Teresa Soto

Stress-induced Response, Localization, and Regulation of the Pmk1 Cell Integrity Pathway in Schizosaccharomyces pombe *

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.

Activation of the cell integrity pathway is channelled through diverse signalling elements in fission yeast

MAPK Pmk1p is the central element of a cascade involved in the maintenance of cell integrity and other functions in Schizosaccharomyces pombe. Pmk1p becomes activated by multiple stressing situations and also during cell separation. GTPase Rho2p acts upstream of the protein kinase C homolog Pck2p to activate the Pmk1 signalling pathway through direct interaction with MAPKKK Mkh1p. In this work we analyzed the functional significance of both Rho2p and Pck2p in the transduction of various stress signals by the cell integrity pathway. The results indicate that basal Pmk1p activity can be positively regulated by alternative mechanisms which are independent on the control by Rho2p and/or Pck2p. Unexpectedly, Pck1p, another protein kinase C homolog, negatively modulates Pmk1p basal activity by an unknown mechanism. Moreover, different elements appear to regulate the stress-induced activation of Pmk1p depending on the nature of the triggering stimuli. Whereas Pmk1p activation induced by hyper- or hypotonic stresses is channeled through Rho2p-Pck2p, other stressors, like glucose deprivation or cell wall disturbance, are transduced via other pathways in addition to that of Rho2p-Pck2p. On the contrary, Pmk1p activation observed during cell separation or after treatment with hydrogen peroxide does not involve Rho2p-Pck2p. Finally, Pck2p function is critical to maintain a Pmk1p basal activity that allows Pmk1p activation induced by heat stress. These data demonstrate the existence of a complex signalling network modulating Pmk1p activation in response to a variety of stresses in fission yeast.

Role for RACK1 Orthologue Cpc2 in the Modulation of Stress Response in Fission Yeast

The receptor of activated C kinase (RACK1) is a protein highly conserved among eukaryotes. In mammalian cells, RACK1 functions as an adaptor to favor protein kinase C (PKC)-mediated phosphorylation and subsequent activation of c-Jun NH(2)-terminal kinase mitogen-activated protein kinase. Cpc2, the RACK1 orthologue in the fission yeast Schizosaccharomyces pombe, is involved in the control of G2/M transition and interacts with Pck2, a PKC-type protein member of the cell integrity Pmk1 mitogen-activated protein kinase (MAPK) pathway. Both RACK1 and Cpc2 are structural components of the 40S ribosomal subunit, and recent data suggest that they might be involved in the control of translation. In this work, we present data supporting that Cpc2 negatively regulates the cell integrity transduction pathway by favoring translation of the tyrosine-phosphatases Pyp1 and Pyp2 that deactivate Pmk1. In addition, Cpc2 positively regulates the synthesis of the stress-responsive transcription factor Atf1 and the cytoplasmic catalase, a detoxificant enzyme induced by treatment with hydrogen peroxide. These results provide for the first time strong evidence that the RACK1-type Cpc2 protein controls from the ribosome the extent of the activation of MAPK cascades, the cellular defense against oxidative stress, and the progression of the cell cycle by regulating positively the translation of specific gene products involved in key biological processes.

Heat-shock response in Schizosaccharomyces pombe cells lacking cyclic AMP-dependent phosphorylation

Abstract Heat sensitivity at 48°C was determined in log-phase cultures of control and pka1-disrupted cells of the fission yeast Schizosaccharomyces pombe grown at 25°C. Cells devoid of protein kinase A exhibited a considerable heat-shock resistance as compared to control cells. Addition of cAMP to control cells prompted a further decrease in viability during heat shock. This effect was not observed with pka1-disrupted cells, suggesting that cAMP-dependent phosphorylation is involved in modulation of the heat-shock response. When control or pka1-disrupted cells were grown at 25°C and then shifted to 37°C they acquired thermo-tolerance to a subsequent treatment at 48°C both in the absence and in the presence of exogenous cAMP. Inhibition of protein synthesis during the adaptive treatment did not block the development of thermo-tolerance. However, the arrest in translation significantly prevented trehalose accumulation in control cells but only slightly affected trehalose increase in pka1-disrupted cells. These data indicate that heat resistance may be established in growing cells of S. pombe by at least two independent post-translational mechanisms: a decrease in cAMP-dependent protein phosphorylation and a hitherto unknown process which may be independent of trehalose accumulation.

Rho1 GTPase and PKC Ortholog Pck1 Are Upstream Activators of the Cell Integrity MAPK Pathway in Fission Yeast

In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.

A role for calcium in the regulation of neutral trehalase activity in the fission yeast Schizosaccharomyces pombe.

Neutral trehalases mobilize trehalose accumulated by fungal cells as a protective and storage carbohydrate. A structural feature of these enzymes is the presence of an EF-like motif similar to that shown by many Ca2+-binding proteins. In this study we provide direct evidence for physical binding of Ca2+ to neutral trehalase (Ntp1p) of the fission yeast Schizosaccharomyces pombe, and show that aspartic residues at positions 97 and 108 in the conserved putative Ca2+-binding motif of Ntp1p appear to be responsible for this interaction. Mutations in these residues do not interfere with the ability of Ntp1p to associate in vivo with trehalose-6-phosphate synthase, but prevent activation of neutral trehalase triggered by the addition of glucose or by subjecting cells to stressing conditions. Strains expressing Ntp1p variants that are unable to bind Ca2+ partially resemble those devoid of the ntp1+ gene in terms of trehalose hyperaccumulation. Gel filtration of cell extracts from wild-type cells after EDTA treatment or from cells containing Ntp1p with mutations in aspartic acid residues within the Ca2+-binding site revealed that Ntp1p eluted mainly in an inactive conformation instead of the dimeric or trimeric active form of the enzyme. These results suggest that activation of S. pombe Ntp1p under different conditions depends upon Ca2+ binding through the Ca2+-binding motif as a prerequisite for correct enzyme oligomerization to its active form. Given the high degree of conservation of the Ca2+ accommodation site, this might be a general mechanism regulating neutral trehalase activity in other yeasts and filamentous fungi.

