Download AMP-activated/SNF1 protein kinases: conserved guardians of

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AMP-activated/SNF1 protein kinases:
conserved guardians of cellular energy
D. Grahame Hardie
Abstract | The SNF1/AMP-activated protein kinase (AMPK) family maintains the balance
between ATP production and consumption in all eukaryotic cells. The kinases are
heterotrimers that comprise a catalytic subunit and regulatory subunits that sense cellular
energy levels. When energy status is compromised, the system activates catabolic pathways
and switches off protein, carbohydrate and lipid biosynthesis, as well as cell growth and
proliferation. Surprisingly, recent results indicate that the AMPK system is also important in
functions that go beyond the regulation of energy homeostasis, such as the maintenance of
cell polarity in epithelial cells.
Non-fermentable carbon
source
A carbon source (for example,
glycerol) that cannot be
metabolized by anaerobic
fermentation in yeast, and is
only metabolized by oxidative,
aerobic metabolism.
Division of Molecular
Physiology, College of Life
Sciences, University
of Dundee, Dow Street,
Dundee, DD1 5EH,
Scotland, UK.
e-mail:
[email protected]
doi:10.1038/nrm2249
Published online
22 August 2007
In 1946, in his book What is Life?, Erwin Schrödinger
wrote: “When is a piece of matter said to be alive? When it
goes on moving and exchanging material with its environ­
ment for a much longer period than we would expect
of an inanimate piece of matter. When a system that is
not alive is isolated or placed in a uniform environ­ment,
all motion usually comes to a standstill very soon; differ­
ences of electric or chemical potential are equalized,
temperature becomes uniform by heat conduction. After
that the whole system fades away into a dead, inert lump
of matter”1.
Living cells are capable of achieving the remarkable
feat described by Schrödinger because they exploit the
environment, constantly taking in energy to maintain a
high ratio of ATP to ADP, analogous to a fully charged
electrical cell or battery. Extending this analogy, catabo­
lism (and photosynthesis in photosynthetic organisms)
‘charges up the battery’ by converting ADP and phosphate
to ATP, whereas almost all other cellular processes require
energy and tend to ‘flatten the battery’ by hydrolysing
ATP to ADP and phosphate (or, in a few cases, to AMP
and pyrophosphate). If the latter reactions were allowed
to reach equilibrium, cells would become Schrödinger’s
“dead, inert lump of matter”. It is therefore obligatory
that this is not allowed to happen, and mechanisms have
evolved that maintain the rates of catabolism and/or
photo­synthesis in balance with rates of ATP consumption.
I would argue that, at least since the evolution of eukaryo­
tic cells, the AMP-activated protein kinase (AMPK), with
its relatives in other eukaryotic kingdoms including the
SNF1 complexes in yeasts and the SNF1-related kinases in
plants (hereafter also referred to as SNF1 complexes), have
been major players in maintaining this balance.
774 | october 2007 | volume 8
AMPK was originally defined as a mammalian pro­
tein kinase that was allosterically activated by AMP2
and was able to phosphorylate and inactivate enzymes
of lipid synthesis3. In Saccharomyces cerevisiae, the SNF1
(sucrose non-fermenting‑1) gene was discovered by a
screen for mutations that caused failure to grow on
sucrose, or on non-fermentable carbon sources such as gly­
cerol and ethanol. Although SNF1 was shown to encode
a protein kinase in 1986 (Ref. 4), it was not realized that it
was the orthologue of AMPK until the catalytic subunit
of the latter was sequenced in 1994 (Refs 5,6).
Mammalian AMPK is sensitive to the cellular AMP:
ATP ratio and is activated by metabolic stresses that
inhibit ATP production (for example, hypoxia, glucose
deprivation, addition of metabolic inhibitors) or those
that stimulate ATP consumption (for example, activation
of motor proteins, ion pumps or channels, or biosynthetic
pathways) (FIG. 1). It is also modulated (FIG. 2) by cytokines
that regulate whole-body energy balance7, including
leptin, adiponectin, ghrelin, cannabinoids, interleukin‑6
(Ref. 8) and ciliary neurotrophic factor (CNTF)9, by drugs
used to treat type 2 diabetes (including metformin10
and thiazolidinediones11), and by natural plant pro­ducts
such as berberine12, resveratrol13 (present in grapes
and red wine) and (–)epigallocatechin‑3-gallate 14
(present in green tea), which are reported to have healthgiving properties that include the prevention of obesity
and insulin resistance, and extension of lifespan. In most
cases, the mechanism by which these agents activate
AMPK remains unclear.
