PHEROMONE-DEPENDENT G(1) 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 h ave indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulat ion. Phosphorylation by the pheromone-stimulated mitogen-activated pro tein (MAP) kinase Fus3 has peen thought to enhance the binding of Farl to G(1)-specific cyclin-dependent kinase (Cdk) complexes, thereby inh ibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside earl y 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 st rongly 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 t o be an important recipient of an activating modification, as substitu tions at this position abolish the G(1) arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the.T306-to-A substitut ion product, can be found in stable association with the Cdc28-Cln2 co mplex. Surprisingly, Far1-associatea: Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase.assays, suggesting unconventional inhibitory mechanisms of Far1.