FUNCTIONAL-ANALYSIS OF THE INTERACTION BETWEEN THE SMALL GTP-BINDING PROTEIN CDC42 AND THE STE20 PROTEIN-KINASE IN YEAST

STE20 encodes a protein kinase related to mammalian p65(Pak) which fun ctions in several signal transduction pathways in yeast, including tho se involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kina se with significant homology to Ste20. It is not clear how the activit y of Ste20 is regulated in response to these different signals in vivo , but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manne r in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective fo r pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kina se activity of such Ste20 mutants was normal when assayed iir vitro. F urthermore, these alleles were able to fully activate the MAP kinase p athway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste2 0 protein was visualized as a crescent at emerging buds during vegetat ive growth and at shmoo tips in cells arrested with a-factor. In contr ast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely t hroughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.