Fig. 4. Biochemical properties of the ankyrin-associated kinase. Ankyrin immunoprecipitates prepared from a whole cell lysate from erythroid cells were incubated in kinase buffer containing [-³²P]-ATP (A and B, lanes 1-3) or [-³²P]-GTP (A, lane 4) for 30 minutes at 37°C. CK2 inhibitors, including 5 mM EDTA (A, lane 2), 50 µg of heparin (A, lane 3), 10 µM TBB (B, lane 2) and 10 µM emodin (B, lane 3), were added to some of the immunoprecipitates during the in vitro kinase reaction. Alternatively, ankyrin precipitates prepared from whole cell lysates from control (C, lanes 1, 3, 5) or calyculin-A-treated erythroid cells (C, lane 2, 4, 6) were either incubated in kinase buffer containing [-³²P]-ATP for 30 minutes at 37°C (C, lanes 1 and 2) or boiled for 2 minutes before the kinase reaction (C, lanes 3 and 4). To some of the heat-treated precipitates, purified CK2 from rat liver was added prior to incubation at 37°C for 30 minutes (C, lanes 5 and 6). Following the kinase reactions, samples were analyzed on 6% SDS polyacrylamide gels. The migration of the 225 kDa, 220 kDa, and 205 kDa ankyrin isoforms are indicated by dashes to the left of each panel. Dashes to the right of the panel C indicate the hyperphosphorylated (225^*) 225 kDa and (205^*) 205 kDa ankyrin isoforms.

Return to article