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

Science

Volumn 278, Issue 5337, 1997, Pages 455-460

  Verma, R ^a   Annan, R S ^b   Huddleston, M J ^b   Carr, S A ^b   Reynard, G ^a   Deshaies, R J ^a  

^a CALIFORNIA INSTITUTE OF TECHNOLOGY   (United States)

^b GLAXOSMITHKLINE   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIN DEPENDENT KINASE INHIBITOR; ANAPHASE PROMOTING COMPLEX; ANAPHASE-PROMOTING COMPLEX; CYCLIN DEPENDENT KINASE; CYCLIN G; CYCLINE; ENZYME INHIBITOR; FUNGAL PROTEIN; HYBRID PROTEIN; LIGASE; PHOSPHOPEPTIDE; SACCHAROMYCES CEREVISIAE PROTEIN; SIC1 PROTEIN, S CEREVISIAE; UBIQUITIN; UBIQUITIN PROTEIN LIGASE;

ARTICLE; CELL CYCLE G1 PHASE; CELL CYCLE S PHASE; DNA SYNTHESIS; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN PHOSPHORYLATION; YEAST; AMINO ACID SEQUENCE; CYTOLOGY; DNA REPLICATION; METABOLISM; MOLECULAR GENETICS; MUTAGENESIS; PHENOTYPE; PHOSPHORYLATION;

AMINO ACID SEQUENCE; CYCLIN-DEPENDENT KINASES; CYCLINS; DNA REPLICATION; ENZYME INHIBITORS; FUNGAL PROTEINS; G1 PHASE; LIGASES; MOLECULAR SEQUENCE DATA; MUTAGENESIS; PHENOTYPE; PHOSPHOPEPTIDES; PHOSPHORYLATION; RECOMBINANT FUSION PROTEINS; S PHASE; SACCHAROMYCES CEREVISIAE PROTEINS; UBIQUITIN-PROTEIN LIGASE COMPLEXES; UBIQUITIN-PROTEIN LIGASES; UBIQUITINS; YEASTS;

EID: 0030612012     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5337.455     Document Type: Article

References (39)

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32. 3-; and so on] (13). When possible, a third dimension of analysis was carried out in the positive-ion mode, in which the complete product-ion mass spectrum was collected, thereby yielding sequence data from the phosphopeptide.
33. mycHis6 was eluted with 0.1% TFA, and the eluted protein was lyophilized in a Speedvac and reconstituted in 50 mM tris (pH 8.8) and 2 M urea.
34. 35S-labeled E. coli cells as described (26).
35. 32P-labeled cells were harvested, vortexed with glass beads (0.5 mm), and then boiled in 2× lysis buffer containing 100 mM tris (pH 7.5), 2% SDS, 200 mM NaCl, 30 mM DTT, and a protease and phosphatase inhibitor cocktail (16). Cell lysates were diluted to a final concentration of 0.2% SDS with an immunoprecipitation buffer containing 50 mM tris (pH 7.5), 500 mM NaCl, 1% Triton X-100, and the phosphatase and protease inhibitor cocktails described above. Diluted lysates were centifuged at 15,000g for 15 min and supplemented with antiserum to Sic1p and protein A beads. Beads were washed three times with immunoprecipitation buffer and twice with 50 mM tris (pH 7.5) and were resuspended in 2× SDS sample buffer.
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39. We thank M. V. Castillo for MBP-SIC1, L. Johnston for Sic1p antiserum, S. Diamond for performing flow cytometry, J. Archer, B. Dunphy, and W. Shou for critically reading the manuscript, and members of the laboratory for helpful discussions. Supported in part by Searle/Chicago Community Trust and Lucille P. Markey Charitable Trust Scholar Awards to R.J.D. and by a grant from NIH (NIH RO1 GM52466-01).