Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo.
In yeast, the pheromone alpha-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.
Gartner
A
A
Institute for Biochemistry and Molecular Cell Biology and Ludwig Boltzmann Forschungsstelle, University of Vienna, Vienna, Austria. gartner@cshl.org
Jovanović
A
A
Jeoung
D I
DI
Bourlat
S
S
Cross
F R
FR
Ammerer
G
G
D000818:Animals; D001665:Binding Sites; D017489:CDC28 Protein Kinase, S cerevisiae / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002455:Cell Division; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D016193:G1 Phase; D016259:Genes, myc; D006131:Growth Inhibitors / Q000494:pharmacology*; D006639:Histidine; D008074:Lipoproteins / Q000494:pharmacology*; D051379:Mice; D016296:Mutagenesis; D010675:Pheromones / Q000494:pharmacology*; D010766:Phosphorylation; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D000091346:Tripeptidyl-Peptidase 1
C082721:CLN2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; C081353:FAR1 protein, S cerevisiae; D005656:Fungal Proteins; D006131:Growth Inhibitors; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D010675:Pheromones; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C000717268:Tpp1 protein, mouse; D000091346:Tripeptidyl-Peptidase 1; D013912:Threonine; D012694:Serine; D006639:Histidine; D017489:CDC28 Protein Kinase, S cerevisiae