scholarly journals The G-Protein β Subunit GPB1 Is Required for Mating and Haploid Fruiting in Cryptococcus neoformans

2000 ◽  
Vol 20 (1) ◽  
pp. 352-362 ◽  
Author(s):  
Ping Wang ◽  
John R. Perfect ◽  
Joseph Heitman
Keyword(s):  
Map Kinase ◽  
Cryptococcus Neoformans ◽  
G Protein ◽  
Nutrient Sensing ◽  
Β Subunit ◽  
A Cells ◽  
Map Kinase Cascade ◽  
Mutant Strains ◽  
Kinase Cascade ◽  
Monokaryotic Fruiting

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle. The gene encoding a heterotrimeric G-protein β subunit, GPB1, was cloned and disrupted.gpb1 mutant strains are sterile, indicating a role for this gene in mating. GPB1 plays an active role in mediating responses to pheromones in early mating steps (conjugation tube formation and cell fusion) and signals via a mitogen-activated protein (MAP) kinase cascade in both MATα and MATa cells. The functions of GPB1 are distinct from those of the Gα protein GPA1, which functions in a nutrient-sensing cyclic AMP (cAMP) pathway required for mating, virulence factor induction, and virulence.gpb1 mutant strains are also defective in monokaryotic fruiting in response to nitrogen starvation. We show thatMATa cells stimulate monokaryotic fruiting ofMATα cells, possibly in response to mating pheromone, which may serve to disperse cells and spores to locate mating partners. In summary, the Gβ subunit GPB1 and the Gα subunit GPA1 function in distinct signaling pathways: one (GPB1) senses pheromones and regulates mating and haploid fruiting via a MAP kinase cascade, and the other (GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.

scholarly journals Mapping of a Yeast G Protein βγ Signaling Interaction

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1407-1417 ◽  
Author(s):  
Simon J Dowell ◽  
Anne L Bishop ◽  
Susan L Dyos ◽  
Andrew J Brown ◽  
Malcolm S Whiteway
Keyword(s):  
Signal Transduction ◽  
Map Kinase ◽  
G Protein ◽  
Coiled Coil ◽  
Receptor Activation ◽  
Temperature Sensitive ◽  
Hydrophobic Residues ◽  
Map Kinase Cascade ◽  
Kinase Cascade

Abstract The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein βγ subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gβ (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gβγ coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Gα) and Ste18p (Gγ) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gβγ coiled-coil in Ste5p binding may set a precedent for Gβγ-effector interactions in more complex organisms.

scholarly journals Requirement of Pyk2 for the activation of the MAP kinase cascade induced by Ca2+ (but not by PKC or G protein) in PC12 cells

FEBS Letters ◽  
1999 ◽  
Vol 461 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Rico Barsacchi ◽  
Harald Heider ◽  
Jean-Antoine Girault ◽  
Jacopo Meldolesi
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Pc12 Cells ◽  
Map Kinase Cascade ◽  
Kinase Cascade

scholarly journals Skh1, the MEK component of the mkh1 signaling pathway in Schizosaccharomyces pombe

2000 ◽  
Vol 113 (1) ◽  
pp. 153-160 ◽  
Author(s):  
R. Loewith ◽  
A. Hubberstey ◽  
D. Young
Keyword(s):  
Map Kinase ◽  
Schizosaccharomyces Pombe ◽  
Genetic Evidence ◽  
Yeast Two Hybrid ◽  
Beta Glucanase ◽  
Map Kinase Cascade ◽  
Mutant Strains ◽  
Kinase Cascade ◽  
Delta Cells ◽  
Two Hybrid

We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to (beta)-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1(DD)protein suppressed these defects in mkh1(delta) cells, and overexpression of Spm1 suppressed these defects in skh1(delta) cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells co-expressing the activated HA-Skh1(DD) protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1(delta) or skh1(delta) cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast.

scholarly journals Loss of sustained Fus3p kinase activity and the G1 arrest response in cells expressing an inappropriate pheromone receptor.

1996 ◽  
Vol 16 (8) ◽  
pp. 4478-4485 ◽  
Author(s):  
A Couve ◽  
J P Hirsch
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
G1 Arrest ◽  
Wild Type ◽  
Pheromone Response Pathway ◽  
Cycle Arrest ◽  
Map Kinase Cascade ◽  
Receptor Inhibition ◽  
Kinase Cascade

