Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11.
The mating-factor response pathway of Saccharomyces cerevisiae employs a set of protein kinase similar to kinases that function in signal transduction pathways of metazoans. We have purified the yeast protein kinases encoded by STE11, STE7, and FUS3 as fusions to glutathione S-transferase (GST) and reconstituted a kinase cascade in which STE11 phosphorylates and activates STE7, which in turn phosphorylates the mitogen-activated protein kinase FUS3. GST-STE11 is active even when purified from cells that have not been treated with alpha-factor. This observation raises the possibility that STE11 activity is governed by an inhibitor which is regulated by pheromone. We also identify a STE11-dependent phosphorylation site in STE7 which is required for activity of STE7. Conservation of this site in the mammalian STE7 homologue MEK and other STE7 relatives suggests that this may be a regulatory phosphorylation site in all MAP kinase kinases.
Neiman
A M
AM
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Herskowitz
I
I
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014157:Transcription Factors
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; C071596:ste11 protein, S pombe; D011494:Protein Kinases; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae; D020929:Mitogen-Activated Protein Kinase Kinases