ANALYSIS OF RASG PHOSPHORYLATION IN REGULATING CHEMOTATIC PATHWAYS IN DICTYOSTELIUM DISCOIDEUM

Abstract

Chemotaxis is a process by which single-cell and multi-cellular organisms are able to migrate in response to different chemical gradients, or chemoattractants. Dictyostelium discoideum, social amoebae used as a common model organism to study eukaryotic chemotactic pathways, have the ability to migrate and aggregate in response to chemoattractants like cAMP. RasG is a protein that is part of the Ras family GTPases, and is activated downstream of the heterotrimeric G-protein in response to cAMP in the Dictyostelium chemotactic pathway. Preliminary data indicates that RasG is possibly regulated by PKA and may be phosphorylated by PKA upon activation. To determine the role of RasG phosphorylation, site directed mutagenesis was used to develop loss of function and phosphomimetic mutations to RasG constructs. The in vivo functionality of these constructs was tested with growth assays, and the expression and activity of RasG upon cAMP stimulation was tested with the Ras activity assay and immunoblotting. There is currently no evidence to confirm that RasG phosphorylation plays a role in RasG activation upon cAMP stimulation. Growth assays suggest that RasG null cells lose their phenotype when kept in culture for long periods of time, thereby questioning the validity of this assay. The Ras activity assay shows no clear pattern between wild type and mutant constructs, suggesting that the phosphorylation sites may not directly affect RasG activity. Further investigation requires a method of testing the in vivo functionality of DNA constructs, while also being able to accurately visualize changes in RasG activity upon cAMP stimulation.