The yeast pheromone response pathway is mediated by two G protein-linked receptors, each of which is expressed only in its specific cell type. The STE3DAF mutation results in inappropriate expression of the a-factor receptor in MATa cells. Expression of this receptor in the inappropriate cell type confers resistance to pheromone-induced G1 arrest, a phenomenon that we have termed receptor inhibition. The ability of STE3DAF cells to cycle in the presence of pheromone was found to correlate with reduced phosphorylation of the cyclin-dependent kinase inhibitor Far1p. Measurement of Fus3p mitogen-activated protein (MAP) kinase activity in wild-type and STE3DAF cells showed that induction of Fus3p activity was the same in both strains at times of up to 1 h after pheromone treatment. However, after 2 or more hours, Fus3p activity declined in STE3DAF cells but remained high in wild-type cells. The level of inducible FUS1 RNA paralleled the changes seen in Fus3p activity. Short-term activation of the Fus3p MAP kinase is therefore sufficient for the early transcriptional induction response to pheromone, but sustained activation is required for cell cycle arrest. Escape from the cell cycle arrest response was not seen in wild-type cells treated with low doses of pheromone, indicating that receptor inhibition is not simply a result of weak signaling but rather acts selectively at late times during the response. STE3DAF was found to inhibit the pheromone response pathway at a step between the G beta subunit and Ste5p, the scaffolding protein that binds the components of the MAP kinase phosphorylation cascade. Overexpression of Ste20p, a kinase thought to act between the G protein and the MAP kinase cascade, suppressed the STE3DAF phenotype. These findings are consistent with a model in which receptor inhibition acts by blocking the signaling pathway downstream of G protein dissociation and upstream of MAP kinase cascade activation, at a step that could directly involve Ste20p.

scholarly journals The G Protein-Coupled Receptor Gpr1 Is a Nutrient Sensor That Regulates Pseudohyphal Differentiation in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 609-622 ◽  
Author(s):  
Michael C Lorenz ◽  
Xuewen Pan ◽  
Toshiaki Harashima ◽  
Maria E Cardenas ◽  
Yong Xue ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Map Kinase ◽  
G Protein ◽  
Nitrogen Starvation ◽  
G Protein Coupled Receptor ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Pseudohyphal Differentiation ◽  
G Protein Coupled

Abstract Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Gα protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Gα protein-cAMP-PKA signal transduction cascade.

scholarly journals Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1

2001 ◽  
Vol 21 (11) ◽  
pp. 3671-3683 ◽  
Author(s):  
John M. Schmitt ◽  
Philip J. S. Stork
Keyword(s):  
Cyclic Amp ◽  
Map Kinase ◽  
G Protein ◽  
Cell Cycle Progression ◽  
3T3 Cells ◽  
Small G Protein ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT In many normal and transformed cell types, the intracellular second messenger cyclic AMP (cAMP) blocks the effects of growth factors and serum on mitogenesis, proliferation, and cell cycle progression. cAMP exerts these growth-inhibitory effects via inhibition of the mitogen-activated protein (MAP) kinase cascade. Here, using Hek293 and NIH 3T3 cells, we show that cAMP's inhibition of the MAP kinase cascade is mediated by the small G protein Rap1. Activation of Rap1 by cAMP induces the association of Rap1 with Raf-1 and limits Ras-dependent activation of ERK. In NIH 3T3 cells, Rap1 is required not only for cAMP's inhibition of ERK activation but for inhibition of cell proliferation and mitogenesis as well.

Tyrosine kinases in activation of the MAP kinase cascade by G-protein-coupled receptors

Nature ◽  
1996 ◽  
Vol 380 (6574) ◽  
pp. 541-544 ◽  
Author(s):  
Yong Wan ◽  
Tomohiro Kurosaki ◽  
Xin-Yun Huang
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Tyrosine Kinases ◽  
G Protein Coupled Receptors ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
G Protein Coupled

scholarly journals The STE12α Homolog Is Required for Haploid Filamentation But Largely Dispensable for Mating and Virulence in Cryptococcus neoformans

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1601-1615 ◽  
Author(s):  
Changli Yue ◽  
Lora M Cavallo ◽  
J Andrew Alspaugh ◽  
Ping Wang ◽  
Gary M Cox ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cryptococcus Neoformans ◽  
Mating Type ◽  
Sexual Cycle ◽  
Type Locus ◽  
Map Kinase Cascade ◽  
Mutant Strains ◽  
Genetic Epistasis ◽  
Kinase Cascade ◽  
Immunocompromised Hosts

Abstract Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised hosts. The organism has a known sexual cycle, and strains of the MATα mating type are more virulent than isogenic MATa strains in mice, and they are more common in the environment and infected hosts. A C. neoformans homolog of the STE12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae and Candida albicans was identified previously, found to be encoded by a novel region of the MATα mating type locus, and shown to enhance filamentous growth when overexpressed. We have disrupted the C. neoformans STE12 gene in a pathogenic serotype A isolate. ste12 mutant strains exhibit a severe defect in filamentation and sporulation (haploid fruiting) in response to nitrogen starvation. In contrast, ste12 mutant strains have only modest mating defects and are fully virulent in two animal models compared to the STE12 wild-type strain. In genetic epistasis experiments, STE12 functions in a MAP kinase cascade to regulate fruiting, but not mating. Thus, the C. neoformans STE12α transcription factor homolog plays a specialized function in haploid fruiting, but it is dispensable or redundant for mating and virulence. The association of the MATα locus with virulence may involve additional genes, and other transcription factors that regulate mating and virulence remain to be identified.

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