Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1.
Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.
Adkins
J N
JN
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
Lumb
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002138:Calibration; D002460:Cell Line; D019925:Cyclin A / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016213:Cyclins / Q000737:chemistry / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera / Q000235:genetics; D019311:Staurosporine / Q000494:pharmacology
C493956:CDKN1A protein, human; D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine