Loss of sustained Fus3p kinase activity and the G1 arrest response in cells expressing an inappropriate pheromone receptor.
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.
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Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York 10029, USA.
Hirsch
J P
JP
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D017931:DNA Primers / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020962:Heterotrimeric GTP-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D000072235:Mating Factor; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000502:physiology*; D043563:Receptors, Pheromone; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D014157:Transcription Factors; D014158:Transcription, Genetic
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D017931:DNA Primers; C081353:FAR1 protein, S cerevisiae; C490024:FUS1 protein, S cerevisiae; D005656:Fungal Proteins; D044387:GTP-Binding Protein beta Subunits; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D043563:Receptors, Pheromone; D012097:Repressor Proteins; C081863:STE3 protein, S cerevisiae; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D014157:Transcription Factors; D000072235:Mating Factor; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins