Figure 2.

Time course of CaMKK-mediated phosphorylation with various nucleotides of GST-CaMKIα (1-293, K49E) or AMPKα. Purified recombinant CaMKKα (left panels) or CaMKKβ (right panels) was incubated with either GST-CaMKIα (1-293, K49E, A) or AMPK (B) at 30°C for the indicated time periods in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(CH₃COO)₂, 1 mM DTT, 1 mM CaCl₂, 10 μM CaM, in the presence of either 1 mM ATP (•), 1 mM GTP (·) or 1 mM UTP (▴) as described in "Methods." After each reaction was terminated, samples were subjected to western blot analysis with either anti-phospho-CaMKI antibody (A, upper images) or anti-phospho-AMPKα antibody (B, upper images) followed by quantitating the immunoreactive bands by densitometric scanning of the scientific imaging film used for detection of chemiluminescence. Phosphate incorporation into each CaMKK target is expressed as a percentage of the value of the reaction for the 5-min time point (30-min time point for AMPK) in the presence of ATP. Results are representative of at least two independent experiments.