In vitro substrate phosphorylation by Ca(2+)/calmodulin-dependent protein kinase kinase using guanosine-5(′)-triphosphate as a phosphate donor

BACKGROUND: Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases — including CaMKI, CaMKIV, and AMPK— to stimulate multiple Ca(2+)-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK. RESULTS: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKIα (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K(m) values of CaMKK isoforms for GTP (400-500 μM) were significantly higher than those for ATP (~15 μM), and a 2- to 4-fold decrease in V(max) was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of ~45 kDa and ~35 kDa whose Ca(2+)/CaM-induced phosphorylation was inhibited by STO-609. CONCLUSIONS: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.