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• W2021129192 abstract "Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential forSalmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes. Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential forSalmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes. guanine nucleotide exchange factor GTPase-activating protein mitogen-activated protein kinase MAPK kinase MAPK kinase kinase high osmolarity glycerol p21-activated kinase Cdc42/Rac-interactive domain synthetic minimal medium SD with 2% galactose instead of glucose SD with 2% raffinose instead of glucose glutathioneS-transferase wild-type Rho-type GTPases (Cdc42, Rac, Rho) play an essential role in regulating cell polarity and actin organization in eukaryotic cells ranging from yeast to mammalian organisms. They act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. The switch is up-regulated by guanine nucleotide exchange factors (GEFs),1which enhance the exchange of bound GDP for GTP, and down-regulated by GTPase-activating proteins (GAPs), which increase the intrinsic rate of hydrolysis of bound GTP. Modulation of Rho-GTPases by these proteins results in reorganizations of the actin cytoskeleton during morphogenesis and cell migration (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar). Many bacterial pathogens have evolved the ability to interfere with the signaling machinery of their host cells by modulating the activity of GTPases as a strategy to develop the pathogenic process. For example,Salmonella typhimurium is able to induce its internalization into non-phagocytic host cells through the activation/inactivation of Rho-GTPases. Via a specialized protein translocation apparatus, the SPI1 type III secretion system, this bacterium directs the delivery of effector proteins that modulate Cdc42 and Rac1 activation. Two of them, the GEF protein SopE2 and the inositol phosphatase SopB, stimulate Cdc42 and/or Rac function, allowing Salmonellainternalization. The protein phosphatase with GAP activity SptP, which reverses the activation of these GTPases, facilitates cell recovery (2Galan J.E. Zhou D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8754-8761Crossref PubMed Scopus (212) Google Scholar). Although no homologs of Rac have been found in Saccharomyces cerevisiae, the CDC42 gene was in fact first identified in this yeast (3Adams A.E. Johnson D.I. Longnecker R.M. Sloat B.F. Pringle J.R. J. Cell Biol. 1990; 111: 131-142Crossref PubMed Scopus (470) Google Scholar, 4Johnson D.I. Pringle J.R. J. Cell Biol. 1990; 111: 143-152Crossref PubMed Scopus (403) Google Scholar). The human CDC42 gene was subsequently cloned by complementation of a yeast cdc42thermosensitive mutant (5Munemitsu S. Innis M.A. Clark R. McCormick F. Ullrich A. Polakis P. Mol. Cell. Biol. 1990; 10: 5977-5982Crossref PubMed Scopus (131) Google Scholar, 6Shinjo K. Koland J.G. Hart M.J. Narasimhan V. Johnson D.I. Evans T. Cerione R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9853-9857Crossref PubMed Scopus (172) Google Scholar), indicating the functional homology between these proteins from both species. In addition to controlling actin rearrangements, Cdc42 has an important function in the regulation of mitogen-activated protein kinase (MAPK) signaling (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar, 7Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (341) Google Scholar). All eukaryotic cells utilize MAPK modules, which consist of a phosphorylation cascade of three conserved kinases, to transduce external signals into appropriate internal responses. The budding yeast S. cerevisiae contains several functionally distinct MAPK cascades involved in the mediation of different physiological responses (for reviews see Refs. 8Banuett F. Microbiol. Mol. Biol. Rev. 1998; 62: 249-274Crossref PubMed Google Scholar and 9Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar). Pheromones activate the mating module, composed of Ste11 (MAPKKK), Ste7 (MAPKK), and Fus3 (MAPK), which allows fusion of the mating partners. The filamentous growth pathway, which is activated by nutrient starvation and induces foraging into agar, also uses Ste11 and Ste7 to activate Kss1 (MAPK). In the high osmolarity glycerol (HOG) pathway that mediates the osmo-adaptive