RECONSTITUTION OF A YEAST PROTEIN-KINASE CASCADE IN-VITRO - ACTIVATION OF THE YEAST MEK HOMOLOG STE7 BY STE11

The mating-factor response pathway of Saccharomyces cerevisiae employs a set of protein kinases similar to kinases that function in signal t ransduction pathways of metazoans. We have purified the yeast protein kinases encoded by STE11, STE7, and FUS3 as fusions to glutathione S-t ransferase (GST) and reconstituted a kinase cascade in which STE11 pho sphorylates and activates STE7, which in turn phosphorylates the mitog en-activated protein kinase FUS3. GST-STE11 is active even when purifi ed from cells that have not been treated with alpha-factor. This obser vation raises the possibility that STE11 activity is governed by an in hibitor which is regulated by pheromone. We also identify a STE11-depe ndent phosphorylation site in STE7 which is required for activity of S TE7. Conservation of this site in the mammalian STE7 homologue MEK and other STE7 relatives suggests that this may be a regulatory phosphory lation site in all MAP kinase kinases.