Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase

• Published in: Science (American Association for the Advancement of Science) Vol. 278; no. 5337; pp. 455 - 460
• Main Authors: VERMA, R, ANNAN, R. S, HUDDLESTON, M. J, CARR, S. A, REYNARD, G, DESHAIES, R. J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for the Advancement of Science 17.10.1997; The American Association for the Advancement of Science
• Subjects: Amino Acid Sequence; Anaphase-Promoting Complex-Cyclosome; Biological and medical sciences; Classical genetics, quantitative genetics, hybrids; Cyclin G; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinases - metabolism; Cyclins - metabolism; Deoxyribonucleic acid; DNA; DNA Replication; Enzyme Inhibitors - metabolism; Fractions; Fundamental and applied biological sciences. Psychology; Fungal Proteins - metabolism; G1 Phase; Genetics of eukaryotes. Biological and molecular evolution; Ligases - metabolism; Mass spectrometry; Molecular biology; Molecular Sequence Data; Mutagenesis; Phenotype; Phosphopeptides - metabolism; Phosphorylation; Recombinant Fusion Proteins - metabolism; S Phase; Saccharomyces cerevisiae Proteins; Scientific imaging; Thallophyta, bryophyta; Ubiquitin-Protein Ligase Complexes; Ubiquitin-Protein Ligases; Ubiquitins - metabolism; Vegetals; Yeast; Yeasts - cytology; Yeasts - metabolism; Phosphorylation; Yeast; Enzyme; Transferases; Enzyme inhibitor; Fungi; Posttranslational modification; G1 Phase; Protein kinase; Proteolysis; S Phase; Cell cycle; Ascomycetes; Mutation; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.278.5337.455

G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast. A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo. A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication. Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction. These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast.