Creencias y Valores

In contrast to S. pombe, S. cerevisiae contains just a single HK, Sln1, acting upstream of the Mpr1-orthologous HPt protein, Ypd1, and the two RRs, Ssk1 and Skn7 (Figure 1.4). The Sln1 HK is a transmembrane protein with an extracellular domain, which senses osmotic and cell wall stress, and a cytoplasmic histidine kinase domain. In contrast, the HKs of S. pombe are entirely cytoplasmic and perceive oxidative stress signals (Quinn et al., 2011). Despite these differences, both the S. cerevisiae and S. pombe phosphorelays dually activate a stress-activated MAPK cascade and a response regulator with DNA-binding activity. Osmotic stress causes a reduction in Sln1 kinase activity and, consequently, a reduction in phosphorylation of Ypd1, Ssk1 and Skn7. Unphosphorylated Ssk1 stimulates the Hog1 MAPK leading to a derepression of Sko1- regulated osmoinducible genes. In contrast, cell wall stress induces an increase in Sln1 kinase activity and renders the Hog1 MAPK pathway inactive (Fassler & West, 2011, Ikner & Shiozaki, 2005). The Skn7 response regulator, like the S. pombe Prr1 homologue, is under the regulation of a His-Asp phosphorelay, but oxidative stress appears to activate Skn7 independently of the phosphorelay system. Phosphorylation of Skn7 by the HPt protein Ypd1 leads to activation of genes involved in cell cycle control and cell wall stress-responsive genes, whereas oxidative activation of Skn7 leads to activation of genes involved in the oxidative stress response (OSR) including

TRX2, TSA1, GPX2, AHP1, CCP1 and CTT1. Many of the oxidative stress response

genes contain not only Skn7 binding sites, but also Yap1 binding sites (Yap1 response elements; YRE) and indeed it has been shown that these two factors coordinately regulate a large set of genes, including CTT1, SOD1, SOD2, TRR1, TRX2 and TSA1 in

,, the OSR regulon. A substantial number of OSR genes, including GLR1, GSH1 and

CYS3, depend on Yap1 alone for expression, however, evidence for Skn7

dependent/Yap1 independent OSR gene expression is lacking (Lee et al., 1999, Mulford & Fassler, 2011). In response to oxidative stress, Yap1 binds weakly to unphosphorylated DNA-bound Skn7. A Skn7 kinase, yet to be identified, is then recruited to the Skn7-Yap1 complex, leading to the phosphorylation of Skn7, which in turn stabilises the complex (He et al., 2009).

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Figure 1.4 S. cerevisiae stress-sensing pathways. The major oxidative stress response pathway in S. cerevisiae involves the transcription factor Yap1. The thiol peroxidase Gpx3 senses and transmits the H2O2 signal to Yap1 resulting in the activation of oxidative stress response genes. Thiol reactive compounds also activate Yap1 via a Gpx3-independent mechanism (See Figure 1.5). Yap1 interacts with the response regulator Skn7 and the Yap1-Skn7 complex recruits a yet to be identified kinase, that phosphorylates Skn7 reinforcing the Skn7-Yap1 interaction resulting in the activation of gene expression. Yap1 also activates a subset of oxidative stress response genes independently of Skn7. The transmembrane histidine kinase Sln1 is autophosphorylated under normal growth conditions. Cell wall stress, induced by cell- wall perturbing agents such as calcofluor white (CFW) and sodium dodecyl sulfate (SDS) induces hyperactivation of the pathway and an accumulation of Ssk1 in a phosphorylated and

Ypd1 Sln1 Ypd1 Skn7ox Gpx3 Yap1ox Yap1ox Ypd Kinase Skn7 7 Skn77 Sko1 Pbs2 Hog1 1 1 1 Ssk2 Ssk22 Ssk1 osmostress wall stress Sln1 1 w S n11

H

O

osmo

stress

wall

stress

Oxidative stress

gene expression response

ative

Cell wall and Osmotic stress

gene expression response

,. inactive state. Osmotic stress, induced by high NaCl and other conditions causing reduced turgor, lead to a reduction in Sln1 kinase activity and a consequential reduction in phosphotransfer to the phosphorelay intermediate Ypd1 and response regulators Ssk1 and Skn7. Accumulation of the unphosphorylated form of Ssk1 activates the Ssk2 and Ssk22 MAP kinase kinase kinases (MAPKKKs) which, in turn, activate the Pbs2-Hog1 MAPKK-MAPK pathway. Hog1 induces gene expression by inactivating the Sko1 transcriptional repressor. Cell wall stress also activates Skn7 via the Sln1-Ypd1 phosphorelay.

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Phosphorelay systems in filamentous fungi

The fungal HK and HPt gene families appear to have expanded in filamentous fungi with 15 HK- and 4 RR-encoding genes identified in the A. nidulans genome along with a single HPt gene (Hagiwara et al., 2007). This is possibly a reflection of the more diverse environmental stresses encountered by these multicellular fungi. The response regulators SskA, a homologue of S. cerevisiae Ssk1 and S. pombe Mcs4, and SrrA, a homologue of S. cerevisiae Skn7 and S. pombe Prr1, have been implicated in oxidative stress and, to a lesser degree, osmotic stress resistance. Conidial germination and hyphal growth of the sskA and srrA mutants are sensitive to peroxide stress induced by tert-butyl hydroperoxide and H2O2 but tolerate oxidative stress generated by thiol-

reactive compounds, menadione and diamide. Furthermore, sskA (but not srrA) conidia are inherently less viable than wild-type conidia. The finding that genes involved in conidial stress-tolerance (catalase A, catA; glycerol-3-phosphate dehydrogenase, gfdB; and trehalose-6-phosphate synthase, tpsA) are downregulated in the sskA mutant supports the notion that this two-component pathway relays signals from receptor to nucleus (Furukawa et al., 2005). While SskA acts upstream of the HogA MAPK cascade, SrrA is thought to be a DNA-binding transcription factor (Furukawa et al., 2005).

1.3.2

Stress-activated MAPK cascades

Mitogen-activated protein kinase (MAPK) cascades constitute a fundamental signalling system universally employed by eukaryotes to regulate responses to various stimuli including pheromones, nitrogen and nutrient starvation, cell wall, osmotic and oxidative stress (Chen & Thorner, 2007). Such cascades consist of a three-tiered hierarchical organisation of sequentially acting kinases: a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK. Additional upstream and downstream components are also required to initiate MAPKKK phosphorylation and translate the phosphorylation signal from the MAPK to a biological response (Chen & Thorner, 2007).