Regulatory Mechanisms for Modulation of Signaling through the Cell Integrity Slt2-mediated Pathway in Saccharomyces cerevisiae

Abstract

Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate-but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7⌬ mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway.

Involvement of Nst1p/YNL091w and Msl1p, a U2B″ splicing factor, in Saccharomyces cerevisiae salt tolerance

“…Ethidium bromide-stained rRNA was used as loading control and Northern analysis with probes for the CTT1 and HAL2 transcripts as positive controls for the NaCl and 3-AT induction, respectively. The fold increase in the ratio of the different mRNA signals to rRNA fluorescence, relative to the value in control SCD medium, is given below the panels 198 A. Goossens, J. Forment and R. Serrano with defects in components mediating maintenance of cell integrity (Gentzsch and Tanner, 1996;Nickas and Yaffe, 1996;Bach et al, 2000) and caffeine has recently been demonstrated to activate the Slt2 MAP kinase pathway regulating cell wall biosynthesis, probably by acting on some intracellular target mediating alterations of the cell surface (Martin et al, 2000). On the other hand, msl1 mutants showed an increased sensitivity to the microtubule-destabilizing compound thiabendazole (Entian et al, 1999).…”

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

“…Cell wall damage caused by the cell wall-perturbing agent zymolyase induces activation of HOG and CWI pathways sequentially (Bermejo et al, 2008;Garcia et al, 2009). These studies (Levin, 2005;Martin et al, 2000;Bermejo et al, 2008;Garcia et al, 2009) suggest that degradation of Fps1p by the HOG pathway is a compensation response to cell stress, and that the lack of Fps1p activates the response of the CWI pathway. When HM-1 perturbs cell wall construction by inhibiting 1,3-beta-glucan synthase as a cell wall-perturbing agent (Kasahara et al, 1994a, b;Kimura et al, 1998;Takasuka et al, 1995;Yamamoto et al, 1986a), both HOG and CWI pathways may be activated to adapt to the stress, as well as to the zymolyase challenge.…”

“…Slt2p is activated by cell wall stress, including heat shock, osmotic shock and the presence of cell wall-perturbing agents, as well as by acetate stress (Levin, 2005;Martin et al, 2000). Cell wall damage caused by the cell wall-perturbing agent zymolyase induces activation of HOG and CWI pathways sequentially (Bermejo et al, 2008;Garcia et al, 2009).…”

Fine regulation of Saccharomyces cerevisiae MAPK pathways by post‐translational modifications

“…However, since this pathway is stimulated upon cell wall alteration, the absence of Slt2 activity could lead to a cell wall biogenesis defect responsible for its own enhanced phosphorylation. Actually, the reduction of Slt2 phosphorylation when kinase-dead mutant cells are stabilized osmotically favours the latter idea, although it does not rule out the existence of a negative feedback [42]. On the other hand, the Slt2-phosphatase Msg5, essential for maintaining a low basal level of signalling through the CWI pathway, can be phosphorylated by this MAPK [17].…”

“…Since this fast-acting negative feedback does not involve transcription or translation, protein phosphatases should be direct targets of MAPK activity. Like in the osmotic and mating pathways, elimination of the kinase activity of the MAPK Slt2 increases its own phosphorylation at the activation loop in the absence of stimulation of the CWI pathway [42]. This could also be explained by a putative negative feedback loop exerted by Slt2, which would attenuate basal signalling.…”