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• W2000551500 abstract "Stress-activated signal transduction pathways, which are largely conserved among a broad spectrum of eukaryotic species, have a crucial role in the survival of many forms of stress. It is therefore important to discover how these pathways are both positively and negatively regulated. Recent genetic studies have implicated protein phosphatase 2C (PP2C) as a novel negative regulator of stress response pathways in both budding and fission yeasts. Moreover, it was hypothesized that PP2C dephosphorylates one or more components of protein kinase cascades that are at the core of stress-activated signal transduction pathways. Herein we present genetic and biochemical studies of the fission yeastSchizosaccharomyces pombe that disprove this hypothesis and indicate that PP2C instead negatively regulates a downstream element of the pathway. First, high expression of PP2C produces phenotypes that are inconsistent with negative regulation of the Wik1-Wis1-Spc1 stress-activated kinase cascade. Second, high expression of PP2C leads to sustained activating tyrosine phosphorylation of Spc1. Third, Spc1-dependent phosphorylation of Atf1, a transcription factor substrate of Spc1, is unaffected by high expression of PP2C. Fourth, high expression of PP2C suppresses Atf1-dependent transcription of a stress-response gene. These studies strongly suggest that PP2C acts downstream of Spc1 kinase in the stress-activated signal transduction pathway. Stress-activated signal transduction pathways, which are largely conserved among a broad spectrum of eukaryotic species, have a crucial role in the survival of many forms of stress. It is therefore important to discover how these pathways are both positively and negatively regulated. Recent genetic studies have implicated protein phosphatase 2C (PP2C) as a novel negative regulator of stress response pathways in both budding and fission yeasts. Moreover, it was hypothesized that PP2C dephosphorylates one or more components of protein kinase cascades that are at the core of stress-activated signal transduction pathways. Herein we present genetic and biochemical studies of the fission yeastSchizosaccharomyces pombe that disprove this hypothesis and indicate that PP2C instead negatively regulates a downstream element of the pathway. First, high expression of PP2C produces phenotypes that are inconsistent with negative regulation of the Wik1-Wis1-Spc1 stress-activated kinase cascade. Second, high expression of PP2C leads to sustained activating tyrosine phosphorylation of Spc1. Third, Spc1-dependent phosphorylation of Atf1, a transcription factor substrate of Spc1, is unaffected by high expression of PP2C. Fourth, high expression of PP2C suppresses Atf1-dependent transcription of a stress-response gene. These studies strongly suggest that PP2C acts downstream of Spc1 kinase in the stress-activated signal transduction pathway. Eukaryotic organisms frequently encounter environmental conditions that cause cytotoxic damage; hence, they have developed sophisticated systems of sensing and responding to physiological stress. Protein kinase cascades are at the core of these stress sensor pathways (1Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar). These cascades follow the paradigm established for mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAP kinase; MAPKKK, MAP kinase kinase; PP2C, protein phosphatase 2C; EMM2, Edinburgh minimal medium; Ni2+-NTA, Ni2+-nitrilotriacetic acid; HA, hemagglutinin; Ha6H, hemagglutinin antigen epitope and six histidines; PTP, protein-tyrosine phosphatase. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAP kinase; MAPKKK, MAP kinase kinase; PP2C, protein phosphatase 2C; EMM2, Edinburgh minimal medium; Ni2+-NTA, Ni2+-nitrilotriacetic acid; HA, hemagglutinin; Ha6H, hemagglutinin antigen epitope and six histidines; PTP, protein-tyrosine phosphatase. cascades: a MAPK kinase kinase (MAPKKK) phosphorylates a MAPK kinase (MAPKK), which in turn phosphorylates a MAPK. The MAPKKK and MAPK components are serine-threonine kinases, whereas MAPKKs are dual specificity enzymes, activating MAPK substrates by phosphorylating threonine and tyrosine residues in a conserved motif (2Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar). It is not understood why protein kinase cascades are used to transmit stress