Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.
Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.
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Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202, USA.
Cook
J G
JG
Thorner
J
J
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology; D005656:Fungal Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D016193:G1 Phase; D020930:MAP Kinase Kinase Kinases; D000072235:Mating Factor; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D005656:Fungal Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D014443:Tyrosine; D000072235:Mating Factor; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C075580:FUS3 protein, S cerevisiae; C541984:KSS1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae; D020929:Mitogen-Activated Protein Kinase Kinases