Structure and regulation of AMPK in the liver
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in human hepatocytes (B. Guigas, unpublished results).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.
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