A Negatively Charged Amino Acid in Skp2 Is Required for Skp2-Cks1 Interaction and Ubiquitination of p27Kip1

Abstract

Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominately in the late G 1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCF Skp2 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 selfubiquitination. 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.

Negative regulation of SCFSkp2 ubiquitin ligase by TGF-β signaling

“…As controls, we also added increasing concentrations of a mutant Cks1 (Cks1N45R) or Cks2, a closely related human homologue that shares 85% homology with Cks1, to the reactions. It has been shown previously that Cks1 but not Cks2 binds Skp2 in vitro and Cks1 N45-R mutation significantly impaired Skp2-Cks1 association (Sitry et al, 2002;Wang et al, 2003). While addition of increasing amounts of Cks1N45R partially inhibits Skp2 self-ubiquitination, adding similar amounts of Cks2 has no effect.…”

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

“…In brief, the SCF Skp2 complex was expressed and purified from insect cells (39) and mixed with in vitro-translated 35 S-labeled p27 that had previously been incubated with cyclin E/Cdk2 along with methylated ubiquitin and ubiquitin aldehyde for 60 min at 30°C. The reaction was stopped with 2ϫ SDS sample buffer and run on polyacrylamide gels.…”

Ubiquitination and Proteolysis of Cancer-Derived Smad4 Mutants by SCFSkp2

“…In vitro ubiquitination assays were performed as previously described (50). Recombinant baculoviruses expressing GST-Skp1, Cul1, Roc1, and Flag-Skp2 were kindly provided by Wade Harper.…”

“…The SCF complex was purified by GST affinity chromatography and stored in QA buffer at Ϫ80°C (50). A typical ubiquitination reaction was carried out in a final volume of 30 l containing 250 ng of E1 (Boston Biochemical), 3 g of 6His-cdc34, 5 g of SCF Skp2 complex, 1 g of ubiquitin, 1.5 g of methylated ubiquitin (Boston Biochem), 1 M ubiquitin aldehyde, 2 l MG132, 2.5 l of 10ϫ ER (50), and 1 mM dithiothreitol. The reaction mixture was incubated at 30°C for 2 h. Anti-Smad4 Western blot analysis was used to evaluate the results.…”