Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.
The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.
Xu
Kui
K
Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.
Belunis
Charles
C
Chu
Wei
W
Weber
David
D
Podlaski
Frank
F
Huang
Kuo-Sen
KS
Reed
Steven I
SI
Vassilev
Lyubomir T
LT
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D044767:Ubiquitin-Protein Ligases; D018844:Cyclin-Dependent Kinases; C439298:p9(Cks1) protein, human; D008025:Ligases