Abstract
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.
Highlights
Three different isoform cdc25 genes have been cloned in mammalian or human cells [14, 15]
We showed that the active site residues residing in the putative phosphate binding loop (P loop) of human Cdc25A and -B proteins play important roles in the catalysis and protein interactions between Cdc25 proteins and various cyclin-Cdk complexes
To test the phosphatase activity of the mutant Cdc25B proteins on cyclin-Cdk complex, the in vivo substrate of Cdc25 proteins, cyclin A-cdc2, or cyclin B-Cdc2 protein kinase complexes was purified from HeLa lysate by immunoprecipiatation with anti-cyclin A and anti-cyclin B antibodies
Summary
Three different isoform cdc genes have been cloned in mammalian or human cells [14, 15]. Crystal structures of human protein-tyrosine phosphatase 1B [28, 31] and Yersinia protein-tyrosine phosphatase [33] have suggested that the conserved active site motif that contains the catalytic cysteine forms a phosphate binding loop (P loop) in a conformation that is similar to each other. Most recently, based on crystal structure of Yersinia protein-tyrosine phosphatase [33] and mutagenesis studies of vaccinia H1-related dual specific phosphatases [32], a highly conserved aspartic acid residue was proposed to act as a general acid to protonate the leaving group in the step of the formation of the enzyme-phosphate intermediate. We showed that the active site residues residing in the putative P loop of human Cdc25A and -B proteins play important roles in the catalysis and protein interactions between Cdc proteins and various cyclin-Cdk complexes. We revealed the in vitro substrate specificity of Cdc25A and -B regarding different cyclin-Cdk complexes
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