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 anal yzed in an in vitro assay system, Ste20p immune complexes exhibited au tophosphorylating activity at serine and threonine residues and specif ically phosphorylated a bacterially expressed glutathione S-transferas e (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 ho mologue) or the beta-subunit of the mating response G-protein and immu noprecipitated Ste5p were not phosphorylated by the Ste20p immune comp lexes. Myelin basic protein was identified as an excellent in. vitro s ubstrate, 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 C a2+-independent. Both the in vivo and in vitro activities were abolish ed by mutational changes of either the conserved lysine residue 649 wi thin the ATP binding site or threonine 777 between the catalytic subdo mains VII and VIII. Wild-type Ste20p and the catalytically inactive T7 77A mutant were identified as phosphoproteins in vivo. The phosphoryla tion occurred at serine and threonine residues independent of pheromon e stimulation. Based on the genetically determined significance of Ste 20p in pheromone signal transduction and on our in vitro studies, we p ropose the model that Ste20p represents a yeast MEK kinase kinase whos e function is to link G-protein-coupled receptors through G(beta gamma ) to a mitogen-activated protein kinase module.