Related Experiment Videos
A Cdc28 mutant uncouples G1 cyclin phosphorylation and ubiquitination from G1 cyclin proteolysis
1Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, F-91191 Gif-sur-Yvette, Cedex, France.
Abstract:
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.
Insights
A genetic screen identified a cak1 mutation that stabilizes yeast G1 cyclins by causing hypophosphorylation. Unexpectedly, a Cdc28 mutant restored phosphorylation but still stabilized these cyclins, revealing complex proteasome degradation pathways.
Area of Science:
- Cellular biology
- Molecular genetics
- Biochemistry
Background:
- Yeast G1 cyclins (Cln1, Cln2) are phosphorylated by Cdc28, ubiquitinated by SCF-Grr1, and degraded by the 26S proteasome.
- This degradation process is crucial for cell cycle progression.
Purpose of the Study:
- To identify genetic factors regulating yeast G1 cyclin stability.
- To elucidate the mechanisms underlying cyclin degradation.
Main Methods:
- Genetic screening to identify mutants stabilizing G1 cyclins.
- Analysis of cyclin phosphorylation, binding to Cdc28, and ubiquitination in mutant strains.
- Utilizing a Cak1-independent Cdc28 mutant (Cdc28-43244) to dissect degradation pathways.
Main Results:
- A cak1 allele was identified, leading to hypophosphorylated and stabilized Cln2HA in cak1 mutants.
- Expression of Cdc28-43244 in cak1 mutants restored Cln2HA phosphorylation but resulted in stabilization of phospho-Cln2HA.
- Polyubiquitinated Cln2HA bound to Cdc28-43244 was recognized by SCF-Grr1, yet degradation was impaired.
Conclusions:
- Cak1 kinase activity is essential for proper G1 cyclin phosphorylation and subsequent degradation.
- The Cdc28-43244 mutant reveals a novel regulatory step or complexity in the proteasomal degradation of ubiquitinated G1 cyclins, independent of initial phosphorylation status.