Fbw7 Isoform Interaction Contributes to Cyclin E Proteolysis

A. Schematic diagram of Fbw7 domains in each splice variant and the deletion mutants used in this study. B. Alignment of the D-domain sequences from Fbw7 and its homologues and a number of other WD40-repeat containing F-box proteins, including βTrCP1 and its homologues. Letters above the alignment, consensus sequence. Black, identical residues; gray, residues of similar functional groups. The alignment was generated using Clustal W and the Megalign program of the Lasergene Navigator Suite.

The domain required for Fbw7 isoform interaction encompasses the D domain. A. The NH₂ terminus is required for the Fbw7 isoform interaction. 293T cells were cotransfected with vectors expressing Flag-tagged full-length Fbw7 isoforms and either Myc-tagged Fbw7-α or Myc-tagged Fbw7 ΔN. Top and middle, Western blot analyzing the expression of the proteins; bottom, immunoprecipitation result. Equal amounts of total protein extracted from each lysate were incubated with 20 μL of anti-Flag agarose beads before SDS-PAGE and Western blot analysis using anti-Myc antibodies. Lanes 7 and 8, empty Flag vector transfected as a negative control. *, nonspecific band recognized by the anti-Myc antibodies. B. Fbw7 ΔDC no longer binds Fbw7 isoforms. 293T cells were transfected with full-length Flag-tagged Fbw7-α or the Fbw7 ΔDC mutant of each isoform together with Myc-tagged Fbw7-α. Top and middle, Western blot analyzing the expression of the proteins; bottom, immunoprecipitation result. Equal amounts of total protein extracted from each lysate were incubated with 20 μL of anti-Flag agarose beads before SDS-PAGE and Western blot analysis. C. The deletion mutants of Fbw7 coimmunoprecipitate with Skp1. 293T cells were transfected with the Fbw7 deletion mutants and HA-tagged Skp1. Top and middle, expression of Flag-tagged full-length Fbw7 isoforms and the ΔDC deletion mutants and HA-tagged Skp1; bottom, immunoprecipitation result. Equal amounts of total protein extracted from each cell lysate were incubated with anti-HA antibody for 2 h followed by incubation with 20 μL prewashed protein A/G agarose beads. Lanes 8 to 14, control experiments in which no HA-Skp1 was transfected. *, the Fbw7 ΔN band, which runs just above the IgG heavy chain. D. The Fbw7 deletion mutants coimmunoprecipitate cyclin E. 293T cells were transfected with Flag-tagged full-length Fbw7 isoforms, ΔDC deletion mutants, or Flag vector together with equal quantities of the cyclin E expression construct. Top and middle, the expression of the indicated forms of Fbw7 and cyclin E; bottom, immunoprecipitation result. Equal amounts of total protein from each cell lysate were incubated with 20 μL of prewashed anti-Flag M2 agarose beads at 4°C overnight.

The homo-oligomeric interaction of Fbw7 isoforms is important for cyclin E proteolysis. A. Examples of cyclin E stability assays. 293T cells were cotransfected with the same amount of cyclin E expression construct together with Flag-tagged Fbw7-α, Fbw7-α ΔDC, Fbw7 ΔN, or empty vector. Thirty-six hours after transfection, cycloheximide (CHX; final concentration, 30 μg/mL) was added to stop protein synthesis (time point 0). Cells were collected at the indicated time points and cyclin E protein abundance was analyzed by Western blot. Anti-GAPDH antibodies were used as a loading control. B. Quantitation of cyclin E stability. The results of the cyclin E stability experiments in A were analyzed by Image J software. The log of the ratio of cyclin E (both bands) to the loading control is plotted versus time. C. The Fbw7-α ΔDC mutant shows similar localization to the full-length protein. 293T cells were grown on coverslips and transfected with Fbw7-α, Fbw7-α ΔDC, and Fbw7 ΔN. Thirty-six hours after transfection, cells were fixed in 3% paraformaldehyde and immunostained with anti-Flag antibodies. DAPI, 4′,6-diamidino-2-phenylindole. D. 293T cells were transfected with Flag-tagged Fbw7-α, Fbw7-α ΔDC, or Fbw7 ΔN. Thirty-six hours after transfection, cycloheximide (final concentration, 30 μg/mL) was added to stop protein synthesis (time point 0). Cells were collected at the indicated time points and Fbw7 protein abundance was analyzed by Western blot using anti-Flag antibodies. Anti-GAPDH antibodies were used as a loading control.