Figure 1
CaMKK can utilize various nucleotides as phosphate donors in vitro. A. Purified recombinant CaMKKα was incubated with GST-CaMKIα (1-293, K49E, 0.5 mg/ml) at 30°C for 10 min in the solution containing 50 mM HEPES (pH 7.5), 1 mM DTT, 1 mM CaCl2, 10 mM Mg(CH3COO)2, and 10 μM CaM in the absence (-) or presence of either 1 mM ATP, UTP, CTP, or GTP. After terminating the reaction, samples were subjected to western blot analysis with either anti-GST antibody (A , upper panel) or anti-phospho-CaMKI antibody (A , lower panel). Similar results were obtained for at least three independent experiments. B. Purified recombinant CaMKKα (+) was incubated with GST-CaMKIα (1-293, K49E, 0.5 mg/ml) at 30°C for 10 min in a solution used in panel A with (+) or without (-) 10 mM Mg(CH3COO)2, in the absence (-) or presence (+) of 1 mM ATP or 1 mM GTP. The reaction mixtures were heated at 68°C for 10 min (heat inactivated +) or kept on ice (-) before initiating the phosphorylation reaction. After each reaction was terminated, samples were subjected to western blot analysis with either anti-GST antibody (B, upper panel) or anti-phospho-CaMKI antibody (B, lower panel).