Phosphorylation controls timing of Cdc6p destruction: A biochemical analysis.
The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell cycle. Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed. First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase. However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p. SCF-Cdc4p- and N-terminal phosphorylation site-dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle. The stable cdc6 mutants show no apparent replication defects in wild-type strains. However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex.
Elsasser
S
S
Braun Laboratories, California Institute of Technology, Pasadena, California 91125, USA.
Chi
Y
Y
Yang
P
P
Campbell
J L
JL
D064173:Anaphase-Promoting Complex-Cyclosome; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D044783:F-Box Proteins; D015966:Gene Expression Regulation, Fungal; D007399:Interphase; D008025:Ligases / Q000378:metabolism; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D010453:Peptide Synthases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
C080443:CDC4 protein, S cerevisiae; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044783:F-Box Proteins; D019913:Nuclear Localization Signals; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D017489:CDC28 Protein Kinase, S cerevisiae; D008025:Ligases; D010453:Peptide Synthases