Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Base Sequence
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Carrier Proteins / physiology*
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GTPase-Activating Proteins / antagonists & inhibitors*
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Glutathione Transferase / genetics
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Glutathione Transferase / isolation & purification
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Heat-Shock Response
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Intracellular Signaling Peptides and Proteins
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Molecular Sequence Data
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Protein Conformation
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Protein Interaction Mapping
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Repressor Proteins / antagonists & inhibitors
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Repressor Proteins / metabolism*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / antagonists & inhibitors*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Saccharomyces cerevisiae Proteins / physiology*
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Sequence Deletion
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Signal Transduction
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Suppression, Genetic
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Two-Hybrid System Techniques
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ras GTPase-Activating Proteins / antagonists & inhibitors*
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ras Proteins / metabolism
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ras-GRF1 / genetics
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ras-GRF1 / metabolism
Substances
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Carrier Proteins
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GTPase-Activating Proteins
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IRA1 protein, S cerevisiae
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IRA2 protein, S cerevisiae
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Intracellular Signaling Peptides and Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Tfs1 protein, S cerevisiae
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ras GTPase-Activating Proteins
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ras-GRF1
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Glutathione Transferase
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ras Proteins