Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast.
Cyclin-dependent kinase inhibitors (CKIs) play key roles in controlling the eukaryotic cell cycle by coordinating cell proliferation and differentiation. Understanding the roles of CKIs requires knowledge of how they are regulated both through the cell cycle and in response to extracellular signals. Here we show that the yeast CKI, Far1p, is controlled by ubiquitin-dependent proteolysis. Wild-type Far1p was stable only in the G1 phase of the cell cycle. Biochemical and genetic evidence indicate that its degradation required the components of the G1-S ubiquitination system, Cdc34p, Cdc4p, Cdc53p, and Skp1p. We isolated a mutant form of Far1p (Far1p-22) that was able to induce cell cycle arrest in the absence of alpha-factor. Cells that overexpress Far1-22p arrested in G1 as large unbudded cells with low Cdc28p-Clnp kinase activity. Wild-type Far1p, but not Far1-22p, was readily ubiquitinated in vitro in a CDC34- and CDC4-dependent manner. Far1-22p harbors a single amino acid change, from serine to proline at residue 87, which alters phosphorylation by Cdc28p-Cln2p in vitro. Our results show that Far1p is regulated by ubiquitin-mediated proteolysis and suggest that phosphorylation of Far1p by the Cdc28p-Clnp kinase is part of the recognition signal for ubiquitination.
Henchoz
S
S
Institut Suisse de Rechereches Expérimentales sur le Cancer (ISREC), 1066 Epalinges/VD Switzerland.
Chi
Y
Y
Catarin
B
B
Herskowitz
I
I
Deshaies
R J
RJ
Peter
M
M
D064173:Anaphase-Promoting Complex-Cyclosome; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism; D000072235:Mating Factor; D009097:Multienzyme Complexes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D044783:F-Box Proteins; C081353:FAR1 protein, S cerevisiae; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D010768:Phosphoserine; D000072235:Mating Factor; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D017489:CDC28 Protein Kinase, S cerevisiae; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases