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.