Cyclin D2 is polyubiquitinated at carboxyl-terminal acceptor sites. (A) Diagram of cyclin D2 deletion mutants constructed. Arrows indicate putative ubiquitin receptor sites. (B) V5-immunoblotting for levels of full-length (FL) or truncated cyclin D2 constructs after cellular expression and culture of cells in the absence (−) or presence (+) of MG132. (C) Cells were transfected with cyclin D2 point mutants before culture in the absence (−) or presence (+) of MG132. Immunoblotting was then performed for detection of accumulated (polyubiquitinated) cyclin D2 proteins in cells. The arrows indicate lack of polyubiquitinated signals after expression of the cyclin D2^K270R construct. (D) In vitro ubiquitination assays. Purified SCF complex components were incubated with V5-cyclin D2 and the full complement of ubiquitination reaction components (second lane from left) showing polyubiquitinated cyclin D2. (E) Cyclin D2 protein levels in cells after cotransfection with either WT cyclin D2 or Lys^270R cyclin D2 with or without ectopic FBXL2 expression. (F) In vitro ubiquitination assays. Purified SCF complex were incubated with WT V5-cyclin D2, or a Lys^270R V5-cyclin D2 mutant and the full complement of ubiquitination reaction components. (G) Cyclin D2 protein half-life determination using cyclohexamide after expression of WT V5-cyclin D2, or Lys^270R (V5-cyclin D2) mutant (data are from n = 2 experiments).