Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiae.
Ras proteins are guanine nucleotide-binding proteins that are highly conserved among eukaryotes. They are involved in signal transduction pathways and are tightly regulated by two sets of antagonistic proteins: GTPase-activating proteins (GAPs) inhibit Ras proteins, whereas guanine exchange factors activate them. In this work, we describe Tfs1p, the first physiological inhibitor of a Ras GAP, Ira2p, in Saccharomyces cerevisiae. TFS1 is a multicopy suppressor of the cdc25-1 mutation in yeast and corresponds to the so-called Ic CPY cytoplasmic inhibitor. Moreover, Tfs1p belongs to the phosphatidylethanolamine-binding protein (PEBP) family, one member of which is RKIP, a kinase and serine protease inhibitor and a metastasis inhibitor in prostate cancer. In this work, the results of (i) a two-hybrid screen of a yeast genomic library, (ii) glutathione S-transferase pulldown experiments, (iii) multicopy suppressor tests of cdc25-1 mutants, and (iv) stress resistance tests to evaluate the activation level of Ras demonstrate that Tfs1p interacts with and inhibits Ira2p. We further show that the conserved ligand-binding pocket of Tfs1-the hallmark of the PEBP family-is important for its inhibitory activity.
Chautard
Hélène
H
Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, UPR 4301, University of Orléans and INSERM, 45071 Orléans Cedex 2, France.
Jacquet
Michel
M
Schoentgen
Françoise
F
Bureaud
Nicole
N
Bénédetti
Hélène
H
D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors*; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D018869:Heat-Shock Response; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017384:Sequence Deletion; D015398:Signal Transduction; D013489:Suppression, Genetic; D020798:Two-Hybrid System Techniques; D020703:ras GTPase-Activating Proteins / Q000037:antagonists & inhibitors*; D018631:ras Proteins / Q000378:metabolism; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; C065725:IRA1 protein, S cerevisiae; C064960:IRA2 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C492432:Tfs1 protein, S cerevisiae; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D005982:Glutathione Transferase; D018631:ras Proteins