ABSTRACT
In yeast, the pheromone α-factor acts as an antiproliferative factor that induces G1 arrest and cellular differentiation. Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation. Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G1-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G1 phase. To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs. Two putative phosphorylation sites that strongly affect Far1 behavior were identified. A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone. In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G1 arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex. Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1.
ACKNOWLEDGMENTS
We are grateful to members of the Nasmyth and Ammerer labs for helpful discussions. Martin Tögel’s help was instrumental for culturing hybridoma cells. We thank Knud Naiz and Alexander Schleiffer for comments. We are especially grateful to Brandt Schneider and Bruce Futcher for advice and for instrumental help in writing the manuscript and to the members of the CSHL graphics facility for helping with the artwork.
We thank Michael Hengartner for paying for the artwork. This study was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung, by the Austrian Jubiläumsfonds der Nationalbank (grants 5760 and 6431), by TMR Network grant ERB 4061 PL95-0662, and by NIH grant GM 49716 to F.C.