Characterization of Autonomous and Ca^2<b>+</b>/Calmodulin-Dependent Activities of CaMKK Isoforms In Vitro and in Mouse Tissues

Ca²⁺/CaM-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream kinases, including CaMKI, CaMKIV, PKB, and AMPK, regulating various cellular functions such as neuronal morphogenesis, metabolic control, and pathophysiological pathways, such as cancer progression. CaMKKα/1 is tightly regulated by an autoinhibitory mechanism. CaMKKβ/2 activity is highly Ca²⁺/CaM-independent (autonomous activity) in vitro and Ca²⁺/CaM-dependent in cultured cells. Whether these two activity states of CaMKKβ/2 exist in vivo and the detailed regulatory mechanisms for the transition of both activity states remain unclear due to the difficulty in distinguishing the two activity states. In this study, we detected Ca²⁺-dependent and autonomous CaMKK activity in HeLa cells and successfully separated both activity states of CaMKKβ/2 in mouse brain and testis extracts using a recently developed CaMKK inhibitor (TIM-063)-coupled sepharose, which binds to the catalytic domain in the active state but not in the autoinhibited state. Furthermore, lambda protein phosphatase treatment converted the Ca²⁺/CaM-dependent form to the autonomous form of CaMKKβ/2, which was not affected by Ala mutation of Ser128, Ser132, and Ser136. The two activity forms of CaMKKβ/2 had equivalent Ca²⁺/CaM-binding ability. The findings demonstrate the presence of autonomous and Ca²⁺/CaM-dependent forms of CaMKKβ/2 independently in mouse tissues and cultured cells. The transition of these states of CaMKKβ/2 may be dynamically regulated by the phosphorylation/dephosphorylation of serine residues in the N-terminal regulatory domain.