The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p-and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast. INTRODUCTIONThe mitogen-activated protein (MAP) kinase (MAPK) signaling pathways are critical for the response of cells to changes in their environment (Marshall, 1994;Herskowitz, 1995;Waskiewicz and Cooper, 1995;Treisman, 1996). They serve to transduce signals generated at the cell surface or in the cytoplasm to the nucleus, where changes in gene expression result. In mammalian cells, multiple distinct MAP kinases have been identified, including a large subset whose members are activated by a variety of environmental stress conditions, DNA-damaging agents, inflammatory cytokines, and certain vasoactive neuropeptides Freshney et al., 1994;Galcheva-Gargova et al., 1994;Han et al., 1994;Kyriakis et al., 1994;Lee et al., 1994;Rouse et al., 1994;Sluss et al., 1994). These stress-activated MAP kinases (SAPKs) fall into two distinct classes, termed the C-Jun N-terminal kinase ( JNK) and p38 kinases, based on their sequences (Davies, 1994;Waskiewicz and Cooper, 1995). A number of transcription factors are phosphorylated in response to SAPK activation; for example, the c-Jun factor is regulated by JNK (Hibi et al., 1993;Derijard et al., 1994;Kyriakis et al., 1994) but not by p38, whereas ATF2 is phosphorylated and regulated by both JNK Livingstone et al., 1995;van Dam et al., 1995) and p38 (Raingeaud et al., 1995). Although a number of MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKKs) that activate the SAPKs have been identified in mammalian cells, very little is known about how these are regulated by stress stimuli (reviewed in Ichijo, 1999;Tibbles and Woodgett, 1999). This is probably due to the multiplicity of SAPK pathways in mammalian cells and the difficulties of genetic analysis in these organisms.Recently, a single member of the SAPK family, called Sty1p (also known as Spc1p or Phh1p), ...

Mitogen-activated protein kinase (MAPK) signaling pathways are critical for the sensing and response of eukaryotic cells to extracellular changes. In Schizosaccharomyces pombe, MAPK Pmk1/ Spm1 has been involved in cell wall construction, morphogenesis, cytokinesis, and ion homeostasis, as part of the so-called cell integrity pathway together with MAPK kinase kinase Mkh1 and MAPK kinase Pek1. We show that Pmk1 is activated in multiple stress situations, including hyper-or hypotonic stress, glucose deprivation, presence of cell wall-damaging compounds, and oxidative stress induced by hydrogen peroxide or pro-oxidants. The stress-induced activation of Pmk1 was completely dependent on Mkh1 and Pek1 function, supporting a nonbranched pathway in the regulation of MAPK activation. Fluorescence microscopy revealed that Mkh1, Pek1, and Pmp1 (a protein phosphatase that inactivates Pmk1) are cytoplasmic proteins. Mkh1 and Pek1 were also found at the septum, whereas Pmk1 localized in both cytoplasm and nucleus as well as in the mitotic spindle and septum during cytokinesis. Interestingly, Pmk1 subcellular localization was unaffected by stress or the absence of Mkh1 and Pek1, suggesting that its activation by the Mkh1-Pek1 cascade takes place at the cytoplasm and/or septum and that the active and inactive forms of this kinase cross the nuclear membrane. Cdc42 GTPase and its effectors, p21-activated kinases Pak2 and Pak1, are not upstream elements controlling the basal level or the stress-induced activation of Pmk1. However, Sty1 MAPK was essential for proper Pmk1 deactivation after hypertonic stress in a process regulated by Atf1 transcription factor. These results provide the first evidence for the existence of cross-talk between two MAPK cascades during the stress response in fission yeast. Mitogen-activated protein kinase (MAPK)5 pathways are signal transduction mechanisms that regulate many cellular processes in eukaryotic organisms, from yeasts to mammals. The basic architecture of each functional cascade is composed of three sequentially acting protein kinases that become activated in response to triggering signals; the MAPK kinase kinases (MAPKKKs) phosphorylate and activate MAPK kinases (MAPKKs), which in turn phosphorylate and activate MAPKs (1, 2). Among other actions, the effector MAPKs control the activity of transcription factors either directly or indirectly. Thus, activation by specific stimuli of MAPK signal transduction pathways is accompanied by changes in gene expression that play a crucial adaptive role in the adjustment of cells to environmental conditions. In contrast to the six or more MAPK cascades present in budding yeast (3), three distinct MAPK signaling cascades have been so far identified in the fission yeast Schizosaccharomyces pombe. These include the mating pheromone-responsive MAPK pathway and the stress-activated protein kinase (SAPK) pathway, whose central elements are MAPKs Spk1 and Sty1/ Spc1, respectively (4, 5). A third pathway, known as the cell integrity pathway, consists of a MAPK ca...

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The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stressactivated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/ Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway.

Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper-or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.