Mutagenic alteration of the distal switch II region of RAS blocks CDC25-dependent signaling functions.
We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange factor CDC25. We first constructed yeast tester strains in which the deletion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination with the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro. Phenotypes included impaired growth at 37 degrees C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations. Tester strains were subsequently used for the reintroduction of various combinations of wild-type and mutated RAS2 and CDC25 genes by genetic techniques, as well as for in vitro reconstitution assays with the corresponding proteins. CDC25 restored both growth and glucose-induced cyclic AMP signaling in the presence, but not in the absence of wild-type RAS2. A gene encoding a RAS2 protein with a mutationally altered switch II region was expressed but was ineffective in reintegrating exchange factor-dependent responses in vivo. Wild-type, but not mutagenically altered, RAS2 proteins were stimulated by exchange factors in vitro. We conclude that the conserved distal switch II region is required for CDC25-dependent activation of RAS.
Mirisola
M G
MG
Dipartimento di Biologia Cellulare e dello Sviluppo, Centro di Oncobiologia Sperimentale, Palermo, Italy.
Seidita
G
G
Verrotti
A C
AC
Di Blasi
F
F
Fasano
O
O
D000262:Adenylyl Cyclases / Q000378:metabolism; D018797:Cell Cycle Proteins; D015825:Chromosomes, Fungal; D000242:Cyclic AMP / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005800:Genes, Fungal; D016153:Genes, Suppressor; D005838:Genotype; D005947:Glucose / Q000494:pharmacology; D007700:Kinetics; D016296:Mutagenesis; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity; D013489:Suppression, Genetic; D018631:ras Proteins; D020666:ras-GRF1
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; C489014:RAS1 protein, S cerevisiae; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases; D005947:Glucose