Pheromone-Dependent G1 Cell Cycle Arrest Requires Far1 Phosphorylation, but May Not Involve Inhibition of Cdc28-Cln2 Kinase, In Vivo

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.