response, the MAPK Hog1 is activated by the MAPKK Pbs2 onto which two activating branches converge. The MAPKKKs Ssk2 and Ssk22 operate in one branch, the promiscuous Ste11 being the MAPKKK used by the other one. The module of the cell integrity pathway is composed of the MAPKKK Bck1, the MAPKKs Mkk1 and Mkk2, and the MAPK Slt2/Mpk1. Different stimuli associated with cell surface alterations activate this pathway, which leads to an appropriate response oriented to maintaining cell wall integrity (10Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (287) Google Scholar,11de Nobel H. Ruiz C. Martin H. Morris W. Brul S. Molina M. Klis F.M. Microbiology. 2000; 146: 2121-2132Crossref PubMed Scopus (221) Google Scholar). Recently, the existence of an additional pathway, the STE vegetative growth pathway, has been proposed; this would use the same MAPK module as the filamentous growth cascade. This pathway would promote vegetative growth and cell wall integrity and would be essential in mutants defective in protein glycosylation (12Lee B.N. Elion E.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12679-12684Crossref PubMed Scopus (127) Google Scholar, 13Cullen P.J. Schultz J. Horecka J. Stevenson B.J. Jigami Y. Sprague G.F. Genetics. 2000; 155: 1005-1018Crossref PubMed Google Scholar). Despite detailed knowledge of signaling through the MAPK modules, the upstream components and mechanisms responsible for their activation remain poorly understood. Among the known upstream components, members of the Rho family of the small G proteins, such as Cdc42, have been identified as important modulators of MAPK cascades in S. cerevisiae (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar). Whereas Cdc24 is believed to be the sole GEF for Cdc42 (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar, 14Sloat B.F. Pringle J.R. Science. 1978; 200: 1171-1173Crossref PubMed Scopus (88) Google Scholar), three proteins have been proposed to be GAPs for Cdc42: Bem3, which has been shown to have GAP activity against Cdc42in vitro (15Bender A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 1295-1305Crossref PubMed Scopus (349) Google Scholar, 16Zheng Y. Cerione R. Bender A. J. Biol. Chem. 1994; 269: 2369-2372Abstract Full Text PDF PubMed Google Scholar), Rga1/Dbm1 (17Stevenson B.J. Ferguson B., De Virgilio C., Bi, E. Pringle J.R. Ammerer G. Sprague G.F., Jr. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar, 18Chen G.C. Zheng L. Chan C.S. Mol. Cell. Biol. 1996; 16: 1376-1390Crossref PubMed Scopus (32) Google Scholar), and Rga2, which is identified by its homology to Rga1 (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar). It has been suggested that the Cdc42 regulation of MAPK modules would be mediated by a conserved family of serine/threonine kinases that have been demonstrated to be activated by this GTPase (19Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1280) Google Scholar, 20Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (299) Google Scholar). These p21-activated kinases (PAKs) function upstream from MAPKKKs, linking Cdc42/Rac GTPases to MAPK signaling. It has been proposed that Cdc42 would activate PAKs by releasing the C-terminal kinase domain from the autoinhibition carried out by the N-terminal regulatory region (21Zhao Z.S. Manser E. Chen X.Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar,22Tu H. Wigler M. Mol. Cell. Biol. 1999; 19: 602-611Crossref PubMed Scopus (78) Google Scholar). This region contains a Cdc42/Rac-interactive (CRIB) domain that appears to be sufficient for the binding of Cdc42 (23Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Three members of the PAK family have been found in S. cerevisiae: Ste20, Cla4, and Skm1 (24Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (343) Google Scholar, 25Cvrckova F., De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar, 26Martin H. Mendoza A. Rodriguez-Pachon J.M. Molina M. Nombela C. Mol. Microbiol. 1997; 23: 431-444Crossref PubMed Scopus (47) Google Scholar), but only Ste20 seems to play a role in MAPK-mediated signaling. In fact, Ste20 is essential for signal transmission through the mating and filamentous growth and in the Ste11-mediated branch of the HOG pathways (24Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (343) Google Scholar, 27Mosch H.U. Roberts R.