In vitro substrate phosphorylation by Ca²⁺/calmodulin-dependent protein kinase kinase using guanosine-5^′-triphosphate as a phosphate donor

Background: Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases - including CaMKI, CaMKIV, and AMPK - to stimulate multiple Ca²⁺-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 (Thr¹⁷⁷) and of AMPK (Thr¹⁷²) 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.