A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1.
Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1. The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure. Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27. Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1. Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination. A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction. Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27.
Wang
Wei
W
Department of Chemistry and Biochemistry, University of Colorado-Boulder, Boulder, Colorado 80309, USA.
Ungermannova
Dana
D
Chen
Lin
L
Liu
Xuedong
X
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C081185:CKS1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27