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5352-5356Crossref PubMed Scopus (296) Google Scholar, 28Raitt D.C. Posas F. Saito H. EMBO J. 2000; 19: 4623-4631Crossref PubMed Scopus (198) Google Scholar). Similarly, Cdc42 has been shown to play a role in activating the MAPK module that operates in the filamentous growth pathway (27Mosch H.U. Roberts R.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5352-5356Crossref PubMed Scopus (296) Google Scholar) and to function in the Ste11 branch of the HOG pathway (28Raitt D.C. Posas F. Saito H. EMBO J. 2000; 19: 4623-4631Crossref PubMed Scopus (198) Google Scholar, 29Reiser V. Salah S.M. Ammerer G. Nat. Cell Biol. 2000; 2: 620-627Crossref PubMed Scopus (117) Google Scholar). Cdc42 also influences signaling in the mating pathway, although the functional relevance of the Cdc42-Ste20 interaction is more controversial (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar, 30Moskow J.J. Gladfelter A.S. Lamson R.E. Pryciak P.M. Lew D.J. Mol. Cell. Biol. 2000; 20: 7559-7571Crossref PubMed Scopus (61) Google Scholar). Finally, although the scheme of sequential activation of Cdc42, Rac, and Rho in mammals has led several authors to describe a hypothetical cascade in which Bud1 would control Cdc42, and this GTPase could control Rho1 (31Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar), there is no evidence for a role of Cdc42 in activation of the Rho1-mediated cell integrity pathway. To further examine the role of Cdc42 in signaling to the different MAPK pathways, here we studied the consequences of Cdc42 activation on MAPK phosphorylation, both by expressing S. typhimurium proteins that regulate Cdc42 in mammalian cells and by removing the S. cerevisiae potential Cdc42-negative regulators Rga1, Rga2, and Bem3. We observed that Cdc42 influences the activation status of the MAPKs operating in the mating, filamentation, and cell integrity pathways. Our data also indicate that S. cerevisiae is a valuable model for studying pathogenic bacterial proteins that influence eukaryotic signaling by acting on Cdc42. The S. cerevisiae strains used in this study are listed in TableI. The S. typhimuriumC53 strain used in this study was kindly provided by F. Norce. Standard procedures were employed for yeast genetic manipulations (32Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2526) Google Scholar).STE20, STE7, FUS3, and KSS1were disrupted or deleted using plasmids p34S2U (26Martin H. Mendoza A. Rodriguez-Pachon J.M. Molina M. Nombela C. Mol. Microbiol. 1997; 23: 431-444Crossref PubMed Scopus (47) Google Scholar), pNC113 (33Company M. Errede B. Mol. Cell. Biol. 1988; 8: 5299-5309Crossref PubMed Scopus (24) Google Scholar), pYEE98 (34Elion E.A. Grisafi P.L. Fink G.R. Cell. 1990; 60: 649-664Abstract Full Text PDF PubMed Scopus (308) Google Scholar), and pBC65 (kindly provided by J. Thorner), respectively, by means of previously described procedures (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For strain YTX35, SLT2 was disrupted with LEU2 as described previously (36Navarro-Garcı́a F. Sánchez M. Pla J. Nombela C. Mol. Cell. Biol. 1995; 15: 2197-2206Crossref PubMed Scopus (153) Google Scholar). A MATα rho1–104strain was obtained as a haploid spore from the diploid strain constructed by crossing HNY21 with the wild-type strain FY834 (37Winston F. Dollard C. Ricupero S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (769) Google Scholar). Strains HM54 and HM55 were progeny of the diploid strain obtained by crossing this MATα rho1–104 strain with YGS51. Yeast transformations were performed with the lithium acetate method (38Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar).Table IStrains used in this workStrainRelevant genotypeSource or reference1783MATatrp1–1 leu2–3112 ura3–52 his4 canR(60Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar)YPH499MATaade2–101 trp1–63 leu2–1 ura3–52 his3-Δ200 lys2–801(26Martin H. Mendoza A. Rodriguez-Pachon J.M. Molina M. Nombela C. Mol. Microbiol. 