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• W2017366722 abstract "Many environmental stresses trigger cellular responses by activating mitogen-activated protein kinase (MAPK) pathways. Once activated, these highly conserved protein kinase cascades can elicit cellular responses such as transcriptional activation of response genes, cytoskeletal rearrangement, and cell cycle arrest. The mechanism of pathway activation by environmental stresses is in most cases unknown. We have analyzed the activation of the budding yeast “cell integrity” MAPK pathway by heat shock, hypoosmotic shock, and actin perturbation, and we report that different stresses regulate this pathway at different steps. In no case can MAPK activation be explained by the prevailing view that stresses simply induce GTP loading of the Rho1p GTPase at the “top” of the pathway. Instead, our findings suggest that the stresses can modulate at least three distinct kinases acting between Rho1p and the MAPK. These findings suggest that stresses provide “lateral” inputs into this regulatory pathway, rather than operating in a linear “top-down” manner. Many environmental stresses trigger cellular responses by activating mitogen-activated protein kinase (MAPK) pathways. Once activated, these highly conserved protein kinase cascades can elicit cellular responses such as transcriptional activation of response genes, cytoskeletal rearrangement, and cell cycle arrest. The mechanism of pathway activation by environmental stresses is in most cases unknown. We have analyzed the activation of the budding yeast “cell integrity” MAPK pathway by heat shock, hypoosmotic shock, and actin perturbation, and we report that different stresses regulate this pathway at different steps. In no case can MAPK activation be explained by the prevailing view that stresses simply induce GTP loading of the Rho1p GTPase at the “top” of the pathway. Instead, our findings suggest that the stresses can modulate at least three distinct kinases acting between Rho1p and the MAPK. These findings suggest that stresses provide “lateral” inputs into this regulatory pathway, rather than operating in a linear “top-down” manner. Both extracellular signaling molecules and intracellular stresses frequently elicit their specific cellular responses through highly conserved signal transduction modules consisting of protein kinase cascades culminating in the activation of mitogen-activated protein kinases (MAPKs) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GAP, GTPase-activating proteing.1The abbreviations used are: MAPK, mitogen-activated protein kinase; GAP, GTPase-activating proteing. (1.Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (534) Google Scholar). MAPKs are a family of serine/threonine protein kinases that are activated by an unusual mechanism involving dual threonine and tyrosine phosphorylation, which is catalyzed by a family of MAPK kinases (MAPKKs). These in turn are phosphorylated and activated by a family of MAPKK kinases (MAPKKKs), and the three kinases of a particular MAPK module are frequently physically associated with each other (sometimes with the help of noncatalytic scaffold proteins) in multimeric complexes (2.Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1127) Google Scholar). In contrast to the highly conserved molecular architecture of the MAPK cascade, the mechanisms whereby various stimuli activate MAPK activity appear to be quite diverse.Perhaps the most extensively investigated instance of MAPK activation involves signal transduction by receptor tyrosine kinases, which activate a MAPK cascade in response to extracellular growth factors (3.van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1241) Google Scholar). Activation of the receptor upon growth factor binding promotes the recruitment of the guanine nucleotide exchange factor Sos to the plasma membrane, where Sos encounters its target GTPase, Ras, leading to GTP loading of Ras. GTP-bound Ras then stimulates a MAPK cascade involving the kinases Raf or Raf-B (MAPKKK), MEK (MAPKK), and ERK (MAPK) (4.Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar).In contrast to the detailed understanding of MAPK cascade activation by growth factors, much less is known about how MAPK pathways are stimulated by cellular stresses. One stress-responsive MAPK that has been extensively studied in Saccharomyces cerevisiae is the Slt2p/Mpk1p MAPK, which is activated by the related (and redundant) MAPKKs Mkk1p and Mkk2p, which are in turn activated by the MAPKKK Bck1p (5.Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (288) Google Scholar). This signaling module has been called the “cell integrity” MAPK cascade, as it is required for proper construction of the cell wall in order to prevent cell lysis (6.Paravicini G. Cooper M. Friedli L. Smith D.J. Carpentier J.L. Klig L.S. Payton M.A. Mol. Cell. Biol. 1992; 12: 4896-4905Crossref PubMed Scopus (181) Google Scholar, 7.Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Crossref PubMed Scopus (302) Google Scholar). Mpk1p is activated in response to various stresses, and Mpk1p activation serves to protect yeast cells from stress by inducing transcription of genes that promote cell wall remodeling and by contributing to the cell cycle arrest triggered by the morphogenesis checkpoint, which delays mitosis until cells have successfully built a bud (5.Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (288) Google Scholar, 8.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google Scholar, 9.Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar).Genetic studies established that Pkc1p, the sole protein kinase C homologue in S. cerevisiae, is absolutely required for any activity in the cell integrity MAPK pathway, and acts upstream of the MAPKKK Bck1p (10.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (421) Google Scholar, 11.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (270) Google Scholar). Several additional functions, independent of the MAPK pathway, have also been ascribed to Pkc1p (12.Perez P. Calonge T.M. J. Biochem. (Tokyo). 2002; 132: 513-517Crossref PubMed Scopus (21) Google Scholar). Pkc1p activity in turn requires the binding of GTP-loaded Rho1p (13.Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 14.Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (303) Google Scholar), and GTP loading of Rho1p is mediated by the partially redundant guanine nucleotide exchange factors Rom1p and Rom2p (15.Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (182) Google Scholar). By analogy to the Ras-Raf-MEK-ERK pathway, it has been suggested that stresses activate the cell integrity pathway by stimulating GTP loading of Rho1p, thereby activating Pkc1p to phosphorylate Bck1p, initiating activation of the kinase cascade (5.Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (288) Google Scholar, 9.Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, 16.Philip B. Levin D.E. Mol. Cell. Biol. 2001; 21: 271-280Crossref PubMed Scopus (240) Google Scholar, 17.Bickle M. Delley P.A. Schmidt A. Hall M.N. EMBO J. 1998; 17: 2235-2245Crossref PubMed Scopus (161) Google Scholar).A multiplicity of stimuli have been shown to promote Mpk1p activation. These include heat shock, hypoosmotic shock, actin depolymerization, and treatment with chlorpromazine, caffeine, vanadate, zymolyase, Congo red, calcofluor, rapamycin, and mating pheromone (8.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google Scholar, 10.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (421) Google Scholar, 18.Krause S.A. Gray J.V. Curr. Biol. 2002; 12: 588-593Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 19.Martin 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 (295) Google Scholar, 20.Davenport K.R. Sohaskey M. Kamada Y. Levin D.E. Gustin M.C. J. Biol. Chem. 1995; 270: 30157-30161Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 21.Zarzov P. Mazzoni C. Mann C. EMBO J. 1996; 15: 83-91Crossref PubMed Scopus (197) Google Scholar). A simplifying hypothesis to accommodate these observations is that there is a common stressful consequence of all of these treatments that causes cells to activate Mpk1p (10.