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是几乎所有有核细胞中细胞增殖的中心调节剂。Ras对生长因子的激活在本质上是短暂的，大量数据已经为Ras信号在细胞命运决策中的时间动态关键作用提供了证据。例如，在PC12神经内分泌细胞中，Ras/Erk信号的持续时间是诱导增殖（瞬时Ras/Erk信号）和分化（延长信号）的决定性因素。类似地，由致癌Ras引发的活性Ras的长时间和持续的放电被假设为驱动Ras转化细胞进入衰老的关键参数。因此，不仅要了解细胞如何协调Ras- gtp的形成，还要了解Ras信号如何终止以限制Ras信号持续时间，这在概念上具有重要意义。Ras激活的过程，即有丝分裂原刺激后Ras- gtp水平的激增，被详细理解。这是由于通过生长因子诱导的核苷酸交换因子（GEF）活性的增强，Ras上的核苷酸交换上调所致。相比之下，Ras失活的机制，即随后Ras- gtp水平的逆转，仍然不清楚。在RU860/4-1的持续研究中，我们揭示了GTP水解酶激活蛋白（GAP）神经纤维蛋白在Ras/MEK/Erk负反馈回路下的激活是Ras失活的机制。这是一个值得注意的发现，因为：首先，它从生物化学角度首次记录了生长因子信号传导中GAP的刺激，其次，它揭示了肿瘤抑制蛋白神经纤维蛋白的反馈信号换能器的作用，神经纤维蛋白是信号网络中未知功能的GAP物种。在这里申请的项目中，我们计划在Ras失活的背景下，详细研究和表征MEK/Erk与神经纤维蛋白刺激的生化过程。我们设想了这种联系的三种潜在机制：第一，Erk直接磷酸化神经纤维蛋白；第二Erk依赖的中间蛋白磷酸化，第三神经纤维蛋白不依赖的Ras微环境变化影响神经纤维蛋白对Ras的作用。实验程序，包括神经纤维蛋白相互作用的公正的蛋白质组学筛选和在几个新开始的合作中的一个背景下的修改，已经被设计为考虑所有这些情况。总之，RU860/4-1的研究结果揭示了一幅全新的有丝分裂瞬时Ras激活的图景，其特征是gef、Ras和神经纤维蛋白GAP的顺序激活。我们在这里申请资源继续这项工作一年多，以获得一个缺乏的信息，这将使我们能够缩小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/失活过程的全图。

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.