Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo

In yeast, the pheromone or-factor acts as an antiproliferative fatter that induces G(1) 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 peen thought to enhance the binding of Farl to G(1)-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 G(1) 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 Farl 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 G(1) 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-associatea: Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase.assays, suggesting unconventional inhibitory mechanisms of Far1.