A Cdc28 mutant uncouples G1 cyclin phosphorylation and ubiquitination from G1 cyclin proteolysis.
Proteolysis of the yeast G(1) cyclins is triggered by their Cdc28-dependent phosphorylation. Phosphorylated Cln1 and Cln2 are ubiquitinated by the SCF-Grr1 complex and then degraded by the 26 S proteasome. In this study, we identified a cak1 allele in a genetic screen for mutants that stabilize the yeast G(1) cyclins. Further characterization showed that Cln2HA was hypophosphorylated, unable to bind Cdc28, and stabilized in cak1 mutants at the restrictive temperature. Hypophosphorylation of Cln2HA could thus explain its stabilization. To test this possibility, we expressed a Cak1-independent mutant of Cdc28 (Cdc28-43244) in cak1 mutants and found that Cln2HA phosphorylation was restored, but surprisingly, the phospho-Cln2HA was stabilized. When bound to Cdc28-43244, Cln2HA was recognized and polyubiquitinated by SCF-Grr1. The Cdc28-43244 mutant thus reveals an unexpected complexity in the degradation of polyubiquitinated Cln2HA by the proteasome.
Ceccarelli
E
E
Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, F-91191 Gif-sur-Yvette, Cedex, France.
Mann
C
C
D048868:Adaptor Proteins, Signal Transducing; D017489:CDC28 Protein Kinase, S cerevisiae / Q000502:physiology*; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056746:Cyclin G; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism*; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D048868:Adaptor Proteins, Signal Transducing; C081185:CKS1 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056746:Cyclin G; D016213:Cyclins; D044783:F-Box Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C071032:GRR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase