Abstract

The Cek1 and Cek2 MAP kinases have been implicated in mating in C. albicans. I investigated the relationships of these MAP kinases and a putative MAP kinase phosphatase Cpp1 in the mating process of this fungal pathogen. Consistent with previous observations, mating type homozygous opaque cells that lack both kinases are sterile. However, several lines of evidence show that the two kinases do not simply provide redundant functions in the mating process. Loss of CEK1 reduces mating significantly, to about 0.3% of wild type strains, and also reduces shmoo formation and pheromone-mediated gene expression. By contrast, loss of CEK2 function has a relatively minor effect on mating, reducing it to approximately one third that of the wild type strain. Intriguingly, CEK2 loss enhances pheromone responsiveness. It increases shmoo formation, pheromone-mediated cell cycle arrest halos, and pheromone-mediated gene expression. The behavior of the cek2 mutants mimics that of cpp1 mutants defective in the putative MAP kinase phosphatase; cpp1 mutants are also hyper responsive to pheromone, generating large halos, high levels of shmoos, and increased pheromone responsive gene expression. Surprisingly, cpp1 mutants are more mating defective than cek2 mutants. The double cek2 cpp1 mutant shows enhanced responsiveness over either single mutant. Analysis of protein phosphorylation shows that Cek1 undergoes pheromone-mediated phosphorylation of the activation loop, and this phosphorylation is enhanced in cells lacking either the Cpp1 phosphatase or the Cek2 kinase. Analysis of GFP localization of Cek1-GFP shows enhanced nuclear localization in response to pheromone treatment, consistent with Cek1 playing a central role in pheromone response. Overall, these results show a complex interaction among the MAP kinases and MAP kinase phosphatase that function in the C. albicans mating pathway.