Roles of active site residues and the NH2-terminal domain in the catalysis and substrate binding of human Cdc25.
Human Cdc25 proteins are dual specific protein phosphatases that play important roles in cell cycle regulation. In this study, the catalytic mechanism and substrate binding specificity of human Cdc25A and -B proteins were investigated by site-directed and deletion mutagenesis methods. Mutations of the cysteine or the arginine residues in the active site motif abolished the Cdc25 phosphatase activity. However, the cysteine mutation in both Cdc25A and -B created enzymes that still retain the ability to bind their substrates. This allowed us to test the ability of Cdc25A and -B to bind various cyclin-Cdk complexes in vitro. While Cdc25A Cys --> Ser could interact with cyclin A-Cdk2, cyclin B-Cdc2, and cyclin E-Cdk2 strongly, Cdc25B mutant was only found to bind to cyclin A-Cdk2 at significant levels. We also identified Arg452 and Ser449 as two crucial residues that could be directly involved in the molecular interactions between Cdc25 and cyclin-Cdk proteins. Deletion mutagenesis data also indicate that the phosphatase catalytic domains of Cdc25A and -B proteins are located within their carboxyl terminus.
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Mitotix Inc., Cambridge, Massachusetts 02135, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D020687:cdc25 Phosphatases
D018797:Cell Cycle Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases; D013917:Thrombin