## 2 The Structure and Activation of AMPK  
AMPK is a critical regulator of cellular energy homeostasis activated under conditions that deplete energy, such as nutrient deprivation, hypoxia, or exposure to mitochondrial toxins [23; 24]. AMPK is composed of a catalytic \(\alpha\) subunit (\(\alpha 1\) and \(\alpha 2\)), a scaffolding \(\beta\) subunit (\(\beta 1\) and \(\beta 2\)), and a regulatory \(\gamma\) subunit (\(\gamma 1\), \(\gamma 2\), and \(\gamma 3\)) [25]. The widely expressed \(\alpha 1\beta 1\gamma 1\) complex is often used as a model for identifying AMPK activators, but relying solely on this complex may not fully represent the diverse physiological roles of AMPK given the distinct functions and tissue-specific distributions of its isoforms [25; 26]. The AMPK\(\alpha\) subunit is crucial for the kinase activity of the complex. It contains a kinase domain (KD), which includes the Thr-172 residue, essential for activation by upstream kinases. This subunit also has an autoinhibitory domain (AID) that interacts with the KD to keep the enzyme in an inactive state. Structural studies have shown that the AID interacts with both the small and large lobes of the KD [27]. The \(\alpha\) subunits also feature two \(\alpha\)-RIMs, which are regulatory subunit-interacting motifs. These motifs trigger conformational changes in response to AMP binding to the AMPK\(\gamma\) subunit. Additionally, the \(\alpha\) subunit has a C-terminal domain (\(\alpha\)-CTD) that binds to the \(\beta\) subunit, facilitating the stability and regulation of the AMPK complex. The AMPK\(\beta\) subunit serves as a scaffold that connects the \(\alpha\) and \(\gamma\) subunits, ensuring the structural integrity of the AMPK complex. It contains a carbohydrate-binding module (CBM), where the Ser108 residue plays a key role in the action of direct AMPK activators, such as thienopyridone (A-769662) and salicate. The \(\beta\) subunit also features a C-terminal domain (\(\beta\)-CTD), which interacts with both the \(\alpha\) and \(\gamma\) subunits, further stabilizing the complex and enabling the regulation of its activity. The AMPK\(\gamma\) subunit is critical for the enzyme's response to cellular energy levels, as it contains cystathione-\(\beta\)-synthase (CBS) domains that bind adenosine nucleotides (AMP, ADP, and ATP). This subunit has four CBS domains that form potential adenine nucleotide-binding sites, labeled sites 1-4. Typically, site 2 remains unoccupied, while site 4 often binds AMP tightly. Sites 1 and 3 are regulatory sites that can bind AMP, ADP, or ATP [27]. Binding of AMP to site 1 initiates the allosteric activation of AMPK, while binding of AMP or ADP to site 3 modulates the phosphorylation state of Thr-172 on the AMPK\(\alpha\) subunit [24; 28]. Although the levels of ADP are generally higher than those of AMP, recent studies have demonstrated that AMP is a key activator, significantly enhancing the LKB1-dependent phosphorylation of Thr-172 in vivo [29]. Physiological activation of AMPK is primarily regulated by the phosphorylation of Thr-172 within the KD's activation loop in the AMPK\(\alpha\) subunit. This phosphorylation is mediated by two upstream kinases, LKB1 and CaMKII(Ca2+/calmodulin-dependentprotein kinase \(\beta\)) [30; 31]. AMP binding to the AMPK subunit significantly enhances the LKB1-dependent phosphorylation of Thr-172, while also inhibiting its dephosphorylation by protein phosphatases such as PP2A and PP2C [32; 33]. Interestingly, the effect of AMP on Thr-172 phosphorylation is influenced by N-terminal myristoylation of the \(\beta\) subunit, although the exact mechanism is not yet fully understood [34]. Unlike the LKB1 complex, CaMKII\(\beta\) can activate AMPK in response to increased intracellular Ca2+ levels, independent of changes in ATP/ADP/AMP ratios. Treatments that deplete cellular ATP do not effectively activate AMPK in LKB1-deficient tumors because the basal activity of CaMKII\(\beta\) is too low to affect Thr-172 phosphorylation, despite increased AMP levels.