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

• Final Report Year: 2016

Final Report Abstract

Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critically required for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the guanine nucleotide exchange factor (GEF) Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but is inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs), the second group of Ras regulatory proteins, participate in controlling the rise and fall of Ras-GTP levels remains largely unknown. In the present project we have monitored nucleotide exchange of Ras in permeabilized cells to understand the exact role played by GEFs and GAP in the growth-factor driven rise and fall of Ras-GTP levels. We find, unexpectedly, that growth factor-induced Ras-GTP levels decline while nucleotide exchange remains high long beyond the time point where Ras-GTP levels have dropped back to their basal level. Together with other findings, these observations strongly point to GAP activation as the main mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of enduringly high nucleotide exchange. To identify the molecular players involved in the stimulation of Ras-GAP activity at later time points of EGF action we used pharmacological and genetic approaches. These data documented that a feedback loop comprising the MEK/Erk pathway and its downstream kinases Rsk1 and Rsk2 was involved in the stimulation of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin. Thus, feedback activation of neurofibromin was responsible for the decline of growth factor-induced Ras-GTP levels and for ensuring transient Ras activation and signaling. Interestingly, our data also rationalize the neurofibromatosis-like phenotype reported sporadically for cases of Coffin-Lowry, a syndrome caused by inactivating mutations in the assumedly pro-mitogenic kinase Rsk2. Moreover, our data do not support the concept that Sos is subject to feedback inhibition in the context of growth-factor signaling,suggesting that the well-established feedback phosphorylation of Sos is relevant for processes other than the control of Ras GTP-loading. Finally, our experiments excluded the participation of SPRED1 and SPRED2 in the control of neurofibromin activity during EGF-induced Ras activation. Suprisingly, our nucleotide exchange measurements disclosed an inhibition of Sos-dependent nucleotide exchange on Ras by SPRED1 and SPRED2, which may explain why SPRED can act as a negative regulator of the Ras/Erk pathway in many biological systems. In conclusion, the findings obtained in this project ascribe for the first time a precise role to neurofibromin in hormonal control of Ras activity. From a conceptual point of view, our data illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange. Beyond the conceptual value of these findings to our understanding of the Ras activation process, our data also offer new concepts to understand and address molecular mechanisms at play in neurofibromatosis type I, Coffin-Lowry and LEGIUS syndromes.

Publications

• (2013) Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling. Biochem J 454, 323-332
  Song SP, Hennig A, Schubert K, Markwart R, Schmidt P, Prior IA, Bohmer FD, Rubio I
  
• (2015) Ras activation revisited: role of GEF and GAP systems. Biol Chem 396, 831-848
  Hennig A, Markwart R, Esparza-Franco MA, Ladds G, Rubio I
  
• (2016) Feedback activation of neurofibromin terminates growth factor-induced Ras activation. Cell Commun Signal 14, 5
  Hennig A, Markwart R, Wolff K, Schubert K, Cui Y, Prior IA, Esparza-Franco MA, Ladds G, Rubio I