signals, although it is likely that the spatial distribution of the cascade elements facilitates rapid signaling from the cell surface to the nucleus where MAPK homologs phosphorylate transcription factor substrates.Recent studies have revealed impressive functional and structural conservation of stress response pathways in yeast, plants, and various metazoan species, including humans (3Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (534) Google Scholar). The fission yeastSchizosaccharomyces pombe has been focus of some of the most interesting investigations, in part because studies of fission yeast have uncovered a link between stress response pathways and cell cycle control (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar). The S. pombe stress-activated kinase cascade consists of Wik1-Wis1-Spc1 kinases (6Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1997; 8: 409-419Crossref PubMed Scopus (110) Google Scholar). The Spc1 MAPK homolog, which is also known as Sty1 and Phh1 (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 7Kato T.J. Okazaki K. Murakami H. Stettler S. Fantes P.A. Okayama H. FEBS Lett. 1996; 378: 207-212Crossref PubMed Scopus (153) Google Scholar), is highly similar to mammalian p38 kinases (8Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2401) Google Scholar) and Hog1p kinase of the budding yeast Saccharomyces cerevisiae (9Brewster J.L. de Valoir T. Dwyer N.D. Winter E. Gustin M.C. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1017) Google Scholar). Like p38 kinase, Spc1 is broadly responsive to many forms of stress, including high osmolarity, oxidative stress, UV irradiation, and heat stress, as well as carbon and nitrogen starvation (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 10Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (255) Google Scholar, 11Shiozaki K. Russell P. Genes & Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (360) Google Scholar). Recent investigations have further revealed that mammalian p38 and fission yeast Spc1 share similar substrates. In the case of Spc1, the substrate is Atf1, a transcription factor containing a bZIP (basic leucine zipper) domain. Remarkably, Atf1 is highly similar to ATF-2, a mammalian transcription factor that is widely believed to be an important substrate of p38 kinase in vivo (11Shiozaki K. Russell P. Genes & Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (360) Google Scholar, 12Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.-C. Toda T. Millar J.B.A. Jones N. Genes & Dev. 1996; 18: 2289-2301Crossref Scopus (307) Google Scholar).It is evident that fission and budding yeasts can serve as useful model systems for uncovering novel modes of regulation of stress-activated protein kinase cascades. This accounts for the interest in recent studies suggesting that stress-activated kinase cascades in yeast are negatively regulated by two types of protein phosphatases: tyrosine-specific enzymes (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 10Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (255) Google Scholar) and serine-threonine phosphatases of the type 2C class (PP2C) (5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 13Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (933) Google Scholar). Evidence for a role of tyrosine-specific phosphatases in the negative regulation of stress-activated kinase cascades first arose from genetic studies ofS. cerevisiae. The gene PTP2 was cloned as a high copy suppressor of mutations that cause lethality due to hyperactivation of the Hog1p kinase cascade. High expression ofPTP2 results in decreased tyrosine phosphorylation of Hog1p, a finding consistent with the conclusion that Ptp2p directly dephosphorylates Hog1p in vivo (13Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (933) Google Scholar). Ptp2p is assumed to be a tyrosine-specific enzyme because it is more closely related to tyrosine-specific phosphatases than to dual specificity tyrosine/threonine phosphatases that have been identified as MAPK phosphatases in mammalian cells, although the enzymatic specificity of Ptp2p has not been examined. In the case of S. pombe, a critical advance was made with the discovery that the synthetic lethal phenotype observed in a strain lacking two tyrosine phosphatase genes,pyp1 and pyp2, was effectively suppressed by null mutations in genes encoding elements of