Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae.
The Ste20p protein kinase was immunopurified from yeast cells and analyzed in an in vitro assay system. Ste20p immune complexes exhibited autophosphorylating activity at serine and threonine residues and specifically phosphorylated a bacterially expressed glutathione S-transferase (GST) fusion of Ste11p (a mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase homologue) at serine and threonine residues. In contrast, GST fusions either of Ste7p (a MEK homologue) or the beta-subunit of the mating response G-protein and immunoprecipitated Ste5p were not phosphorylated by the Ste20p immune complexes. Myelin basic protein was identified as an excellent in vitro substrate, whereas histone H1 was only poorly phosphorylated. Evidence was obtained that autophosphorylation might play a regulatory role for the in vitro kinase activity. The in vitro activity was found to be Ca(2+)-independent. Both the in vivo and in vitro activities were abolished by mutational changes of either the conserved lysine residue 649 within the ATP binding site or threonine 777 between the catalytic subdomains VII and VIII. Wild-type Ste20p and the catalytically inactive T777A mutant were identified as phosphoproteins in vivo. The phosphorylation occurred at serine and threonine residues independent of pheromone stimulation. Based on the genetically determined significance of Ste20p in pheromone signal transduction and on our in vitro studies, we propose the model that Ste20p represents a yeast MEK kinase kinase whose function is to link G-protein-coupled receptors through G beta gamma to a mitogen-activated protein kinase module.
Wu
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Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Whiteway
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Thomas
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Leberer
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002846:Chromatography, Affinity; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D008954:Models, Biological; D008969:Molecular Sequence Data; D010675:Pheromones / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010675:Pheromones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C071596:ste11 protein, S pombe; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; D020929:Mitogen-Activated Protein Kinase Kinases