1997; 23: 431-444Crossref PubMed Scopus (47) Google Scholar)SY2002MATahis3∷FUS1-HIS3 mfa2-Δ1∷FUS1-lacZ ura3 leu2 trp1 his3 ade1G. SpragueYGS2Isogenic to SY2002,rga1∷URA3G. SpragueYGS7Isogenic to SY2002, rga2∷TRP1G. SpragueYGS50Isogenic to SY2002,bem3∷TRP1G. SpragueYGS72Isogenic to SY2002, rga1∷URA3 rga2∷TRP1G. SpragueYGS51Isogenic to SY2002, rga1∷URA3 bem3∷TRP1G. SpragueYGS56Isogenic to SY2002, rga2∷TRP1 bem3∷TRP1G. SpragueYGS57Isogenic to SY2002, rga1∷URA3 rga2∷TRP1 bem3∷TRP1G. SpragueYTX10Isogenic to SY2002, ste7∷LEU2This workYTX21Isogenic to YGS72,ste7∷LEU2This workYTX22Isogenic to YGS51, ste7∷LEU2This workYTX23Isogenic to YGS56,ste7∷LEU2This workYTX24Isogenic to YGS57, ste7∷LEU2This workYTX25Isogenic to SY2002,ste20Δ∷URA3This workYTX26Isogenic to YGS56,ste20Δ∷URA3This workYTX27Isogenic to SY2002,kss1Δ∷URA3This workYTX28Isogenic to YGS56, kss1Δ∷URA3This workYTX31Isogenic to SY2002,fus3Δ∷LEU2This workYTX32Isogenic to YGS56, fus3Δ∷LEU2This workYTX33Isogenic to SY2002, kss1Δ∷URA3 fus3Δ∷LEU2This workYTX34Isogenic to YGS56, kss1Δ∷URA3 fus3Δ∷LEU2This workYTX35Isogenic to YGS56, slt2∷LEU2This workOHNYMATa ura3 his3 trp1 leu2 ade2(61Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (302) Google Scholar)HNY21Isogenic to OHNY,rho1–104(62Yamochi W. Tanaka K. Nonaka H. Maeda A. Musha T. Takai Y. J. Cell Biol. 1994; 125: 1077-1093Crossref PubMed Scopus (209) Google Scholar)HM54MATα ura3 his3 trp1 leu2 ade2 rho1–104This workHM55MATa ura3 his3 trp1 leu2 ade2 rho1–104 rga1∷URA3 bem3∷TRP1This work Open table in a new tab General DNA methods were employed, using standard techniques (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The plasmids used in this study were as follows: pKGSOPE2, pKGSPTP, and pKGSPGAP, expressing GST-SopE2, GST-SptP, and GST-SptPΔC (residues 1–338) fusion proteins, were constructed by PCR amplification from thesopE2 and sptP open reading frames fromS. typhimurium C53 genomic DNA, using oligonucleotides U-SOPE2 (5′-CCGGATCCGTGACTAACATAACACTATC-3′) and L-SOPE2 (5′-CCAAGCTTTCAGGACCGATTCTGAAG-3′), U-SPTP (5′-CCGGATCCATGCTAAAGTATGAGGAG-3′) and L-SPTP (5′-CCAAGCTTCAGCTTGCCGTCGTC-3′), and U-SPTP and L-SPGAP (5′-CCAAGCTTTCAAGAGTTAACGTATTCAC-3′), cleavage withBamHI-HindIII, and cloning in the correct orientation into the corresponding site of pEG(KG) (URA3 GAL1-GST leu2-d 2μ) (39Mitchell D.A. Marshall T.K. Deschenes R.J. Yeast. 1993; 9: 715-722Crossref PubMed Scopus (265) Google Scholar). Amplified DNA was verified by DNA sequencing. YPD (1% yeast extract, 2% peptone, and 2% glucose) broth or agar was the complete medium used for growing the yeast strains. Synthetic minimal medium (SD) contained 0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, and 2% glucose and was supplemented with appropriate amino acids and nucleic acid bases (32Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2526) Google Scholar). SG and SR were SD with 2% galactose or raffinose, respectively, instead of glucose. Where indicated,d-sorbitol was added to the media at a final concentration of 1 m. Galactose induction experiments in liquid media were performed by growing cells in SR minimal medium to log phase and then adding galactose to 2% for 8 h. Samples were observed under an Olympus BH-2 microscope connected to a Panasonic WV-CL310 video camera. For scanning electron microscopy, cells were prepared essentially as described in a previous study (40Williams S. Veldkamp C. Trans. Br. Mycol. 1974; 63: 409-412Google Scholar) and visualized using a JEOL JSM-6400 microscope. The procedures employed in yeast cell growth, collection, and lysis, collection of proteins, fractionation by SDS-polyacrylamide gel electrophoresis, and transfer to nitrocellulose membranes have been previously described (41Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Anti-phospho-p44/p42 MAPK (Thr202/Tyr204) antibody (New England Biolabs) was used to detect dually phosphorylated Slt2, Kss1, and Fus3 MAPKs. Slt2 protein was detected using an anti-GST-Slt2 antibody (42Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar). GST fusion proteins, Fus3 and Kss1, were detected with anti-GST, anti-Fus3, and anti-Kss1 polyclonal antibodies (Santa Cruz Biotechnology). We wished to test whether S. cerevisiae could be used as a model system to gain insight into the role of the previously described S. typhimurium Cdc42-modulating proteins SopE2 and SptP. To this end, we first transformed the yeast wild-type strains SY2002, YPH499, and 1783 with the multicopy GAL1-inducible expression plasmid pKG-SopE2 to overexpress this protein fused to GST at the N terminus. As a first