Ras proteins function as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDPbound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). Oncogenic mutants of Ras are constitutively activated through continuous association with GTP and the incidence of Ras in human cancer is approximately 30%. The three Ras isoforms were once thought to be functionally redundant. Evidence to date is now suggesting that the activation of different Ras isoforms leads to different cellular consequences. Consequently, understanding the Ras proteins and the proteins that lead to their activation is critical to the synthesis of anti-cancer therapies.
The cloning and characterizing of a novel RasGEF is described in this thesis. RasGRP2 is the longer alternatively spliced isoform of the RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of NH2-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and K-ras, but not H-ras. RasGRP2 also catalyzes nucleotide exchange on Rapl, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 towards N-ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH 3T3 cells transfected with wild type N-ras results in an accelerated growth rate but not morphological transformation. Thus, under appropriate growth conditions, RasGRP2 and CalDAG-GEFI are dual specificity Ras and Rap exchange factors.
Characterization of the diacylglycerol binding domain (DGB) of RasGRP2 and CalDAG-GEFI, and the NH2-terminal plasma membrane targeting motif of RasGRP2 determined that the DGB domain is critical to the regulation of RasGRP2 and CalDAG-GEFI. Deletion of the DGB domain abolishes PC 12 cell differentiation driven by RasGRP2 and CalDAG-GEFI. Without the DGB domain, CalDAG-GEFI does not translocate to the plasma membrane in response to phorbol esters and the constitutive localization of RasGRP2 is decreased when the DGB domain is absent. Loss of the DGB domain impairs the regulation of the guanine nucleotide exchange activity of RasGRP2 and CalDAG-GEFI towards N-ras and Rap 1, and the responses of the two exchange factors to TPA and calcium ionophor are compromised. The dual acylation motif of RasGRP2 facilitates plasma membrane localization of RasGRP2. The combination of palmitoylation and myristoylation is essential for plasma membrane targeting; myristoylation by itself is not capable of targeting RasGRP2 to the plasma membrane. The RasGRP2 myristoylation consensus signal is atypical and restoration of a typical consensus sequence improves the plasma membrane localization of RasGRP2 in the presence of posttranslational palmitoylation. The dual acylation motif can target RasGRP2 to the plasma membrane in the absence of the DGB domain but is insufficient to restore regulation of the RasGEF. These results indicate that the DGB domain is essential for specific plasma membrane microdomain localization of RasGRP2.
Investigations into Ras isoform activation by growth factors in BHK cells indicate that growth factors preferentially activate Ras isoforms. Whilst EGF leads to the endogenous activation of all three Ras isoforms, PDGF preferentially activates H-ras and insulin preferentially activates N-ras followed by K-ras. Further investigation into Ras isoform activation determined that RasGEFs also selectively activate Ras isoforms and can act together to synergistically increase RasGTP loading. Replacement of the H-ras Hypervariable region (HVR) with the N-ras HVR forces the GTP loading profile of N-ras on the H-ras chimera, indicating that the HVR clearly influences the preferential activation of Ras isoforms. The Ras COOH-terminal anchors are primarily responsible for membrane micro-localization but recent work has shown that interaction of H-ras with lipid rafts is modulated by GTP-loading via a mechanism that requires the HVR. Analysis of the H-ras HVR in this thesis identified two regions in the HVR linker domain that regulate H-ras plasma membrane association. Release of activated H-ras from lipid rafts is blocked by deleting amino acids 166-172 or 173-179. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific NH2-terminal sequences of the linker domain operate in concert with a more COOHterminal spacer domain to regulate H-ras raft association. Mutations in the linker domain which confine activated H-rasG12V to lipid rafts inhibit PC 12 cell differentiation.
The HVR linker domain also influences Ras interaction with exchange factors. Mutations in the linker domain also suppress the dominant negative phenotype of H-rasS17N in vivo, and the Sos1-induced GTP loading of H-ras linker domain mutants is impaired in intact cells, indicating that HVR sequences are essential for efficient interaction of H-ras with exchange factors. Caveolins are involved in cholesterol transport and signal transduction. The ectopic expression of a caveolin-3 (cav-3) mutant, cav-3C71W inhibited H-rasG12V signalling as measured in a biological PC 12 cell differentiation assay. Cav-3C71W must be plasma membranelocalized to inhibit H-rasG12V signalling as mutations that localize it to the Golgi region rescue H-rasG12V-mediated PCI2 cell differentiation. Mutation of cysteine to cav-3C71W has no effect upon H-rasG 12V-mediated PC 12 cell differentiation, indicating that cav-3C71W gains its phenotype due the acquisition of tryptophan rather than the loss of cysteine. Finally, mutations in the H-rasG12V HVR linker domain and the cav-3C71Wmutation may also impair H-rasG12V trafficking. Taken together, these results indicate that Ras microlocalization within the plasma membrane is critical for activation and signalling function. The possible physiological relevance of the activation of Ras by RasGRP2 and the preferential activation of Ras isoforms is discussed in this thesis.