Ca²⁺/Calmodulin-dependent Kinase Kinase β (CaMKKβ) is a serine/threonine directed kinase that activates the key metabolic regulator AMP-activated protein kinase (AMPK) by phosphorylation of Thr172 of the α-catalytic subunit activation loop in response to increases in intracellular Ca²⁺. CaMKKβ consists of a Ser/Thr-directed kinase domain followed by a C-terminal autoregulatory domain containing overlapping autoinhibitory and calmodulin (CaM) binding regions. Binding of Ca²⁺/CaM to the CaM binding region removes autoinhibition and causes maximal catalytic activity. Autonomous activity in the absence of Ca²⁺/CaM is minimised by constitutive phosphorylation of three sites in the N-terminus: Ser129, Ser133 and Ser137. Alanine mutants of these residues show increased Ca²⁺/CaM autonomous activity, and analysis by mass spectrometry has revealed that these sites are sequentially phosphorylated, with phosphorylation of Ser137 priming for phosphorylation of Ser133, and phosphorylation of Ser133 priming for phosphorylation of Ser129. Treatment of cells with the Casein Kinase I (CKI) inhibitor IC-261 and the Glycogen Synthase Kinase 3 (GSK3) inhibitor GSK3 XV led to increases in the Ca²⁺/CaM autonomous activity of CaMKKβ, comparable to the autonomous activity seen in the Ser133Ala and Ser137Ala mutants. These data indicate that CKI and GSK3 may act as upstream kinases that sequentially phosphorylate CaMKKβ at these 3 sites. Ongoing experiments are being conducted to further elucidate the identity of these upstream regulatory kinases that may reveal signaling pathways that can modulate CaMKKβ activity independent of Ca²⁺ signaling.