Figure 8.

CK2 phosphorylation of A-, B- and c-Myb DBDs. (A) Recombinant NR₁₂₃ domains of A-Myb (lane 1), B-Myb (lane 2), c-Myb (lane 3) and the S11D/S112D mutant of c-Myb (C^DD; lane 4) were phosphorylated in vitro with [γ-³²P]ATP and protein kinase CK2 at 30°C for 30 min before separation by SDS–PAGE and autoradiography (as described in Materials and Methods). The S11D/S12D mutant was constructed as previously described (22). The lower panel shows an SDS–PAGE analysis of the four NR₁₂₃ domains from A-Myb (lane 1), B-Myb (lane 2), c-Myb (lane 3) and C^DD (lane 4) (1.5 µg of each), purified as described, and visualized by staining with Coomassie brilliant blue. The theoretical molecular weights of the NR₁₂₃ domains are 22.6, 22.0 and 23.1 kDa for A-, B- and c-Myb, respectively. (B) Recombinant NR₁₂₃ domain of c-Myb (200 µg) was phosphorylated in vitro with 0.1 mM ATP (and trace amounts of [γ-³²P]ATP) and protein kinase CK2 at 30°C for 30 min before separation on a SP Sepharose Fast Flow (Amersham Pharmacia) ion exchange column using a 150–1500 mM NaCl gradient. Equal amounts of non-phosphorylated NR₁₂₃ domain of c-Myb and protein from the peak fraction corresponding to phosphorylated c-Myb were analysed by EMSA (as described in Materials and Methods). The degree of phosphorylation in the purified fraction was determined as described (75) and estimated to be 70%.