title: Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae.
abstract: The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Colombo
authors.forename: Daniela
authors.initials: D
authors.lastname: Ronchetti
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein
authors.forename: Joris
authors.initials: J
authors.lastname: Winderickx
authors.forename: Enzo
authors.initials: E
authors.lastname: Martegani
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000483:Alleles; D049109:Cell Proliferation; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D017353:Gene Deletion; D005838:Genotype; D005947:Glucose / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D018631:ras Proteins / Q000378:metabolism*
chemical_list: C109334:GPR1 protein, S cerevisiae; D044385:GTP-Binding Protein alpha Subunits; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C083907:Gpa2 protein, S cerevisiae; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases; D005947:Glucose