Figure 6
Thr-572 of c-Myb is phosphorylated in vivo. (a) c-Myb, either wild type or mutant, was transiently expressed in HEK293 cells. In order to inhibit proteolysis and dephosphorylation of c-Myb, cells were treated with 20 μM MG132 and 20 nM Okadaic acid for 5 h before harvest. Cell lysates were prepared with lysis buffer, which contained phosphatase inhibitors, then subjected to immunoblotting (IB) analysis with antibodies against c-Myb or P-T572 c-Myb. (b) To reconfirm that Thr-572 of c-Myb is phosphorylated in vivo, HEK293 cells were transfected with wild-type c-Myb, again. Cells were treated with 20 μM MG132 in the absence or presence of 20 nM Okadaic acid for 5 h before harvest. Cell lysates were prepared with lysis buffer, which contained phosphatase inhibitors, then subjected to IB analysis with antibodies against P-T572 c-Myb, c-Myb or α-tubulin. (c) Phosphorylation of synthetic peptide by GSK3 kinase. Indicated synthetic peptides were incubated with [γ-32P]ATP in the absence or presence of glycogen synthase kinase 3 (GSK3) kinase for the indicated time as described in the Materials and methods section. The levels of incorporation of 32P are means±s.d. of results of triplicate assays. Each peptide sequence was indicated below. Opened boxes are putative GSK3 phosphorylation sites. (d) Wild-type c-Myb protein was phosphorylated by GSK3β in vitro. Myc tagged wild type or T572A mutant c-Myb or empty vector plasmid was transfected in HEK293 cells. Cells were treated with MG132 for 5 h starting at 43 h after transfection and then harvested. Cell lysates were prepared in the absence of phosphatase inhibitors, and subjected to immunoprecipitation with anti Myc antibody. The precipitates were incubated with or without recombinant GSK3β kinase at 30 °C for 30 min, and then subjected to immunoblot analysis. Mouse c-Myb phosphorylated Thr-572 and the total level of precipitated c-Myb was detected with P-T572 c-Myb antibody and anti c-Myb antibody, respectively.
