Activation of SAD kinase by Ca²⁺/calmodulin-dependent protein kinase kinase

To search for the downstream target protein kinases of Ca ²⁺/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKα catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5′-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr¹⁸⁹ in the activation loop. Phosphorylation of Thr¹⁸⁹ by CaMKKα induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr¹⁸⁹ phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKα (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKα is capable of activating SAD-B through phosphorylation of Thr¹⁸⁹ both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.