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.

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.

The fission yeast Sty1/Spc1 mitogen‐activated protein (MAP) kinase is a member of the eukaryotic stress‐activated 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.

In Schizosaccharomyces pombe, glucose concentrations below a certain threshold trigger the stress-activated protein kinase (SAPK) signal transduction pathway and promote increased transcription of Atf1-dependent genes coding for the general stress response. Removal of glucose specifically induces the nuclear accumulation of green fluorescent protein-labeled Pap1 (GFP-Pap1) and the expression of genes dependent on this transcription factor. In contrast, depletion of the nitrogen source triggers the SAPK pathway but does not activate Pap1-dependent gene transcription, indicating that carbon stress rather than growth arrest leads to an endogenous oxidative condition that favors nuclear accumulation of Pap1. The reductant agents glutathione or N-acetylcysteine suppress the nuclear accumulation of GFP-Pap1 induced by glucose deprivation without inhibiting the activation of the MAPK Sty1. In addition, cells expressing a mutant GFP-Pap1 unable to accumulate into the nucleus upon hydrogen peroxide-mediated oxidative stress failed to show this protein into the nucleus in the absence of glucose. These results support the concept of a concerted action between the SAPK pathway and the Pap1 transcription factor during glucose exhaustion by which glucose limitation induces activation of the SAPK pathway prior to the oxidative stress caused by glucose deprivation. The ensuing induction of Atf1-dependent genes (catalase) decreases the level of hydroperoxides allowing Pap1 nuclear accumulation and function. Congruent with this interpretation, glucose-depleted cells show higher adaptive response to exogenous oxidative stress than those maintained in the presence of glucose.

Fission yeast PKC ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, MAP kinase Pmk1, in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the PDK ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role for Pck2 catalytic activation and downstream signaling. These events together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2 stabilize and render Pck2 competent to exert its biological functions. Remarkably, the TORC2 complex does not participate in catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level and add a new regulatory layer to MAPK signaling cascades.