Figure 5
Analysis of the required sites in c-Myb for Fbw7-mediated ubiquitylation. (a) Effect of C-terminal deletion on ubiquitylation of c-Myb by Fbw7. Schematic representation of mouse c-Myb mutants. DBS, DNA-binding sequences; TA, transactivation domain; Leu Zipper, leucine zipper; NRD, negative regulatory domain. HEK293 cells were transfected with HA-Ubiquitin and wild type (WT) or the deletion mutant of Myc-tagged c-Myb in the absence or presence of FLAG-Fbw7, and were then incubated with MG132 for 5 h. To detect ubiquitylation of c-Myb protein and expression of Fbw7, lysates were subjected to immunoblotting (IB) with antibodies against Myc and FLAG, respectively. Results of ubiquitylation of deleted c-Myb by Fbw7 were also summarized. (b) Consensus sequence for phosphorylation by glycogen synthase kinase 3 (GSK3) and Cdc4 phosphodegron (CPD) are indicated. In the C-terminal region of c-Myb, aa numbers indicates the sites of aa exchanged to Ala in the mutant c-Myb proteins which were used in (c). Four putative GSK3 sites (opened box) and two putative CPD sites (bold under bar) in the C-terminal region of mouse c-Myb are indicated. (c, d) HEK293 cells were transfected with HA-ubiquitin and either wild type or mutant c-Myb, in the absence or presence of FLAG-Fbw7. Ubiquitylation of c-Myb and expression of Fbw7 in cell lysates were determined by IB analysis with antibodies against Myc or FLAG.
