MAP Kinase Regulation of theCandida albicansPheromone Pathway

ABSTRACTWe investigated the relationships of the Cek1 and Cek2 mitogen-activated protein (MAP) kinases and the putative MAP kinase phosphatase Cpp1 in the mating process ofCandida albicans. Mutants of theCPP1gene are hyperresponsive to pheromone, generating large halos, high levels of projections, and an increase in pheromone-responsive gene expression. Mating-type-homozygous opaque cells that lack both kinases are sterile, consistent with previous observations, although several lines of evidence show that the two kinases do not simply provide redundant functions in the mating process. Loss ofCEK1reduces mating significantly, to about 0.3% of wild-type strains, and also reduces projection formation and pheromone-mediated gene expression. In contrast, loss ofCEK2has less of an effect, reducing mating to approximately one-third that of the wild-type strain and moderately reducing projection formation but having little influence on the induction of gene expression. However, loss of Cek2 function reduces adaptation to pheromone-mediated arrest. The mutation enhances pheromone response halos to a level similar to that ofcpp1mutants, although thecpp1mutants are considerably more mating defective than thecek2mutant. The doublecek2 cpp1mutant shows enhanced responsiveness relative to either single mutant in terms of gene expression and halo formation, suggesting the kinase and phosphatase roles in the adaptation process are independent. 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. In addition, Cek1-GFP shows enhanced nuclear localization in response to pheromone treatment. In contrast, Cek2 shows no evidence for pheromone-mediated phosphorylation or pheromone-mediated nuclear localization. Intriguingly, however, deletion ofCPP1enhances both the phosphorylation state and the nuclear localization of Cek2-GFP. Overall, these results identify a complex interaction among the MAP kinases and MAP kinase phosphatase that function in theC. albicansmating pathway.IMPORTANCEMAP kinases and their regulators are critical components of eukaryotic signaling pathways implicated in normal cell behavior as well as abnormal behaviors linked to diseases such as cancer. The mating pathway of the yeastSaccharomycescerevisiaewas central in establishing the MAP kinase paradigm. Here we investigate the mating pathway in a different ascomycete, the fungal pathogenC. albicans. In this dimorphic fungus MAP kinases are also implicated in the mating response, with two MAP kinases apparently playing redundant roles in the mating process. This work establishes that while some level of mating can occur in the presence of a single kinase, the Cek1 kinase is most important for mating, while the Cek2 kinase is involved in adaptation to signaling. While both kinases appear to be themselves regulated by dephosphorylation through the action of the Cpp1 phosphatase, this process appears important for mating only in the case of Cek1.