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

• Applicant: Privatdozent Dr. Ignacio Rubio
• Subject Area: Cell Biology ; General Genetics and Functional Genome Biology ; Biochemistry
• Term: from 2011 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 209439699

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.

• DFG Programme: Research Grants