step, transformants were assayed for growth phenotypes under inducing and non-inducing conditions. All transformants were able to grow at similar rates when growing on solid glucose-based medium. However, in contrast to cells carrying the empty vector, transformants bearing pKG-SopE2 were not able to form colonies on galactose plates; only transformants from the SY2002 strain displayed a reduced growth on this medium (Fig.1A). Therefore, the expression of GST-SopE2 confers a severe inhibitory growth phenotype in yeast cells. Microscopic observation of yeast haploid cells of strain SY2002 overproducing SopE2 revealed elongated cell morphologies and the formation of chains of cells (Fig. 1B) similar to the pseudohyphal growth displayed by diploid cells under conditions of nitrogen starvation. Therefore, although SopE2 expression eventually leads to cell growth inhibition, in the SY2002 strain, in which this growth phenotype is less intense, SopE2 overexpression promotes pseudohyphal differentiation. This suggests the existence of a stimulatory effect of this Salmonella protein on the filamentous growth pathway. The differences in the intensity of the phenotype among strains could be due to a different level of GAL1-inducible expression from plasmid pKG-SopE2 in each strain. To test this possibility, extracts from the wild-type strains SY2002, YPH499, and 1783 expressing GST or GST-SopE2 were analyzed by immunoblotting, using anti-GST antibody. As shown in Fig. 1C, the bands corresponding to GST and GST-SopE2 are weaker in the extracts from strain SY2002 than in those from the other strains. Thus, the lower expression of theGAL1 promoter in this strain could account for the less intense growth phenotype observed. Cdc42 is a potent regulator of filamentous growth in S. cerevisiae (27Mosch H.U. Roberts R.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5352-5356Crossref PubMed Scopus (296) Google Scholar). Therefore, the pseudofilamentation phenotype suggests that SopE2 effectively targets Cdc42. Because Kss1 is the MAPK that operates in the filamentation pathway, if SopE2 activates Cdc42, then the expression of this bacterial protein should lead to Kss1 activation. To investigate the activation status of Kss1 we used a commercial antibody raised against dually phosphorylated (Thr202/Tyr204)-p44/42 MAPK as a tool to monitor the phosphorylation level of Kss1 and hence its activation. As shown in Fig. 2, whereas extracts from the wild-type strain SY2002 expressing GST elicited a weak immunoreactive band at the expected mobility for Kss1 (∼43 kDa), the expression of GST-SopE2 resulted in a strong increase in the intensity of this band. This band was confirmed to be Kss1 (see Fig.4F). We next disrupted STE7, the gene encoding the MAPKK of the filamentous growth pathway responsible for Kss1 activation. As expected, this band was absent in ste7mutants (Fig. 2). Furthermore, disruption of STE7 prevented GST-SopE2-induced filament formation and partially restored cell growth (not shown).Figure 4Signal transduction across pheromone response and filamentous growth pathways is necessary for Cdc42-GAP mutant-induced Slt2 activation. Effect on MAPK activation of the elimination of Ste20 (A), Ste7 (B), Fus3 (C), Kss1 (D), and both Fus3 and Kss1 (E) in double and triple Cdc42-GAP mutants. SY2002 and the isogenic mutant strains YTX10 (ste7), YGS72 (rga1 rga2) and YTX21 (rga1 rga2 ste7), YGS51 (rga1 bem3) and YTX22 (rga1 bem3 ste7), YGS56 (rga2 bem3) and YTX23 (rga2 bem3 ste7), YGS57 (rga1 rga2 bem3) and YTX24 (rga1 rga2 bem3 ste7), YTX25 (ste20) and YTX26 (rga2 bem3 ste20), YTX31 (fus3) and YTX32 (rga2 bem3 fus3), YTX27 (kss1) and YTX28 (rga2 bem3 kss1), and YTX33 (fus3 kss1) and YTX34 (rga2 bem3 fus3 kss1) were grown in YPD to mid-log phase at 24 °C. Immunoblot analysis was performed as in Fig. 2. rga1, rga2, andbem3 are indicated as 1, 2, and3, respectively. F, verification of the identity of the phospho-MAPKs revealed with the anti-phospho-p44/p42 MAPK antibodies using anti-Slt2, anti-Kss1, and anti-Fus3 antibodies for immunodetection of the corresponding proteins in cell extracts from the isogenic strains YGS56 (rga2 bem3), YTX35 (rga2 bem3 slt2), YTX32 (rga2 bem3 fus3), and YTX28 (rga2 bem3 kss1).View Large Image Figure ViewerDownload Hi-res image Download (PPT) These Western blotting experiments also revealed that SopE2 expression resulted in the appearance of a weak ∼40-kDa immunoreactive band (Fig. 2) at the expected mobility of Fus3. This band was confirmed to be Fus3 (see Fig. 4F). Elimination of the MAPKK Ste7, which also phosphorylates Fus3 in the mating pathway, led to the disappearance of this band (Fig. 2). Taken together, these results indicate that the expression of SopE2 stimulates signaling through the filamentation and mating pathways, resulting in a strong and a weak activation of Kss1 and Fus3, respectively. Kss1 activation is probably responsible for the filamentation phenotype displayed by cells overexpressing GST-SopE2. Furthermore, the results strongly suggest that SopE2 displays GEF activity against Cdc42, functioning in a similar way in both mammalian and yeast systems. Interestingly, these experiments also showed that SopE2 overexpression results in the appearance of an ∼62-kDa immunoreactive band (Fig. 2) that corresponds to the MAPK Slt2 (41Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) (see Fig. 4F). These observations indicate that the expression of SopE2 also results in the activation of the cell integrity pathway. It has been previously reported that mating pheromone stimulates Slt2 activation, and signal transduction through the mating pathway has been shown to be necessary for this activation (43Buehrer B.M. Errede B. Mol. Cell. Biol. 1997; 17: 6517-6525Crossref PubMed Scopus (123) Google Scholar). Therefore, SopE2-induced activation of the cell integrity pathway might also occur subsequent to the activation of the mating or filamentation pathways. However, as is evident from Fig.2, Slt2 activation was independent of the presence of Ste7. Two feasible possibilities were suggested. First, Slt2 activation could be a consequence of the direct effect of SopE2 on Rho1, the Rho-GTPase that operates in the cell integrity pathway. Second, Cdc42 could be acting on Rho1 in a mating/filamentation pathway-independent manner. It is also not possible to rule out the simultaneous function of these two mechanisms. To distinguish between these possibilities and in general to gain insight into the role of Cdc42 on MAPK-mediated signaling, further experimentation was carried out. To investigate these aspects, we next studied the morphology and MAPK signaling in yeast cells expressing activated Cdc42. To this end, we analyzed the effect of the lack of the different potential GAPs for Cdc42: Rga1, Rga2, and Bem3. Because Cdc42 plays a key role in controlling cell morphogenesis, we first examined the cell morphology of the different rga1, rga2, andbem3 mutants (Fig.3A). Consistent with previous data (17Stevenson B.J. Ferguson B., De Virgilio C., Bi, E. Pringle J.R. Ammerer G. Sprague G.F., Jr. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar), microscopic examination of rga1 cells revealed an elongated morphology. Whereas rga2 mutants appeared normal, double rga1 rga2 cells showed the same phenotype as singlerga1 cells. In contrast with the data from previous studies (16Zheng Y. Cerione R. Bender A. J. Biol. Chem. 1994; 269: 2369-2372Abstract Full Text PDF PubMed Google Scholar) bem3 mutants displayed buds that were elongated. Simultaneous lack of Rga1 and Bem3 resulted in a combined phenotype. Triple rga1 rga2 bem3 showed the most intense morphological alterations, with 98% of the cells displaying irregular, elongated, and misshapen cells. To address the issue of whether this phenotype was a consequence of increased signaling through the mating or filamentous growth pathways, STE7 was disrupted in the triplerga1 rga2 bem3 mutant and the resulting phenotype was observed microscopically. Deletion ofSTE7 in this strain resulted in the appearance of a normal morphology" @default.
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• W2021129192 date "2002-07-01" @default.
• W2021129192 modified "2023-10-01" @default.
• W2021129192 title "A Novel Connection between the Yeast Cdc42 GTPase and the Slt2-mediated Cell Integrity Pathway Identified through the Effect of Secreted Salmonella GTPase Modulators" @default.
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• W2021129192 doi "https://doi.org/10.1074/jbc.m201527200" @default.
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