Substrate recognition and ubiquitination of SCFSkp2/Cks1 ubiquitin-protein isopeptide ligase.
p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.
Xu
Shuichan
S
Department of Biochemistry and Biomarker Development, Signal Pharmaceuticals, LLC, San Diego, California 92121, USA. sxu@celgene.com
Abbasian
Mahan
M
Patel
Palka
P
Jensen-Pergakes
Kristen
K
Lombardo
Christian R
CR
Cathers
Brian E
BE
Xie
Weilin
W
Mercurio
Frank
F
Pagano
Michele
M
Giegel
David
D
Cox
Sarah
S
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D019925:Cyclin A / Q000737:chemistry / Q000378:metabolism; D019927:Cyclin E / Q000737:chemistry / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000737:chemistry / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016196:S Phase / Q000502:physiology; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism
C093090:CKS1B protein, human; D002352:Carrier Proteins; D019925:Cyclin A; D019927:Cyclin E; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D044767:Ubiquitin-Protein Ligases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases