Dataset Information

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway.

ABSTRACT: The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11-Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11-Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2.

SUBMITTER: Tatebayashi K

PROVIDER: S-EPMC1500976 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway.

Tatebayashi Kazuo K Yamamoto Katsuyoshi K Tanaka Keiichiro K Tomida Taichiro T Maruoka Takashi T Kasukawa Eri E Saito Haruo H

The EMBO journal 20060615 13

The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and ST ...[more]

PMID: 16778768

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Project description:Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

Project description:BackgroundCandida albicans is a commensal fungus that inhabits the oral mucosal surface and causes oral and systemic candidiasis. Oral candidiasis most commonly occurs in patients with AIDS, denture wearers and newborn children. Systemic candidiasis occurs mainly in immunocompromised patients and patients admitted to hospitals for prolonged periods. C. albicans homologous genes, DFG5 and DCW1, encode for two closely related cell wall proteins with putative glycosyltransferase enzyme activity and C-terminal GPI-anchors. Past studies have shown that individual DFG5 and DCW1 mutations are viable but simultaneous deletion of DFG5 and DCW1 in C. albicans results in lethality. However, the exact functions of these cell wall based enzymes, which represent potential drug targets, are not understood.MethodsC. albicans DFG5/DCW1 heterologous and conditional double mutant strains were assessed for growth and biofilm formation in comparison to wild type and parental strains. Cell wall and heat stress susceptibility of the mutant and control strains were assessed using agar spotting assays. Growth was assessed under normal and osmotic stress conditions along with light microscopy imaging. Biofilm dry weight and microscopic imaging analysis of biofilms was performed. Hypha formation in response to serum was analyzed using light microscopy imaging. Western blot analysis of mutant strains and control strains was performed to assess Hog1 basal levels and phosphorylation status.ResultsAnalysis of the heterologous mutants indicated that Dfg5p is more important for growth while Dcw1p appeared to play a role in cell wall integrity response. The conditional double mutant was observed to be less resistant to cell wall stress. However, growth of the mutants was similar under control and osmotic stress conditions. The mutants were also able to grow similar to wild type under heat stress. Biofilm formation was reduced in the mutants where DFG5 was deleted or suppressed. Hyphal morphogenesis was reduced although germ tube formation was observed in the biofilms of the mutant strains. Basal Hog1 protein levels were reduced or absent in the DFG5 and DCW1 mutants. However, osmotic stress was able to induce Hog1 protein levels comparable to wild type. Hog1 phosphorylation appeared to be slightly reduced although not significantly. In addition to biofilm assays, serum dose response imaging analysis indicated that hyphae formation in DFG5 and DCW1 mutants was defective.ConclusionsThese data indicate that DFG5 and DCW1 are required for hyphal morphogenesis and biofilm formation in C. albicans. These functions may be regulated via basal Hog1 MAPK which is required for transcriptional regulation of chitin synthesis.

Dataset Information

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway.

Publications

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway.

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