Regulation of the Ras exchange factor Ras-GRF2

grantor: University of TorontoThe Ras GTPases play a pivotal role in cellular proliferation and differentiation and become activated in response to a variety of extracellular signals, including growth and differentiation factors, hormones, immune antigens and calcium influx. Ras requires the action of guanine nucleotide exchange factors (GEFs) in order to become GTP-bound, enabling it to transmit a signal to downstream signaling components. Ras-GRF2 (GRF2) is a calcium-responsive Ras exchange factor which contains many recognizable protein domains, including an ilimaquinone (IQ) motif and a destruction box (DB). The IQ motif of a homologous protein, Ras-GRF1, binds calmodulin (CaM) and it was postulated that this binding activated GRF1. I demonstrated that the role of calcium and CaM in GRF2 function is not the same as that proposed for GRF1. GRF2 does indeed bind calmodulin but the IQ motif is not the sole determinant of CaM binding, and mutant proteins that cannot bind CaM are still calcium-responsive. Interestingly, an IQ-deleted protein can activate Ras but cannot activate the effector molecular Raf. I postulate that exchange factors may play a role in determining effector interactions and therefore play a larger role than previously thought in Ras signaling. The varied responses to different extracellular signals that all activate Ras could be a result of the individual GEF that was activated upstream of Ras. Because the DB is a motif that targets proteins for ubiquitin-mediated proteolysis, I examined the proteolysis of GRF2. I demonstrated that activated GRF2 is an unstable protein and that binding to Ras targets GRF2 for destruction. Deletion of the DB protects GRF2 from Ras-triggered degradation. I established that GRF2 is ubiquitinated 'in vivo' and that it becomes ubiquitinated only upon binding to Ras; downstream signaling is not required for this degradation, suggesting it is merely binding that triggers this degradation. Point mutants that cannot bind Ras are not ubiquitinated. GRF2 accumulates in a highly ubiquitinated state in the presence of proteasome inhibitors. I have reported the first evidence of 'in vivo' ubiquitination of a mammalian exchange factor and have provided a mechanism as to how the protein is targeted for proteolysis.Ph.D