Investigation of the mechanisms terminating growth factor induced Ras activation
Transient Ras activation induced by growth factors is crucial for normal cell proliferation. Ras activity is regulated by opposing actions of two classes of Ras regulatory enzymes. Guanine nucleotide exchange factors (GEFs) promote GTP-bound Ras state by enhancing exchange of GDP with GTP. GTPase activating proteins (GAPs) enhance the otherwise slow intrinsic Ras GTPase activity rate, promoting conversion into the inactive GDP-bound state of Ras. The rise in RasGTP levels following exposure to growth factors is well understood. The mechanism of the ensuing deactivation of Ras is not yet fully understood, but is inferred to involve feedback inhibition of Sos, a GEF which drives the accumulation of active, GTP-loaded Ras. This study focuses on understanding of how Ras signalling is terminated in the context of a mitogenic environment to limit and control Ras signalling in time. Growth factors induce a transient Ras activation in HeLa cells. Strikingly, GEF activity is continuously high at all time points tested, even if RasGTP levels have dropped back to the basal state. Finally we identified a crucial activation of the GAP protein NF1 to counteract GEF activity and to deactivate Ras. Furthermore, Rsk1/2 inhibition led to a sustained Ras activation whereas the GEF activity was not affected. Collectively, the findings emerged from this thesis identified a new molecular mechanism terminating growth factor induced Ras activation characterised by the involvement of Rsk and NF1 in a feedback loop. For the first time a biochemical stimulation of the GAP activity associated with growth factor signalling pathways was demonstrated. Secondly, the acquired data revealed a central role of the tumour suppressor NF1 as an executing target protein of a MAPK induced feedback pathway. Prospectively it is of medical importance to figure out the biochemical link between Rsk and NF1 activation in growth factor induced Ras deactivation to improve therapeutic strategies for patients with RASopathy syndromes.
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