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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Molecular biology reports 26 (1999), S. 53-57 
    ISSN: 1573-4978
    Keywords: Cdc25B ; cell cycle ; cyclin-dependent kinase ; phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The CDC25 dual specificity phosphatase is a universal cell cycle regulator. The evolutionary conservation of this enzyme from yeast to man bears witness to its major role in the control of cyclin-dependent kinases (CDK) activity that are central regulators of the cell cycle machinery. CDC25 phosphatase both dephosphorylates and activates CDKs. Three human CDC25s have been identified. CDC25A is involved in the control of G1/S, and CDC25C at G2/M throught the activation of CDK1-cyclin B. The exact function of CDC25B however remains elusive. We have found that CDC25B is degraded by the proteasome pathway in vitro and in vivo. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex, but not by CDK1-cyclin B. Together with the observations of others made in yeast and mammals, our results suggest that CDC25B might act as a ‘mitotic starter’ triggering the activation of an auto-amplification loop before being degraded.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; cell cycle ; Cdc2 kinase ; GST ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione-Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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