. This is a remarkable finding because: 1st, it documents biochemically for the first time a stimulation of GAPs in growth factor signalling, and 2nd, it reveals a role as a feedback signal transducer for the tumour suppressor protein neurofibromin, a GAP species of unaccounted function in the signalling network.In the project applied for here we plan to investigate and characterize in detail the biochemical process linking MEK/Erk to neurofibromin stimulation in the context of Ras deactivation. We envisage three potential mechanisms for this connection: 1st direct phosphorylation of neurofibromin by Erk; 2nd Erk dependent phosphorylation of intermediary proteins, and 3rd neurofibromin-independent changes to the Ras microenvironment that affect neurofibromin action on Ras. The experimental program, including an unbiased proteomic screen of neurofibromin interactors and modifications in the context of one out of several newly started cooperations, has been designed to account for all these scenarios. In sum, our findings from RU860/4-1 reveal a fundamentally new picture of mitogenic transient Ras activation characterized by the sequential activation of GEFs, Ras, and the neurofibromin GAP. We apply here for resources to continue this work one more year in order to obtain one lacking piece of information that will allow us to close the mechanistic gap between Erk and neurofibromin and will help us obtain the full picture of the mitogenic Ras activation/deactivation process.