the Wis1-Spc1 kinase cascade. Moreover, the stress-sensitive and G2 cell cycle delay phenotypes exhibited by spc1 and wis1 mutants were replicated by high expression of Pyp1 and Pyp2 enzymes (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar). These genetic studies were followed by definitive biochemical findings showing that Pyp1 and Pyp2 directly dephosphorylated tyrosine 173 of Spc1 both in vivo and in vitro (5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 10Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (255) Google Scholar). Interestingly, pyp2 + expression is induced in response to stress by a process that requires the Wis1-Spc1 kinase cascade and Atf1 transcription factor, indicating a mechanism of negative feedback regulation of the Wik1-Wis1-Spc1 signal transduction pathway (11Shiozaki K. Russell P. Genes & Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (360) Google Scholar, 12Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.-C. Toda T. Millar J.B.A. Jones N. Genes & Dev. 1996; 18: 2289-2301Crossref Scopus (307) Google Scholar).In contrast to the situation with tyrosine-specific phosphatases, the role of PP2C in the negative regulation of stress-activated kinase cascades is poorly understood. The initial insights came from studies of S. cerevisiae that revealed that two PP2C genes,PTC1 and PTC3, were multicopy suppressors of mutations that caused hyperactivation of the Hog1p kinase cascade (13Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (933) Google Scholar). The suggestion that PP2C negatively regulates the Hog1p stress-activated kinase cascade was further supported by the discovery of a synthetic lethal interaction involving ptp2 andptc1 mutations, which was suppressed by hog1mutations (14Maeda T. Tsai A.Y.M. Saito H. Mol. Cell. Biol. 1993; 13: 5408-5417Crossref PubMed Scopus (150) Google Scholar). These findings led to the hypothesis that PP2C negatively regulates the stress signaling pathway by dephosphorylating Hog1p or Pbs2p, the MAPKK homolog that activates Hog1p. Independent evidence for a role of PP2C in the negative regulation of stress-activated kinase cascades came from studies of S. pombe. In S. pombe the genesptc1 +, ptc2 +, andptc3 + account for ∼90% of the total PP2C activity (15Shiozaki K. Akhavan-Niaki H. McGowan C.H. Russell P. Mol. Cell. Biol. 1994; 14: 3743-3751Crossref Scopus (56) Google Scholar, 16Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (150) Google Scholar). Mutants carrying deletions of two or three of these genes are hypersensitive to increased levels of calcium. These defects are rescued by wis1 and spc1 mutations (16Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (150) Google Scholar). These findings are consistent with the notion that PP2C negatively regulates the Wis1-Spc1 kinase cascade in S. pombe, possibly via direct dephosphorylation of Spc1 and/or Wis1.Studies of PP2C in yeast are consistent with an increasing body of evidence suggesting a role for PP2C in the regulation of signal transduction systems. In mammals, PP2C is believed to play a role in Ca2+-dependent signal transduction in the brain (17Fukunaga K. Kobayashi T. Tamura S. Miyamoto E. J. Biol. Chem. 1993; 268: 133-137Abstract Full Text PDF PubMed Google Scholar). Findings implicating PP2C in Ca2+-related signal transduction have also arisen from studies of Arabidopsis thaliana, whose abi1 gene, which is essential for the response to the plant hormone abscisic acid, encodes a protein homologous to PP2C that also has a putative Ca2+ binding site (18Meyer K. Leube M.P. Grill E. Science. 1994; 264: 1452-1455Crossref PubMed Scopus (600) Google Scholar, 19Leung J. Bouvier-Durand M. Morris P.-C. Guerrier D. Chefdor F. Giraudat J. Science. 