DH5 [(NalBL21(DE3) [gal dcm (DE3)] strain was used to produce 6His-Tfs1, and SCS1 ((1987-2358)-DBD, NLSMoBiTecpEG202TpEG202, (1983-2432)See textpJG4-5cpJG4-5, (1992-2444)See textpJG4-5fpJG4-5, (1920-2347)See textpJGIra2TBDpJG4-5, 2232-2347See textpJGIra1pJG4-5, (1987-2358)See textpJGTpJG4-5, was amplified by PCR using the primers HB1 and HB2

DH5 [(NalBL21(DE3) [gal dcm (DE3)] strain was used to produce 6His-Tfs1, and SCS1 ((1987-2358)-DBD, NLSMoBiTecpEG202TpEG202, (1983-2432)See textpJG4-5cpJG4-5, (1992-2444)See textpJG4-5fpJG4-5, (1920-2347)See textpJGIra2TBDpJG4-5, 2232-2347See textpJGIra1pJG4-5, (1987-2358)See textpJGTpJG4-5, was amplified by PCR using the primers HB1 and HB2. small guanine nucleotide-binding proteins that serve as molecular switches in signal transduction pathways and thus control cell growth and differentiation (35). Ras proteins cycle between an active GTP-bound form and an inactive GDP-bound form. The GDP/GTP exchange reaction that converts inactive Ras into its active form is enhanced by guanine exchange factors (GEFs) (4). The GTP-bound form of Ras is converted into its GDP-bound form by a GTPase activity stimulated by GTPase-activating proteins (GAPs) (4). Ras proteins are highly conserved among eukaryotes. Furthermore, the fact that the functional domains of GEF and GAP are homologous suggests that their Tirasemtiv (CK-2017357) activity is regulated by similar mechanisms. Indeed, possesses two Ras proteins, Ras1 and Ras2, which are activated by two GEFs, Cdc25 and Sdc25 (12, 13), and inhibited by two GAPs, Ira1 and Ira2 (43, 44). All of these proteins display sequence and functional homology with their mammalian counterparts (18, 26, 30, 42, 53). In yeast, Ras proteins activate the cyclic AMP (cAMP)/protein kinase A (PKA) pathway, which controls metabolism, stress resistance, growth, and meiosis (10, 45, 46). In this pathway, cAMP is synthesized by adenylate cyclase (27, 48). cAMP then activates the cAMP-dependent PKA by AGO binding to its regulatory subunit (47). The numerous downstream targets of PKA include enzymes involved in intermediate metabolism, proteins that are important for cell cycle control, and transcription factors involved in the expression of stress response element (STRE)-controlled genes and ribosomal protein genes (29, 32, 34, 41). In this work, we describe the first physiological inhibitor of Ras GAP activity: that of the yeast protein, Ira2. The inhibitor protein is named Tfs1p (for Twenty-Five suppressor) and was previously isolated by Robinson and Tatchell (33) as a multicopy suppressor of the mutant. is an STRE-regulated gene. It is overexpressed in many stress conditions such as oxidative stress, diauxic shift, and heat shock (19, 8, 7). Tfs1p has also been shown to inhibit CPY (11), and it has recently been shown that the acetylation of its N terminus is required for this activity (28). Tfs1p belongs to the conserved phosphatidylethanolamine-binding protein (PEBP) family (present in many organisms such as mammals, plants, worms, mutation, and (iii) that Tfs1p directly affects the activation level of the Ras/cAMP/PKA Tirasemtiv (CK-2017357) pathway. We also demonstrate that conserved residues (involved in the formation of the cavity in members of the PEBP family) are required for Tfs1p function. MATERIALS AND METHODS Strains, press, and general methods. DH5 [(NalBL21(DE3) [gal dcm (DE3)] strain was used to Tirasemtiv (CK-2017357) produce 6His-Tfs1, and SCS1 ((1987-2358)-DBD, NLSMoBiTecpEG202TpEG202, (1983-2432)Observe textpJG4-5cpJG4-5, (1992-2444)Observe textpJG4-5fpJG4-5, (1920-2347)Observe textpJGIra2TBDpJG4-5, 2232-2347See textpJGIra1pJG4-5, (1987-2358)Observe textpJGTpJG4-5, was amplified by PCR using the primers HB1 and Tirasemtiv (CK-2017357) HB2. After digestion with NdeI and XhoI, the gene was put into pET14-b that had been opened with the same enzymes. The producing construct, pET14T, contains the entire coding sequence of Tfs1 put in framework downstream of a six-His tag. The and open reading frames were amplified by PCR with Tirasemtiv (CK-2017357) primer pair HB13 and HB2 and primer pair HB14 and HB15, respectively. After cleavage with EcoRI and XhoI, the PCR fragments were ligated into the EcoRI and XhoI sites of both pEG202 and pJG4-5. This generated pEG202T and pEG202Y, which contain the entire coding sequences of and and sequences, respectively, put in framework downstream of a set of three DNA sequences encoding the simian disease 40 nuclear localization transmission, the B42 activator, and the hemagglutinin (HA) epitope tag. A DNA fragment encoding amino acids (aa) 1983 to 2347 of Ira2 was PCR amplified with the primer pair HB7 and HB8. After cleavage with EcoRI and XhoI, the PCR fragments were ligated into the EcoRI and XhoI sites of pEG202 and pJG4-5. The fragments were inserted in framework downstream of LexA in pEG202 and.