The study investigates the role of Ca²⁺/calmodulin-dependent protein kinase kinases (CaMKKs) as AMP-activated protein kinase (AMPK) kinases (AMPKKs) in cell lines deficient in LKB1, specifically HeLa, A549, and murine embryo fibroblasts derived from LKB1−/− mice. The researchers found that CaMKKs, particularly CaMKKα and CaMKKβ, are capable of activating AMPK by phosphorylating AMPKαThr-172. This activation is significantly inhibited by the CaMKK inhibitor STO-609, both in vitro and in intact cells. Additionally, knockdown of CaMKKs using small interfering RNAs (siRNAs) further reduced AMPK activation and phosphorylation of downstream targets such as acetyl-CoA carboxylase. The study also observed that the activation of AMPK by ionomycin and 2-deoxyglucose was not impaired in LKB1−/− cells, suggesting that CaMKKs may play a more significant role in AMPK regulation in these cells. These findings indicate that CaMKKs function as AMPKKs in intact cells, highlighting their potential roles in regulating AMPK activity in vivo.The study investigates the role of Ca²⁺/calmodulin-dependent protein kinase kinases (CaMKKs) as AMP-activated protein kinase (AMPK) kinases (AMPKKs) in cell lines deficient in LKB1, specifically HeLa, A549, and murine embryo fibroblasts derived from LKB1−/− mice. The researchers found that CaMKKs, particularly CaMKKα and CaMKKβ, are capable of activating AMPK by phosphorylating AMPKαThr-172. This activation is significantly inhibited by the CaMKK inhibitor STO-609, both in vitro and in intact cells. Additionally, knockdown of CaMKKs using small interfering RNAs (siRNAs) further reduced AMPK activation and phosphorylation of downstream targets such as acetyl-CoA carboxylase. The study also observed that the activation of AMPK by ionomycin and 2-deoxyglucose was not impaired in LKB1−/− cells, suggesting that CaMKKs may play a more significant role in AMPK regulation in these cells. These findings indicate that CaMKKs function as AMPKKs in intact cells, highlighting their potential roles in regulating AMPK activity in vivo.