1994; 264: 1448-1452Crossref PubMed Scopus (657) Google Scholar). PP2C also appears to be important for cell maturation and development, since its expression and activity is reported to be up-regulated during the monocytic differentiation evoked by vitamin D3 in the human leukemic HL-60 cells (20Nishikawa M. Omay S.B. Nakai K. Kihira H. Kobayashi T. Tamura S. Shiku H. FEBS Lett. 1995; 375: 299-303Crossref PubMed Scopus (12) Google Scholar). Moreover, a recent study demonstrated that the FEM-2 gene ofCaenorhabditis elegans encodes a PP2C enzyme that is required to promote male development (21Chin-Sang I.D. Spence A.M. Genes & Dev. 1996; 10: 2314-2325Crossref PubMed Scopus (71) Google Scholar).Although genetic studies of fission and budding yeasts point toward a role for PP2C in regulating stress-activated protein kinase cascades, as of yet there have been no studies that directly test this hypothesis. Herein we describe such studies carried out with S. pombe. We report that expression of one of the PP2C genes,ptc1 +, is induced after osmotic stress by a mechanism that requires Spc1 and Atf1. Contrary to the hypothesis, we have found that high expression of ptc1 + does not reduce activating tyrosine phosphorylation of Spc1 or the phosphorylation of Atf1 that is catalyzed by Spc1. In fact, high expression of ptc1 + causes stress-induced tyrosine phosphorylation of Spc1 to be sustained, suggesting that Ptc1 acts downstream of Spc1 to negatively regulate the stress response. This finding is consistent with the observation that the level of stress-induced transcription of several genes is reduced in cells that overproduce Ptc1. Eukaryotic organisms frequently encounter environmental conditions that cause cytotoxic damage; hence, they have developed sophisticated systems of sensing and responding to physiological stress. Protein kinase cascades are at the core of these stress sensor pathways (1Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (958) Google Scholar). These cascades follow the paradigm established for mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAP kinase; MAPKKK, MAP kinase kinase; PP2C, protein phosphatase 2C; EMM2, Edinburgh minimal medium; Ni2+-NTA, Ni2+-nitrilotriacetic acid; HA, hemagglutinin; Ha6H, hemagglutinin antigen epitope and six histidines; PTP, protein-tyrosine phosphatase. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAP kinase; MAPKKK, MAP kinase kinase; PP2C, protein phosphatase 2C; EMM2, Edinburgh minimal medium; Ni2+-NTA, Ni2+-nitrilotriacetic acid; HA, hemagglutinin; Ha6H, hemagglutinin antigen epitope and six histidines; PTP, protein-tyrosine phosphatase. cascades: a MAPK kinase kinase (MAPKKK) phosphorylates a MAPK kinase (MAPKK), which in turn phosphorylates a MAPK. The MAPKKK and MAPK components are serine-threonine kinases, whereas MAPKKs are dual specificity enzymes, activating MAPK substrates by phosphorylating threonine and tyrosine residues in a conserved motif (2Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar). It is not understood why protein kinase cascades are used to transmit stress signals, although it is likely that the spatial distribution of the cascade elements facilitates rapid signaling from the cell surface to the nucleus where MAPK homologs phosphorylate transcription factor substrates. Recent studies have revealed impressive functional and structural conservation of stress response pathways in yeast, plants, and various metazoan species, including humans (3Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (534) Google Scholar). The fission yeastSchizosaccharomyces pombe has been focus of some of the most interesting investigations, in part because studies of fission yeast have uncovered a link between stress response pathways and cell cycle control (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar). The S. pombe stress-activated kinase cascade consists of Wik1-Wis1-Spc1 kinases (6Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1997; 8: 409-419Crossref PubMed Scopus (110) Google Scholar). The Spc1 MAPK homolog, which is also known as Sty1 and Phh1 (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 7Kato T.J. Okazaki K. Murakami H. Stettler S. Fantes P.A. Okayama H. FEBS Lett. 1996; 378: 207-212Crossref PubMed Scopus (153) Google Scholar), is highly similar to mammalian p38 kinases (8Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2401) Google Scholar) and Hog1p kinase of the budding yeast Saccharomyces cerevisiae (9Brewster J.L. de Valoir T. Dwyer N.D. Winter E. Gustin M.C. