Structure and regulation of AMPK in the liver

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AMPK exists as a heterotrimeric complex consisting of a catalytic subunit a and two regulatory subunits b and g involved in heterotrimer formation and ligand sensing. The conventional serine/threonine kinase activity of AMPK is supported by the a subunit which is characterized by the presence of a threonine residue within the activation loop of the kinase domain. The C-terminal region of a subunit is required for the association with the regulatory b and g subunits. The b subunit contains a C-terminal region acting as scaffold binding a and g subunits and a central region that allowed AMPK complex to bind glycogen but this has been recently disputed in the liver (Parker et al., 2007). The g subunit contains four tandem repeats known as cystathionine b-synthase (CBS) motifs which bind AMP or ATP molecules in a competitive manner. Recent structural studies revealed that two sites on the g subunit bind either AMP or Mg.ATP, a third site contains a tightly bound AMP that does not exchange (Xiao et al., 2007). Isoforms of all three subunits encoding by different genes (a1, a2, b1, b2, g1, g2, g3) have been identified in mammals that allow for the generation of 12 different heterotrimeric combinations. These heterotrimeric AMPK complexes show relative tissue specificity. AMPKa1 and a2-containing complexes account each for about half of total AMPK activity in rodent liver (Cheung et al., 2000) but AMPKa1-containing complexes are predominant in human hepatocytes (B. Guigas, unpublished results). Expression of the b1 and g1 regulatory subunits predominates in rodent liver (Cheung et al., 2000). To the best of our knowledge, no selective association between catalytic a1 and a2 and regulatory b and g subunits or differences in the activity of the various hepatic AMPK complexes combination have been reported. Nevertheless, it should be noted that AMPK complexes distribution can be regulated intracellularly with AMPKa2-containing complexes present in both nucleus and cytoplasm raising the possibility of direct regulation of gene transcription (Salt et al., 1998) and with AMPKa1-containing complexes localized in the cytoplasm but also at the plasma membrane (Evans et al., 2005, Hallows et al., 2003). Changes in the subcellular localization of AMPKa2 in response to specific stimuli appear to be conserved from yeast to mammals via a mechanism involving the interaction with the regulatory b subunits (Suzuki et al., 2007, Vincent et al., 2001). In mammalian muscle cells, AMPKa2 bound to the b2 subunit translocates to the nucleus in a manner dependent on a nuclear localization signal that is present in AMPKa2 but not in AMPKa1 subunit. AMPKa2 bound to the b1 subunit is anchored into the cytoplasm at the outer mitochondrial membrane through the myristoylation of b1 subunit. These data suggest that activation of AMPK complexes may elicit distinct metabolic effects in tissues and cells depending on the expression of the different a- and b-subunit isoforms and illustrate the complexity of the molecular mechanisms by which energy metabolism can be regulated by AMPK.

Regulation of AMPK activity involves both direct allosteric activation and reversible phosphorylation. Activation of AMPK requires phosphorylation on Thr-172 within the catalytic subunit and three upstream kinases have been identified corresponding to the tumor suppressor LKB1 kinase, CaMKKb (Ca2+/calmodulin-dependent protein kinase kinase b) and possibly TAK1 (TGFb-activated kinase-1, a member of the mitogen-activated protein kinase kinase family). LKB1 is mainly involved in Thr-172 phosphorylation following change in AMP:ATP ratio (Hawley et al., 2003, Shaw et al., 2004, Woods et al., 2003). It has become evident in the last years that LKB1 plays a crucial role in activating AMPK to control glucose and lipid metabolism in the liver (Shaw et al., 2005, Imai et al., 2006). It has been suggested that LKB1 may be constitutively active (Lizcano et al., 2004, Sakamoto et al., 2004) but recent studies indicates that cytosolic localization and activity of LKB1 can be governed by LKB1 acetylation status in the liver (Lan et al., 2008). CaMKKb is viewed as an alternate upstream kinase that could also phosphorylate Thr-172 and activate AMPK in intact cells by an AMP-independent manner in response to increased intracellular Ca²⁺ concentrations (Hawley et al., 2005, Hurley et al., 2005, Woods et al., 2005). However, CaMKKb is highly expressed in neural tissue and the role for Ca²⁺-mediated AMPK activation in the liver remains to be investigated. The third potential upstream kinase is TAK1, which activates the S. cerevisiae homologue of AMPK, the SNF1 complex, when expressed in the yeast (Momcilovic et al., 2006), and could also phosphorylate Thr-172 and activate AMPK in mouse embryonic fibroblasts (Xie et al., 2006) but its role as an upstream kinase remains controversial (Goransson et al., 2007). In addition to phosphorylation, AMPK is allosterically activated by AMP which binds to the regulatory g subunit. Binding of AMP to AMPK induces a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr-172 (Riek et al., 2008), probably catalysed by a form of protein phosphatase-2C (Sanders et al., 2007). The combination of the allosteric and phosphorylation effects causes >1000-fold increase in kinase activity (Suter et al., 2006) allowing to respond to small changes in cellular energy status in a highly sensitive manner.