Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p

In budding yeast cells, the cytoskeletal polarization and depolarization ev ents that shape the bud are triggered at specific times during the cell cyc le by the cyclin-dependent kinase Cdc28p, Polarity establishment also requi res the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechan ism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here w e show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p, However, the role of Cdc28p is indirect, and the phos phorylation appears to be catalyzed by the pal-activated kinase family memb er Cla4p and also depends on Cdc42p and the scaffold protein Bem1p, Express ion of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange; stimula ted Cdc24p phosphorylation independent of cell cycle cues, raising the poss ibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can m ediate complex formation between these proteins in vitro. We suggest that B em1p acts to concentrate polarity establishment proteins at a discrete site , facilitating polarization and promoting Cdc24p phosphorylation at specifi c times during the cell cycle.