Reconstitution of cyclin-dependent cdc2 and cdk2 kinase activities in vitro.
The genes that encode human cdc2 and cdk2 proteins are essential for cell cycle progression. In this report, we describe the purification of cyclin-associated cdc2 and cdk2 kinases as well as cyclin-free cdc2 and cdk2 protein preparations from HeLa cells. The cdc2-cyclin B kinase complex that we have isolated, consisting of two polypeptides of p60 (cyclin B) and p34 (cdc2), phosphorylated both the p34 and p70 subunits of the three-subunit human single-stranded DNA-binding protein (also called RP-A), a DNA replication and repair factor. We also partially purified a histone H1 kinase activity that is associated with the cdk2 and cyclin A proteins. Purified human cyclins A and B1, overproduced in bacteria, complemented a cellular fraction enriched in cdc2 and cdk2 proteins to reconstitute histone H1 kinase activity. Using this complementation system, human cdc2 and cdk2 proteins were purified and separated from one another. Glycerol gradient analyses demonstrated that the purified cdk2 (p33) protein co-sedimented with a cyclin A-dependent H1 kinase activity. Thus, cdk2 and cyclin A proteins are components that assemble to yield a kinase complex that catalyzes the phosphorylation of histone H1.
Pan
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Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Hurwitz
J
J
D016203:CDC2 Protein Kinase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D042846:CDC2-CDC28 Kinases; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D006367:HeLa Cells; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D006656:Protamine Kinase / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases
D016213:Cyclins; D006657:Histones; D011494:Protein Kinases; D006656:Protamine Kinase; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases