Noncatalytic requirement for cyclin A-cdk2 in p27 turnover.
Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27(kip1) is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G(1) cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF(skp2) complex.
Zhu
Xin-Hua
XH
Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Nguyen
Hoang
H
Halicka
H Dorota
HD
Traganos
Frank
F
Koff
Andrew
A
D042846:CDC2-CDC28 Kinases / Q000032:analysis / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D019925:Cyclin A / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D019927:Cyclin E / Q000032:analysis; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005434:Flow Cytometry; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2