Biochemistry and Molecular Pharmacology
Program in Molecular Medicine
First Thesis Advisor
Dr. Michael P. Czech
Son of Sevenless Protein, Drosophila, Drosophila, Ras Proteins
The questions outlined in this thesis dissertation were proposed in order to provide insight regarding the mechanism by which the Drosophila Son of sevenless (dSOS) protein activates Ras. Ras proteins are GTP-binding proteins which bind guanine nucleotides very tightly and cycle between the inactive GDP-bound state and the active GTP-bound state. To address the mechanism by which the dSOS proteins activates Ras, a structure-function analysis of the dSOS protein was performed using truncation and deletion mutants of dSOS. In vivo Ras activation experiments using transiently transfected cells revealed that the NH2-terminal domain of dSOS is required in order for the catalytic domain of dSOS to exhibit exchange activity in cultured mammalian cells. The COOH-terminal GRB2 (Growth Factor Receptor Binding Protein) binding domain on the otherhand was insufficient to confer Ras exchange activity to the dSOS catalytic domain. Further analysis of the NH2-terminal domain of the dSOS protein demonstrated that the function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin (PH) and DBL (Diffuse B-cell Lymphoma) homology sequences. Fractionation studies of cells transiently transfected with various dSOS mutant proteins demonstrated that the NH2-terminus of dSOS is also necessary for membrane association. These findings suggested that the model proposing that the recruitment of SOS via the adaptor protein GRB2 to the membrane is the main mechanism by which SOS activates Ras is unlikely to be the only mechanism by which SOS can activate Ras. From our data, a model can be proposed which postulates that SOS can activate Ras as a consequence of at least two steps. One step involves the SOS/GRB2 interaction and the second step involves the NH2-terminal domain of SOS associating with unidentified cellular elements.
McCollam-Guilani, LS. Analysis of the Mechanism of Ras Activation: Mapping of Important Functional Domains of the Son of Sevenless Protein. (1998). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 90. DOI: 10.13028/dajk-k043. https://escholarship.umassmed.edu/gsbs_diss/90
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