Skp2 contains a novel cyclin A binding domain that directly protects cyclin A from inhibition by p27Kip1.
Skp2 is well known as the F-box protein of the SCF(Skp2) x Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpression, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A interaction is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins. Furthermore, a short peptide derived from the mapped cyclin A binding sequences of Skp2 can block Skp2-cyclin A interaction but not p27-cyclin A interaction, whereas a previously identified RXL peptide can block p27-cyclin A interaction but not Skp2-cyclin A interaction. Functionally, Skp2-cyclin A interaction is separable from Skp2 ability to mediate p27 ubiquitylation. Rather, Skp2-cyclin A interaction serves to directly protect cyclin A-Cdk2 from inhibition by p27 through competitive binding. Finally, we show that disruption of cyclin A binding with point mutations in the cyclin A binding domain of Skp2 compromises the ability of overexpressed Skp2 to counter cell cycle arrest by a p53/p21-mediated cell cycle checkpoint without affecting its ability to cause degradation of cellular p27 and p21. These findings reveal a new functional mechanism of Skp2 and a new regulatory mechanism of cyclin A.
Ji
Peng
P
Department of Developmental and Molecular Biology, the Albert Einstein Comprehensive Cancer Center and Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Goldin
Luba
L
Ren
Hao
H
Sun
Daqian
D
Guardavaccaro
Daniele
D
Pagano
Michele
M
Zhu
Liang
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D049109:Cell Proliferation; D019925:Cyclin A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology; D010455:Peptides / Q000502:physiology; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044786:S-Phase Kinase-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism
C436291:CDKN1B protein, human; D019925:Cyclin A; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; D044786:S-Phase Kinase-Associated Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D051357:Cyclin-Dependent Kinase 2