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

Abstract

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...

The Rho1p Exchange Factor Rgf1p Signals Upstream from the Pmk1 Mitogen-activated Protein Kinase Pathway in Fission Yeast

“…It has been reported that MAP kinase Pmk1p activation is induced by multiple stressing situations, including hyper-and hypotonic conditions, the presence of cell wall damaging compounds, heat shock, glucose deprivation, and oxidative stress (Madrid et al, 2006). The catalytic activity of this family of kinases depends on the phosphorylation of both the Thr-186 and the Tyr-188 residues, and can be detected by Western blotting with polyclonal anti-phospho-p42/44 antibody (see Materials and Methods;Zaitsevskaya-Carter and Cooper, 1997;Loewith et al, 2000).…”

“…First, we looked at Pmk1p activation after osmotic stress caused by KCl or sorbitol. To this end, cells from wild-type (MI200) and mutant rgf1⌬ (PG285) strains with the genomic copy of pmk1 ϩ tagged with HA6H (Madrid et al, 2006) were grown at 28°C to the early log-phase. Extracts were obtained from samples taken before and after the addition of 0.6 M KCl or 1 M sorbitol for the times indicated.…”

“…In fission yeast, the Pmk1p MAPK pathway also regulates morphogenesis and ion homeostasis and becomes activated under multiple stresses, including hyper-or hypotonic conditions, the presence of cell wall-damaging compounds, glucose deprivation, and oxidative stress (Madrid et al, 2006). The MAPK module is composed of MAPKKK Mkh1p (Sengar et al, 1997), MAPKK Pek1p/Shk1p (Sugiura et al, 1999;Loewith et al, 2000), and MAPK Pmk1p/Spm1p (Toda et al, 1996b;Zaitsevskaya-Carter and Cooper, 1997).…”

“…A polarized actin cytoskeleton targets secretion to the growth sites, where probably specialized multienzyme complexes, consisting of both synthases and hydrolases, assemble the cell wall during the cell cycle (Motegi et al, 2001;Win et al, 2001;Mulvihill et al, 2006). Both the cytoskeleton and the cell wall are dynamic structures that are constantly remodelled and reorganized in response to growth signals and environmental stresses in order to ensure the integrity of the yeast cell (Levin, 2005;Madrid et al, 2006).…”

A Measurable Activation of the bZIP Transcription Factor Atf1 in a Fission Yeast Strain Devoid of Stress-activated and Cell Integrity Mitogen-activated Protein Kinase (MAPK) Activities

“…In fission yeast, the Pmk1 MAPK signaling pathway is also activated under multiple stresses, including heat shock, cell wall damage, and low glucose (15,24,25), and recent studies have reported that Pmk1 MAPK pathway plays a key role in cell wall integrity by regulating Atf1 (26). These results led us to investigate the Renilla reporter response upon treatment with the ROS-generating agents in ⌬pmk1 single and ⌬sty1⌬pmk1 double mutants.…”