Chemoattractant-induced Ras activation during Dictyostelium aggregation.
Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.
Kae
Helmut
H
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3.
Lim
Chinten James
CJ
Spiegelman
George B
GB
Weeks
Gerald
G
D000818:Animals; D001681:Biological Assay; D002449:Cell Aggregation; D002460:Cell Line; D002630:Chemotactic Factors / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D002630:Chemotactic Factors; D015800:Protozoan Proteins; D029702:Schizosaccharomyces pombe Proteins; C424065:rasG protein, Dictyostelium; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; C077892:BYR2 protein, S pombe; D020930:MAP Kinase Kinase Kinases; D018631:ras Proteins