Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.
The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.
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Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Amin
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Gibbs
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Niu
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Hurwitz
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D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism* / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051716:Replication Protein A
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases