Heat Stress Activates Fission Yeast Spc1/StyI MAPK by a MEKK-Independent Mechanism

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Δpyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Δwis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Δpyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.     

TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases

The coupling of growth to cell cycle progression allows eukaryotic cells to divide at particular sizes depending on nutrient availability. In fission yeast, this coupling involves the Spc1/Sty1 mitogen-activated protein kinase (MAPK) pathway working through Polo kinase recruitment to the spindle pole bodies (SPBs). Here we report that changes in nutrients influence TOR signalling, which modulates Spc1/Sty1 activity. Rapamycin-induced inhibition of TOR signalling advanced mitotic onset, mimicking the reduction in cell size at division seen after shifts to poor nitrogen sources. Gcn2, an effector of TOR signalling and modulator of translation, regulates the Pyp2 phosphatase that in turn modulates Spc1/Sty1 activity. Rapamycin- or nutrient-induced stimulation of Spc1/Sty1 activity promotes Polo kinase SPB recruitment and Cdc2 activation to advance mitotic onset. This advanced mitotic onset is abolished in cells depleted of Gcn2, Pyp2, or Spc1/Sty1 or on blockage of Spc1/Sty1-dependent Polo SPB recruitment. Therefore, TOR signalling modulates mitotic onset through the stress MAPK pathway via the Pyp2 phosphatase.     

Pyp3 PTPase acts as a mitotic inducer in fission yeast.

The p34cdc2 M-phase kinase is regulated by inhibitory phosphorylation of Tyr15, largely through the actions of the p107wee1 tyrosine kinase and p80cdc25 protein tyrosine phosphatase (PTPase). In this study we demonstrate that a second PTPase, encoded by pyp3, also contributes to tyrosyl dephosphorylation of p34cdc2. Pyp3 was identified as a high copy suppressor of a cdc25- mutation. The pyp3 gene encodes a 33 kDa PTPase that is more closely related to human PTP1B and fission yeast pyp1 and pyp2 PTPases than to cdc25. Pyp3 does not share an essential overlapping function with pyp1 or pyp2. We demonstrate that disruption of pyp3 causes a mitotic delay that is greatly exacerbated in cells that are partially defective for cdc25 function and that pyp3 function is essential in cdc25-disruption wee1- strains. Pyp3 PTPase effectively dephosphorylates and activates the p34cdc2 kinase in vitro. We conclude that the pyp3 PTPase acts cooperatively with p80cdc25 to dephosphorylate Tyr15 of p34cdc2.     

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca²⁺-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-β-d-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 ⁺ with concomitant resumption of the mutant cells’ growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.     

Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade.

Spc1 in Schizosaccharomyces pombe is a member of the stress-activated protein kinase family, an evolutionary conserved subfamily of mitogen-activated protein kinases (MAPKs). Spc1 is activated by a MAPK kinase homologue, Wis1, and negatively regulated by Pyp1 and Pyp2 tyrosine phosphatases. Mutations in the spc1+ and wis1+ genes cause a G2 cell cycle delay that is exacerbated during stress. Herein, we describe two upstream regulators of the Wis1-Spc1 cascade. wik1+ (Wis1 kinase) was identified from its homology to budding yeast SSK2, which encodes a MAPKK kinase that regulates the HOG1 osmosensing pathway. Delta wik1 cells are impaired in stress-induced activation of Spc1 and show a G2 cell cycle delay and osmosensitive growth. Moreover, overproduction of a constitutively active form of Wik1 induces hyperactivation of Spc1 in wis1(+)-dependent manner, suggesting that Wik1 regulates Spc1 through activation of Wis1. A mutation of mcs4+ (mitotic catastrophe suppressor) was originally isolated as a suppressor of the mitotic catastrophe phenotype of a cdc2-3w wee1-50 double mutant. We have found that mcs4- cells are defective at activation of Spc1 in response to various forms of stress. Epistasis analysis has placed Mcs4-upstream of Wik1 in the Spc1 activation cascade. These results indicate that Mcs4 is part of a sensor system for multiple environmental signals that modulates the timing of entry into mitosis by regulating the Wik1-Wis1-Spc1 kinase cascade. Inactivation of the sensor system delays the onset of mitosis and rescues lethal premature mitosis in cdc2-3w wee1-50 cells.     

Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway,and protein tyrosine phosphatases are essential under heat stress

The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1Δ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways.     

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.     

Atf1 is a target of the mitogen-activated protein kinase Pmk1 and regulates cell integrity in fission yeast

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.     

Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK

The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG‐specific Kss1 MAPK is activated by a combination of an O‐glycosylation defect caused by disruption of the gene encoding the protein O‐mannosyltransferase Pmt4, and an N‐glycosylation defect induced by tunicamycin. The O‐glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG‐specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

SakA MAP kinase is involved in stress signal transduction,sexual development and spore viability in Aspergillus nidulans

In eukaryotic cells, environmental stress signals are transmitted by evolutionarily conserved MAPKs, such as Hog1 in the budding yeast Saccharomyces cerevisiae, Spc1 in the fission yeast Schizosaccharomyces pombe and p38/JNK in mammalian cells. Here, we report the identification of the Aspergillus nidulans sakA gene, which encodes a member of the stress MAPK family. The sakA gene is able to complement the S. pombe spc1- defects in both osmo-regulation and cell cycle progression. Moreover, SakA MAPK is activated in response to osmotic and oxidative stress in both S. pombe and A. nidulans. However, in contrast to hog1 and spc1 mutants, the sakA null mutant is not sensitive to high osmolarity stress, indicating a different regulation of the osmostress response in this fungus. On the other hand, the DeltasakA mutant shows development and cell-specific phenotypes. First, it displays premature steA-dependent sexual development. Second, DeltasakA mutant produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage. Indeed, SakA is transiently activated early after induction of conidiation. Our results indicate that SakA MAPK is involved in stress signal transduction and repression of sexual development, and is required for spore stress resistance and survival.