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (421) Google Scholar). A number of plasma membrane glycoproteins (Wsc1p-3p, Mid2p) that influence Mpk1p activity have been identified, and it is thought that these proteins might act as “stress sensors” that somehow detect cellular stress and transduce a signal to activate Rom1p/Rom2p (16.Philip B. Levin D.E. Mol. Cell. Biol. 2001; 21: 271-280Crossref PubMed Scopus (240) Google Scholar, 22.Gray J.V. Ogas J.P. Kamada Y. Stone M. Levin D.E. Herskowitz I. EMBO J. 1997; 16: 4924-4937Crossref PubMed Scopus (203) Google Scholar, 23.Verna J. Lodder A. Lee K. Vagts A. Ballester R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13804-13809Crossref PubMed Scopus (317) Google Scholar, 24.Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar). However, these putative stress sensors appear to be dispensable for Mpk1p activation in response to actin depolymerization (8.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google Scholar) or rapamycin (18.Krause S.A. Gray J.V. Curr. Biol. 2002; 12: 588-593Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), raising the possibility that different stresses might employ distinct pathways to activate Mpk1p. Precedent for stress-specific regulation of MAPK pathways is provided by findings on the Schizosaccharomyces pombe Sty1p pathway, in which it appears that although other stresses activate Sty1p through regulation of its upstream kinases, heat shock activates Sty1p primarily through regulation of Sty1p-directed phosphatases (25.Shieh J.C. Martin H. Millar J.B. J. Cell Sci. 1998; 111: 2799-2807PubMed Google Scholar, 26.Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1998; 9: 1339-1349Crossref PubMed Scopus (96) Google Scholar, 27.Nguyen A.N. Shiozaki K. Genes Dev. 1999; 13: 1653-1663Crossref PubMed Scopus (105) Google Scholar).In this report, we have dissected the requirements for activation of the cell integrity pathway by different stresses. Our findings are inconsistent with the view that all stresses activate the pathway in a “top-down” manner by stimulating Rho1p GTP loading, resulting in the activation of a linear Pkc1p/Bck1p/Mkk1,2p/Mpk1p kinase cascade. We find that different stresses can activate the pathway by different mechanisms, in one case regulating the core kinases Mkk1,2p/Mpk1p themselves rather than Rho1p or Pkc1p. Thus, different stresses activate Mpk1p through signaling pathways that impact the MAPK cascade at different levels.MATERIALS AND METHODSMedia, Growth Conditions, Stresses, and Drug Treatments—Yeast media (YEPD rich media, synthetic media lacking specific nutrients, and sporulation media) have been described (49.Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 1-933PubMed Google Scholar). For heat shock cells were grown to mid-log phase in media supplemented with 1 m sorbitol at 24 °C and then shifted to 39 °C for 35 min. For Lat-B treatment cells were grown to mid-log phase in media supplemented with 1 m sorbitol at 30 °C and then treated with Lat-B (BioMol Research Laboratories, Inc., Plymouth Meeting, PA) at 100 μm for 2 h. For hypoosmotic shock, cells were grown to mid-log phase in media supplemented with 1 m sorbitol at 24 °C, diluted 10-fold into water for ∼1 min, and immediately pelleted and frozen at –80 °C.Strains, Plasmids, and PCR Manipulations—Standard media and methods were used for plasmid manipulations (50.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1995Google Scholar) and yeast genetic manipulations (49.Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 1-933PubMed Google Scholar). The yeast strains used in this study are listed in Table I.Table IStrains used in this studyaAll strains are in the BF264-15D (55) strain background (ade1 his2 ura3 leu2 trp1).Ref.DLY1a bar1 ade1 his2 ura3 leu2 trp155.Sia R.A. Herald H.A. Lew D.J. Mol. Biol. Cell. 1996; 7: 1657-1666Crossref PubMed Scopus (127) Google ScholarDLY455a bck1ΔLEU2 BCK1-20::URA3This studyDLY456α pkc1ΔLEU2 galPKC1::TRP1 BCK1-20::URA3This studyDLY3962α pkc1ΔLEU2 galPKC1::TRP18.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google ScholarDLY3994a bck1::LEU28.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google ScholarDLY3996a bar1 msg5::LEU2This studyDLY4351a mkk1::LEU2 mkk2::URA38.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google ScholarDLY4497a bar1 rho2::kanR8.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google ScholarDLY4509a bar1 RHO1::RHO1Q68H::LEU2This studyDLY6473a bar1 RHO1::RHO1Q68H::LEU2 rho2::kanRThis studyDLY6474α ptp2::hygRThis studyDLY6475a bar1 sdp1::kanR msg5::LEU2This studyDLY6506α ptp3::URA3This studyDLY6508a sdp1::kanR msg5::LEU2 ptp2::hygRThis studyDLY6509a msg5::LEU2 ptp2::hygRThis studyDLY6510a sdp1::kanR ptp2::hygRThis studyDLY6519a msg5::LEU2 ptp3::URA3This studyDLY6520a sdp1::kanR ptp3::URA3This studyDLY6521α sdp1::kanR ptp3::URA3 ptp2::hygRThis studyDLY6522α sdp1::kanR msg5::LEU2 ptp3::URA3 ptp2::hygRThis studyDLY6523a sdp1::kanR msg5::LEU2 ptp3::URA3This studyDLY6524a ptp3::URA3 ptp2::hygRThis studyDLY6526a bar1 sdp1::kanRThis studyDLY6536a msg5::LEU2 ptp3::URA3 ptp2::hygRThis studya All strains are in the BF264-15D (55.Sia R.A. Herald H.A. Lew D.J. Mol. Biol. Cell. 1996; 7: 1657-1666Crossref PubMed Scopus (127) Google Scholar) strain background (ade1 his2 ura3 leu2 trp1). Open table in a new tab To express Rho1pQ68H in yeast, a 2-kb HindIII/SacI fragment containing RHO1Q68H (29.Madaule P. Axel R. Myers A.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 779-783Crossref PubMed Scopus (181) Google Scholar) was isolated from p68LEUe (gift from A. Myers) and ligated into the corresponding sites in pRS305 (51.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), yielding pDLB2257. Integration at the RHO1 genomic locus was targeted by digestion with MluI leaving the endogenous RHO1 gene intact.To express Bck1–20p in yeast, a 6.5-kb XhoI/NotI fragment from pRS314BCK1–20 (11.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (270) Google Scholar) was cloned into pRS306 (51.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), yielding pDLB1774. Integration at the BCK1 genomic locus was targeted by digestion with MluI. Because the MluI site is in the BCK1 promoter, this plasmid can also be integrated next to bck1ΔLEU2.To analyze cells lacking Pkc1p activity, we employed strains in which the only copy of Pkc1p was expressed from the GAL1 promoter (8.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google Scholar). These strains were grown in osmotically stabilized dextrose-containing media for at least 36 h to repress Pkc1p.To express Mkk1p in yeast, the MKK1 open reading frame was amplified using primers H83 (5′-CATGCCATGGCTTCACTGTTCAGACCCCCAGAATCTGCG-3′) and H140 (5′-GATCGAGCTCGTATGAATCTTGTATGGAG-3′), digested with NcoI and SacI (sites underlined in primers), and cloned into the corresponding sites in pDLB1610. This expression vector contains a pRS304 backbone (51.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), CDC42 promoter (52.Moskow 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), a start codon preceded by a good Kozak sequence, and sequences encoding 12 tandem HA epitope tags upstream of the NcoI and SacI cloning sites. EcoRI/SacI fragments containing the promoter, epitope tags, and MKK1 sequences were then transferred to pRS314 yielding pDLB823 (CEN HA-MKK1).To create the MKK1DD mutant, altering residues Ser-377 and Thr-381 to Asp, we employed an overlap PCR strategy with the end primers H83 and H140 (described above) and complementary “overlap” primers H141 (5′-CCGTTAACGACCTAGCCACAGACTTCACGG-3′) and H142 (5′-CCGTGAAGTCTGTGGCTAGGTCGTTAACGG-3′). The MKK1DD PCR product was subcloned as described above to make pDLB824 (CEN HA-MKK1DD). Overexpression of activated Pkc1p was performed using pDL242 (53.Watanabe M. Chen C.Y. Levin D.E. J. Biol. Chem. 1994; 269: 16829-16836Abstract Full Text PDF PubMed Google Scholar).To disrupt MSG5, the LEU2 gene from pRS305 was amplified by PCR using primers MSG55′KO (5′-ATGCAATTTCACTCAGATAAGCAGCATTTGGACAGTAAAACCGACATCGATTTCAAGCCACAGATTGTACTGAGAGTGC-3′) and MSG53′KO (5′-AACATCATCTGTTCCGGGGCAGTAGATATTGATTCGTTGTCCACAGAAGCTTCCAGTGAACCTTACGCATCTGTGCGG-3′), the linear product was transformed into yeast, and Leu+ transformants were screened by PCR using primers MSG53′KO and MSG5 UTR (5′-CAAGAGTGAGGGTTATGC-3′) to detect homologous recombination yielding msg5::LEU2.To disrupt SDP1, the deletion allele yil113w::kanR from the genome knockout strain collection (obtained through Research Genetics, Inc.) was amplified by PCR using primers Z286 (5′-CTATTATCTAAAATAACACATAC-3′) and Z287 (5′-CAATAAAGCCTCATTGAATGCTATATC-3′), the linear product was transformed into yeast, and G418-resistant transformants were screened by PCR using primers Z287 and Z294 (5′-CTTCTGGAACAGGCAACTTC-3′) to detect homologous recombination yielding sdp1::kanR.To disrupt PTP3, the URA3 gene from pRS306 was amplified by PCR using primers Z290 (5′-GTATTGTATCTCCCTTCTCCATCATGCAACACAGATCTACTTATCATATAGAACGCGCGTTTCGGTGATGAC-3′) and Z291 (5′-GGGGTTTCGTATTAATAAAATAGAGATCAAATACATTCATATTAAGCCTAACCTGATGCGGTATTTTCTCCT-3′), the linear product was transformed into yeast, and Ura+ transformants were screened by PCR using primers Z291 and Z293 (5′-CATTGACCTTAGCGGTTTTC-3′) to detect homologous recombination yielding ptp3::URA3.To disrupt PTP2, the hygromycin B resistance gene (54.Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1356) Google Scholar) was amplified by PCR using primers Z288 (5′-CCCCAGTGCTATTAATAGTTTACAATAAAATAGGATCGACGTTGCTATTGCAGCTGAAGCTTCGTACGC-3′) and Z289 (5′-GTAATTTATCAAAACGAAAAGTGTTTGTATAATAGGAGAAAAACAATTCTAGCATAGGCCACTAGTGGATCTG-3′), the linear product was transformed into yeast, and hygromycin-resistant transformants were screened by PCR using primers Z289 and Z292 (5′-CATCTTTCTTTGAACACCGC-3′) to detect homologous recombination yielding ptp2::hygR.Biochemical Procedures—Procedures for cell lysis, SDS-PAGE, and Western blotting were as described (8.Harrison J.C. Bardes E.S. Ohya Y. Lew D.J. Nat. Cell Biol. 2001; 3: 417-420Crossref PubMed Scopus (117) Google Scholar).RESULTSEffect of RHO1Q68H on Mpk1p Activity—As mentioned in the Introduction, the activity of Pkc1p and subsequent kinases in the cell integrity pathway requires GTP-bound Rho1p. Although it is thought that stresses trigger an increase in GTP loading of Rho1p and in Pkc1p activity, technical limitations have thus far precluded direct measurement of the amount of Rho1p-GTP or of the activity of Pkc1p in vivo. In the case of the mitogenic growth factors that promote GTP loading of Ras, one of the major findings that established the paradigm was that oncogenic mutants of Ras that lock the protein in its GTP-bound state mimic constitutive signaling by the receptors, leading to constitutive ERK activation and uncontrolled proliferation (28.Der C.J. Finkel T. Cooper G.M. Cell. 1986; 44: 167-176Abstract Full Text PDF PubMed Scopus (400) Google Scholar). By analogy, we reasoned that expression of a GTP-locked mutant of RHO1 should lead to constitutive Mpk1p activation. Mpk1p activity can be monitored using a phosphospecific antibody that recognizes only the doubly phosphorylated (threonine and tyrosine) form of Mpk1p. Using this reagent, we compared Mpk1p activity in wild-type cells to that in cells that expressed the GTP-locked RHO1Q68H allele (29.Madaule P. Axel R. Myers A.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 779-783Crossref PubMed Scopus (181) Google Scholar), analogous to the GTP-locked oncogenic Ras mutant RasQ61H (28.Der C.J. Finkel T. Cooper G.M. Cell. 1986; 44: 167-176Abstract Full Text PDF PubMed Scopus (400) Google Scholar).Previous studies showed that overexpression of RHO1Q68H from the strong GAL1 promoter triggered the accumulation of multiple transcripts known to be responsive to Mpk1p activity (30.Roberts C.J. Nelson B. Marton M.J. Stoughton R. Meyer M.R. Bennett H.A. He Y.D. Dai H. Walker W.L. Hughes T.R. Tyers M. Boone C. Friend S.H. Science. 2000; 287: 873-880Crossref PubMed Scopus (718) Google Scholar), indicating that Mpk1p had indeed been activated by excess GTP-Rho1p. However, GAL1-RHO1Q68H also caused dramatic actin depolarization, through a pathway that required Pkc1p but not Mpk1p (31.Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (242) Google Scholar). This result raised the possibility that Mpk1p activation in GAL1-RHO1Q68H strains was the result of actin stress induced by overexpression. To assess whether more physiological (and therefore less disruptive) activation of Rho1p would activate Mpk1p, we introduced a single integrated copy of RHO1Q68H expressed from the RHO1 promoter into a wild-type strain (in addition to wild-type RHO1). Single-copy RHO1Q68H caused detectable but much less severe actin depolarization than GAL1-RHO1Q68H (data not shown), suggesting that limited hyperactivation of Pkc1p took place, but the cells were viable and proliferated normally. However, the activity of Mpk1p in cells with a single integrated copy of RHO1Q68H was indistinguishable from that in wild-type cells (Fig. 1A). The simplest interpretation of this result is that additional GTP loading of Rho1p (without overexpression) does not automatically elevate Mpk1p activity. Alternatively, constitutive activation of Rho1p might provoke adaptive desensitization of the pathway, returning Mpk1p activation to basal levels. If these cells were desensitized, then we would expect that they would no longer activate Mpk1p in response to stress. However, activation of Mpk1p in response to stress from heat shock, hypoosmotic shock, or actin depolymerization was completely unaffected by RHO1Q68H (Fig. 1B). This stress responsiveness was not because of Rho2p (which has some functional overlap with Rho1p (15.Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (182) Google Scholar, 32.Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar)), because similar results were obtained in cells lacking Rho2p (Fig. 1B). These results are inconsistent with the hypothesis that Mpk1p regulation occurs simply by modulating Rho1p GTP loading, and imply that significant regulation of the cell integrity pathway must occur downstream from Rho1p, presumably by regulating the activity of one or more of the downstream kinases.Regulatory or Permissive Roles for the Cell Integrity Kinases?—For each of the kinases upstream of Mpk1p in the cell integrity pathway, one can ask whether regulation of that kinase is important for the activation of Mpk1p in response to a given stress. Previous studies demonstrated that these kinases are each absolutely required for Mpk1p activity (10.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (421) Google Scholar, 11.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (270) Google Scholar, 13.Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 14.Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (303) Google Scholar, 33.Irie K. Takase M. Lee K.S. Levin D.E. Araki H. Matsumoto K. Oshima Y. Mol. Cell. Biol. 1993; 13: 3076-3083Crossref PubMed Scopus (259) Google Scholar), but that requirement does not discriminate whether a given kinase plays an active role in stress-mediated Mpk1p activation or a passive role providing a constitutive pathway input that is then modulated at a level downstream of that kinase by stress-responsive signaling pathways. In the latter scenario, it should be possible to dispense with a particular kinase entirely yet retain stress-responsive Mpk1p activity if an alternative source for the constitutive pathway input could be found. In the studies reported below, we have employed mutant alleles of BCK1 and MKK1 to provide a constitutive pathway input in the absence of the normally essential upstream kinases (Pkc1p and Bck1p, respectively).To provide a constitutive pathway input in the absence of Pkc1p, we employed the previously described BCK1–20 allele (11.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (270) Google Scholar). The lethality of the PKC1 deletion is suppressed by the dominant BCK1–20 allele, a result that we confirmed in our strain background (Fig. 2A). Bck1p is a large 1478-amino acid kinase with a C-terminal catalytic domain (residues 1175 to 1440), and Bck1–20p contains a missense mutation immediately upstream of the kinase domain (Ala-1174 to Pro) (11.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (270) Google Scholar). Bck1–20p is thought to encode a constitutively activated, Pkc1p-independent form of Bck1p that mimics Pkc1p-phosphorylated Bck1p, although the Pkc1p-targeted phosphorylation sites on Bck1p have not yet been mapped (11.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (270) Google Scholar).Fig. 2Suppression of pkc1Δ and bck1Δ phenotypes by activated alleles of Bck1p and Mkk1p. A, wild type (DLY1), pkc1Δ (DLY3962), and pkc1Δ BCK1–20 (DLY456) strains were grown on YEPD plates supplemented with 1 m sorbitol for 2 days at 24 °C and then replica plated to YEPD plates for 2 additional days of growth at 24 °C. B, wild type (WT) (DLY1), bck1Δ (DLY3994), and BCK1–20 (DLY455) cells carrying pDL242 (CEN URA3 GAL1-PKC1*) were grown in non-inducing sucrose media, Pkc1p* was induced by addition of galactose to the culture for the indicated time, and Mpk1p activity was monitored by Western blot. C, characterization of the dominant active MKK1DD allele. Wild type (DLY1), mkk1Δ mkk2Δ (DLY4351), or bck1Δ (DLY3994) cells carrying vector alone (pRS314), MKK1 (pDLB823), or MKK1DD (pDLB824) plasmids as indicated were streaked onto –trp media (left plate) and also onto a YEPD plate containing 10 mm caffeine (right plate). Cells lacking activity of the Pkc1p/Mpk1p pathway cannot grow on this media (see text). In the absence of Bck1p, wild type Mkk1p is inactive and cannot support pathway activity. bck1Δ cells carrying MKK1DD, however, are viable on caffeine suggesting that Mkk1pDD is activated and does not require Bck1p for its function.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To provide a constitutive pathway input in the absence of Bck1p, we constructed the MKK1DD allele. In other MAPK cascades, MAPKK activation by the MAPKKK involves the phosphorylation of two conserved serine residues on the MAPKK (34.Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar, 35.Zheng C.F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (297) Google Scholar). Mutation of these residues to aspartic acid mimics phosphorylation, yielding constitutively active MAP-KKs (26.Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1998; 9: 1339-1349Crossref PubMed Scopus (96) Google Scholar, 34.Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar). By analogy to these earlier studies we generated a mutant, MKK1DD, predicted to encode an activated version of Mkk1p. Mkk1pDD was expressed at levels comparable with those of wild-type Mkk1p (data not shown), and it was functional by the criterion that it could rescue the caffeine sensitivity of mkk1Δ mkk2Δ cells (Fig. 2C) (yeast cells lacking components of the cell integrity pathway are unable to grow on medium containing 10 mm caffeine, presumably because of thei" @default.
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• W2017366722 title "Stress-specific Activation Mechanisms for the “Cell Integrity” MAPK Pathway" @default.
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