Glucose-induced, cyclic-AMP-independent signalling pathway for activation of neutral trehalase in the fission yeast Schizosaccharomyces pombe

Summary: The addition of glucose to derepressed cells of Schizosaccharomyces pombe provokes a cAMP signal and activation of the cytoplasmic neutral trehalase. This transduction pathway does not require functional RAS protein since RAS1-disrupted cells exhibited a glucose response similar to that shown by control cells. Treatment of activated trehalase by alkaline phosphatase resulted in enzyme deactivation suggesting that trehalase may be modulated in vivo by reversible phosphorylation through cAMP-dependent protein kinase (PKA1). However, the addition of glucose to derepressed growing cells of Schiz. pombe lacking the catalytic subunit of protein kinase A (Δpka1::URA4+ strains) induced stimulation of trehalase as well as phosphorylation of the enzyme protein. This glucose-induced response was absent in PKA1-deficient cells from resting cultures. Addition of exogenous cAMP activated trehalase in normal growing cells but failed to produce any effect on trehalase in PKA1-disrupted growing cells. These results confirm the occurrence of a PKA1-dependent pathway for trehalase activation and imply the existence of another glucose-induced phosphorylation pathway capable of activating trehalase during growth by a distinct, cAMP-independent protein kinase. At least one of the upstream components playing a role in the transduction of this alternative signal is either absent or inactive in cells from stationary phase and sporulated cultures. Cells harbouring the disrupted PKA1 gene responded also to a heat-shock signal by increasing trehalase activity, thus revealing that this enzyme may be a target common to various signalling pathways in the fission yeast.

Rga4 Modulates the Activity of the Fission Yeast Cell Integrity MAPK Pathway by Acting as a Rho2 GTPase-activating Protein

Rho GTPase-activating proteins (GAPs) are responsible for the inactivation of Rho GTPases, which are involved in the regulation of critical biological responses in eukaryotic cells, ranging from cell cycle control to cellular morphogenesis. The genome of fission yeast Schizosaccharomyces pombe contains six genes coding for putative Rho GTPases, whereas nine genes code for predicted Rho GAPs (Rga1 to Rga9). One of them, Rga4, has been recently described as a Cdc42 GAP, involved in the control of cell diameter and symmetry in fission yeast. In this work we show that Rga4 is also a Rho2 GAP that negatively modulates the activity of the cell integrity pathway and its main effector, MAPK Pmk1. The DYRK-type protein kinase Pom1, which regulates both the localization and phosphorylation state of Rga4, is also a negative regulator of the Pmk1 pathway, but this control is not dependent upon the Rga4 role as a Rho2-GAP. Hence, two subsets of Rga4 negatively regulate Cdc42 and Rho2 functions in a specific and unrelated way. Finally, we show that Rga7, another Rho2 GAP, down-regulates the Pmk1 pathway in addition to Rga4. These results reinforce the notion of the existence of complex mechanisms determining the selectivity of Rho GAPs toward Rho GTPases and their functions.

Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe.

Trehalase activity is markedly enhanced upon addition of glucose and a nitrogen source to cells of the fission yeast Schizosaccharomyces pombe. This increase corresponds to a post-translational activation of the enzyme, which is controlled by cAMP-dependent and cAMP-independent pathways. Recent work has shown that overexpression of SCK1 in Schiz. pombe is able to suppress mutations that result in reduced Pka1 (cAMP-dependent protein kinase A activity, suggesting that Sck1 (suppressor of loss of cAMP-dependent protein kinase) might be a functional analogue of Pka1 in the fission yeast. Here, an analysis of the possible role of Sck1 in the activation of trehalase triggered by glucose and a nitrogen source is reported in cells that were deficient in either Pka1, Sck1 or both protein kinases. The results showed that, except in repressed cells, Sck1 probably mediates a cAMP-independent activation of trehalase following the signal(s) triggered by glucose and the nitrogen source. The absence of functional Sck1 in depressed cells renders trehalase insensitive to activation by glucose and the nitrogen source even in the presence of Pka1, indicating that the Sck1-dependent, cAMP-independent pathway is the main signalling pathway controlling trehalase activation under derepression conditions. It is proposed that, during the activation of trehalase induced by glucose or a nitrogen source, the cAMP-Pka1 activation pathway previously characterized is to some extent parallel to this newly described one which includes Sck1 as phosphorylating enzyme. Neither of these two pathways, however, plays a key role in the heat-induced increase in trehalase activity.

ANALYSIS OF THE NTP1+ GENE, ENCODING NEUTRAL TREHALASE IN THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE

We have cloned and sequenced the ntp1+ gene that codes for neutral trehalase in the fission yeast Schizosaccharomyces pombe. The ntp1+ gene product (Ntp1p) showed a 45-55% identity with neutral trehalases from other yeasts at the amino acid sequence level. However, in clear contrast to other neutral yeast trehalases so far characterized (which show two cAMP phospho-sites), only one consensus site for cAMP-dependent protein phosphorylation was found in Ntp1p. Northern blot hybridization experiments demonstrated that the Wis-Phh1/Sty1 MAP kinase cascade regulates ntp1+ expression during osmostress.