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1017) Google Scholar). Like p38 kinase, Spc1 is broadly responsive to many forms of stress, including high osmolarity, oxidative stress, UV irradiation, and heat stress, as well as carbon and nitrogen starvation (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 10Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (255) Google Scholar, 11Shiozaki K. Russell P. Genes & Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (360) Google Scholar). Recent investigations have further revealed that mammalian p38 and fission yeast Spc1 share similar substrates. In the case of Spc1, the substrate is Atf1, a transcription factor containing a bZIP (basic leucine zipper) domain. Remarkably, Atf1 is highly similar to ATF-2, a mammalian transcription factor that is widely believed to be an important substrate of p38 kinase in vivo (11Shiozaki K. Russell P. Genes & Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (360) Google Scholar, 12Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.-C. Toda T. Millar J.B.A. Jones N. Genes & Dev. 1996; 18: 2289-2301Crossref Scopus (307) Google Scholar). It is evident that fission and budding yeasts can serve as useful model systems for uncovering novel modes of regulation of stress-activated protein kinase cascades. This accounts for the interest in recent studies suggesting that stress-activated kinase cascades in yeast are negatively regulated by two types of protein phosphatases: tyrosine-specific enzymes (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 10Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (255) Google Scholar) and serine-threonine phosphatases of the type 2C class (PP2C) (5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 13Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (933) Google Scholar). Evidence for a role of tyrosine-specific phosphatases in the negative regulation of stress-activated kinase cascades first arose from genetic studies ofS. cerevisiae. The gene PTP2 was cloned as a high copy suppressor of mutations that cause lethality due to hyperactivation of the Hog1p kinase cascade. High expression ofPTP2 results in decreased tyrosine phosphorylation of Hog1p, a finding consistent with the conclusion that Ptp2p directly dephosphorylates Hog1p in vivo (13Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (933) Google Scholar). Ptp2p is assumed to be a tyrosine-specific enzyme because it is more closely related to tyrosine-specific phosphatases than to dual specificity tyrosine/threonine phosphatases that have been identified as MAPK phosphatases in mammalian cells, although the enzymatic specificity of Ptp2p has not been examined. In the case of S. pombe, a critical advance was made with the discovery that the synthetic lethal phenotype observed in a strain lacking two tyrosine phosphatase genes,pyp1 and pyp2, was effectively suppressed by null mutations in genes encoding elements of the Wis1-Spc1 kinase cascade. Moreover, the stress-sensitive and G2 cell cycle delay phenotypes exhibited by spc1 and wis1 mutants were replicated by high expression of Pyp1 and Pyp2 enzymes (4Millar J.B.A. Buck V. Wilkinson M.G. Genes & Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (308) Google Scholar, 5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar). These genetic studies were followed by definitive biochemical findings showing that Pyp1 and Pyp2 directly dephosphorylated tyrosine 173 of Spc1 both in vivo and in vitro (5Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (392) Google Scholar, 10Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (255) Google Scholar). Interestingly, pyp2 + expression is induced in response to stress by a process that requires the Wis1-Spc1 kinase cascade and Atf1 transcription factor, indicating a mechanism of negative feedback regulation of the Wik1-Wis1-Spc1 signal transduction pathway (11Shiozaki K. Russell P. Genes & Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (360) Google Scholar, 12Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.-C. Toda T. Millar J.B.A. Jones N. Genes & Dev. 1996; 18: 2289-2301Crossref Scopus (307) Google Scholar). In contrast to the situation with tyrosine-specific phosphatases, the role of PP2C in the negative regulation of stress-activated kinase cascades is poorly understood. The initial insights came from studies of S. cerevisiae that revealed that two PP2C genes,PTC1 and PTC3, were multicopy suppressors of mutations that caused hyperactivation of the Hog1p kinase cascade (13Maeda T. Wurgler-Murphy S.M. Saito H. Nature. 