Ste5 is a Zn2+ finger-like protein thought to function before three kinases, Ste11 (a MEKK), Ste7 (a MEK), and Fus3 (a MAPK), in a conserved MAP kinase cascade required for mating in S. cerevisiae. Here, we present evidence that Ste5 forms a multikinase complex that joins these kinases for efficient Fus3 activation. By two-hybrid analysis, Ste11, Ste7, and Fus3 associate with different domains of Ste5, while Kss1, another MAPK, associates with the same domain as Fus3, thus implying that Ste5 simultaneously binds a MEKK, MEK, and MAPK. Ste5 copurifies with Ste11, Fus3, and a hypophosphorylated form of Ste7, and all four proteins cosediment in a glycerol gradient as if in a large complex. Ste5 also increases the amount of Ste11 complexed to Ste7 and Fus3 and is required for Ste11 to function. These results substantiate a novel signal transduction component that physically links multiple kinases within a single cascade.
Bibliographical noteFunding Information:
Correspondence should be addressed to E. A. E. We thank B. Errede, E. Leberer, and P. Bartel for plasmids and strains, B. Braell for discussion and advice with the glycerol gradients, M. Wigler and S. Marcus, and J. Printen and G. Sprague for communicating results prior to publication, and M. Cobb for helpful discussions. This work was supported by the National Institutes of Health (grant ROl-GM46962-01) and an American Cancer Society Junior Faculty Award to E. A. E.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)