Figure 7

Fbw7 recognized phosphorylation of Thr-572, which could be mediated by glycogen synthase kinase 3 (GSK3). (a) GSK3 inhibitors repressed c-Myb ubiquitylation which was mediated by Fbw7. HEK293 cells were transfected with FLAG-Fbw7, HA-Ub and c-Myb, as indicated, and were treated with the indicated GSK3 inhibitors for 24 h, and were incubated in the presence of MG132 during the final 5 h. Cell lysates were subjected to immunoblotting (IB) with antibodies to c-Myb or Fbw7. (b) GSK3 was involved in stability of c-Myb protein. HeLa cells were transfected with c-Myb in the absence or presence of FLAG-Fbw7 with or without a dominant negative kinase, GSK3βK58R. For the cycloheximide (CHX) analysis, cells were prepared at the indicated times of the chase incubation and subjected to IB with antibodies against c-Myb or αTubulin. The percentage of c-Myb remaining after the various chase times was quantitated by image analysis. Percent of c-Myb protein levels were calculated as the mean±s.d., from three independent experiments. (c) Depletion of GSK3β inhibited phosphorylation of Thr-572. HEK293 cells were transfected with c-Myb expression plasmid and control or GSK3β of siRNA oligonucleotides. After 43 h transfection, cells were treated with Okadaic acid and MG132 for 5 h. Cell lysates were prepared and subjected to IB analysis with antibodies against P-T572 c-Myb, c-Myb, GSK3β or α-tubulin. (d) Interaction of Fbw7 with a synthetic peptide was analysed by an in vitro ‘pull down’ assay. HEK293 cells were transfected with FLAG-Fbw7. The cell lysates were subsequently subjected to a ‘pull-down’ assay with beads conjugated with nonphosphorylated (NP-T572) peptide or phosphorylated Thr-572 (P-T572) peptide corresponding to aa 567–577 of c-Myb. To detect FLAG-Fbw7 bound to peptides, the resulting precipitates and 5% of the input cell lysates were subjected to IB analysis with antibodies against FLAG. (e) T572A mutant was resistant to Fbw7-promoted degradation. HeLa cells were transfected with c-Myb, either wild type or T572A, in the absence or presence of FLAG-Fbw7. For the CHX analysis, the cells were prepared at the indicated times of the chase incubation and subjected to IB with antibodies against c-Myb, α-tubulin or FLAG. The percentage of c-Myb remaining after the various chase times was quantitated by image analysis. Immunoblots show the representative data, and the percentage of c-Myb protein levels was calculated as the mean±s.d. from three independent experiments.