Figure 2

Fbw7-dependent ubiquitylation of deleted or site-specific amino acid-substituted mutants of human c-Myb. (A) The consensus motif for phosphorylation by GSK3 is indicated in the upper panel. The human c-Myb sequence is compared with that of mouse c-Myb surrounding Thr-572, which is phosphorylated by GSK3 and then recognized by Fbw7, in the lower panel. Only one putative Cdc4-phosphodegron (CPD) site in human c-Myb is described in parallel. (B) Fbw7-dependent ubiquitylation of mouse and human c-Myb mutants. For analyses of mouse c-Myb ubiquitylation, HA-ubiquitin and Myc-tagged wild-type (WT) or mutant (T568A or T572A) mouse c-Myb in the absence or presence of mouse Fbw7 were transfected into HEK293 cells. For analyses of human c-Myb ubiquitylation, HA-ubiquitin and WT or mutant (Δ558, S560A or 13A) human c-Myb in the absence or presence of human Fbw7 were transfected into HEK293 cells. The transfected cells were then incubated with MG132. Cell lysates were subjected to immunoblotting with an anti-Myc-tag antibody to detect ubiquitylation of the mouse c-Myb protein or with an anti-c-Myb antibody to detect ubiquitylation of the human c-Myb protein. The results for the ubiquitylation of the various mutant human c-Myb proteins by Fbw7 are summarized in (C). (C) Schematic representations of the human c-Myb mutants. The asterisks and vertical bars indicate the putative GSK3 and NLK phosphorylation sites, respectively. DBS, DNA-binding sequence; TA, transactivation domain; Leu Zipper, leucine zipper; NRD, negative regulatory domain. (D) Effects of mutations at the putative GSK3 recognition sites on Fbw7-mediated degradation of c-Myb. HeLa cells were transfected with the human c-Myb mutant 13A in the absence or presence of HA-human Fbw7. For the cycloheximide (CHX) assay, cells were prepared after the indicated times of chase incubation and subjected to immunoblotting. The percentages of c-Myb remaining after the various chase times were quantified by image analysis.