Ubiquitination of p27Kip1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2.
p27Kip1 is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G1/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCF(Skp2), and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCF(Skp2). Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCF(Skp2). In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCF(Skp2).
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Dana
D
Ungermannova
Yuefeng
Y
Gao
Xuedong
X
Liu
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D042846:CDC2-CDC28 Kinases / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry*; D019927:Cyclin E / Q000737:chemistry* / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007313:Insecta; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D025521:Tumor Suppressor Proteins / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D010710:Phosphates; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D001120:Arginine; D005982:Glutathione Transferase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2