1994; 369: 242-245Crossref PubMed Scopus (933) Google Scholar). The suggestion that PP2C negatively regulates the Hog1p stress-activated kinase cascade was further supported by the discovery of a synthetic lethal interaction involving ptp2 andptc1 mutations, which was suppressed by hog1mutations (14Maeda T. Tsai A.Y.M. Saito H. Mol. Cell. Biol. 1993; 13: 5408-5417Crossref PubMed Scopus (150) Google Scholar). These findings led to the hypothesis that PP2C negatively regulates the stress signaling pathway by dephosphorylating Hog1p or Pbs2p, the MAPKK homolog that activates Hog1p. Independent evidence for a role of PP2C in the negative regulation of stress-activated kinase cascades came from studies of S. pombe. In S. pombe the genesptc1 +, ptc2 +, andptc3 + account for ∼90% of the total PP2C activity (15Shiozaki K. Akhavan-Niaki H. McGowan C.H. Russell P. Mol. Cell. Biol. 1994; 14: 3743-3751Crossref Scopus (56) Google Scholar, 16Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (150) Google Scholar). Mutants carrying deletions of two or three of these genes are hypersensitive to increased levels of calcium. These defects are rescued by wis1 and spc1 mutations (16Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (150) Google Scholar). These findings are consistent with the notion that PP2C negatively regulates the Wis1-Spc1 kinase cascade in S. pombe, possibly via direct dephosphorylation of Spc1 and/or Wis1. Studies of PP2C in yeast are consistent with an increasing body of evidence suggesting a role for PP2C in the regulation of signal transduction systems. In mammals, PP2C is believed to play a role in Ca2+-dependent signal transduction in the brain (17Fukunaga K. Kobayashi T. Tamura S. Miyamoto E. J. Biol. Chem. 1993; 268: 133-137Abstract Full Text PDF PubMed Google Scholar). Findings implicating PP2C in Ca2+-related signal transduction have also arisen from studies of Arabidopsis thaliana, whose abi1 gene, which is essential for the response to the plant hormone abscisic acid, encodes a protein homologous to PP2C that also has a putative Ca2+ binding site (18Meyer K. Leube M.P. Grill E. Science. 1994; 264: 1452-1455Crossref PubMed Scopus (600) Google Scholar, 19Leung J. Bouvier-Durand M. Morris P.-C. Guerrier D. Chefdor F. Giraudat J. Science. 1994; 264: 1448-1452Crossref PubMed Scopus (657) Google Scholar). PP2C also appears to be important for cell maturation and development, since its expression and activity is reported to be up-regulated during the monocytic differentiation evoked by vitamin D3 in the human leukemic HL-60 cells (20Nishikawa M. Omay S.B. Nakai K. Kihira H. Kobayashi T. Tamura S. Shiku H. FEBS Lett. 1995; 375: 299-303Crossref PubMed Scopus (12) Google Scholar). Moreover, a recent study demonstrated that the FEM-2 gene ofCaenorhabditis elegans encodes a PP2C enzyme that is required to promote male development (21Chin-Sang I.D. Spence A.M. Genes & Dev. 1996; 10: 2314-2325Crossref PubMed Scopus (71) Google Scholar). Although genetic studies of fission and budding yeasts point toward a role for PP2C in regulating stress-activated protein kinase cascades, as of yet there have been no studies that directly test this hypothesis. Herein we describe such studies carried out with S. pombe. We report that expression of one of the PP2C genes,ptc1 +, is induced after osmotic stress by a mechanism that requires Spc1 and Atf1. Contrary to the hypothesis, we have found that high expression of ptc1 + does not reduce activating tyrosine phosphorylation of Spc1 or the phosphorylation of Atf1 that is catalyzed by Spc1. In fact, high expression of ptc1 + causes stress-induced tyrosine phosphorylation of Spc1 to be sustained, suggesting that Ptc1 acts downstream of Spc1 to negatively regulate the stress response. This finding is consistent with the observation that the level of stress-induced transcription of several genes is reduced in cells that overproduce Ptc1. We thank the cell cycle labs at Scripps for support and encouragement, with particular thanks going to Geneviéve Degols, Odile Mondésert, and Clare McGowan." @default.
• W2000551500 created "2016-06-24" @default.
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• W2000551500 date "1997-07-01" @default.
• W2000551500 modified "2023-09-30" @default.
• W2000551500 title "Protein Phosphatase 2C Acts Independently of Stress-activated Kinase Cascade to Regulate the Stress Response in Fission Yeast" @default.
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• W2000551500 doi "https://doi.org/10.1074/jbc.272.28.17873" @default.
• W2000551500 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9211944" @default.
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