Once activated by metabolic stresses, cytokines or
drugs, AMPK switches on catabolic pathways that gener­
ate ATP such as the uptake and metabolism of glucose
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Metabolic stress
(for example, hypoxia, glucose deprivation, metformin)
Catabolism
ADP
Adenylate
kinase
AMP
AMPK
ATP
ATP
ATP consumption
Increased ATP consumption (for example,
cell growth and division, activation of motor proteins)
Figure 1 | Regulation of energy homeostasis by the
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Cell Biology
AMPK system. Under
unstressed
carbon source and oxygen, optimal environment),
catabolism maintains a high ratio of ATP:ADP. This drives
the adenylate kinase reaction in favour of ADP synthesis
and, consequently, the cellular AMP:ATP ratio is low and
AMP-activated protein kinase (AMPK) is inactive.
However, if the cell is subjected to a metabolic stress that
interferes with ATP synthesis (for example, hypoxia,
glucose deprivation or a metabolic inhibitor such as
metformin) or a stress that accelerates ATP consumption
(for example, activation of motor proteins, ion pumps or
channels, or biosynthetic pathways), the ADP:ATP ratio
tends to increase. This is amplified by adenylate kinase
into a much larger increase in the AMP:ATP ratio that
then switches on AMPK. In turn, AMPK restores energy
homeostasis by promoting catabolism and inhibiting
ATP-consuming processes.
Adenylate kinase
An enzyme that catalyses the
reversible interconversion of
ATP, ADP and AMP via the
reaction 2ADP ↔ ATP + AMP.
Ser/Thr kinase domain
A kinase domain is a region of
~300 amino acids that folds
into a structure that catalyses
the phosphorylation of
proteins. Ser/Thr kinases are
specific for phosphorylation of
Ser and Thr side chains.
Activation loop
A feature that is conserved in
many protein kinases. In many
cases, phosphorylation of the
activation loop is required for
the kinase to be active.
and fatty acids, while switching off ATP-consuming,
anabolic pathways such as the synthesis of fatty acids,
cholesterol, glycogen and proteins (FIGS 1,2). It achieves
this by rapid phosphorylation of metabolic enzymes
and by phosphorylation of transcription factors and
co-activators that regulate gene expression. Regulation
of AMPK in the hypothalamus of the brain by cytokines
and other agents also controls food intake. For example,
its activation by hypoglycaemia (low glucose) stimulates
feeding behaviour, consistent with the idea that AMPK
represents an ancient starvation response system.
Here, I focus on recent insights into the regulation of
the mammalian and fungal AMPK/SNF1 systems that
have been provided by structural studies. I also discuss
selected recent findings that concern the downstream
processes regulated by mammalian AMPK, especially
glucose transport, transcription, cell growth and pro­
liferation, and the establishment and maintenance
of cell polarity. Insights gained from studies in other
eukaryotes such as plants, nematode worms and insects
are also considered. The role of AMPK in the metabolic
responses to exercise, in the regulation of whole-body
energy balance, or as a target for drugs used in the
treatment of metabolic disorders such as obesity and
diabetes, have been recently reviewed elsewhere7,15–17.
nature reviews | molecular cell biology
Regulation and structure of AMPK
All AMPK/SNF1 kinases appear to exist as hetero­
trimeric complexes comprising catalytic α-subunits
and regulatory β- and γ-subunits (FIG. 3). The mamma­
lian kinases are activated by AMP in two ways. First, the
kinase activity that resides in the α-subunit is stimulated
by the binding of AMP to the γ-subunit. Second, AMP
binding to the γ-subunit also promotes phosphoryla­
tion of a Thr residue within the kinase domain (Thr172
in the rat α-subunits), the phosphorylation of which
is essential for activity18. The combination of the two
effects causes >1000-fold increase in kinase activity19.
Although it was previously thought that AMP promoted
phosphorylation20 and inhibited dephosphorylation21,
recent work suggests that it works entirely by inhibiting
the dephosphorylation of Thr172, probably catalysed by
a form of protein phosphatase-2C22. Whatever the exact
mechanism, the effects of AMP to activate the kinase
directly and to promote phosphorylation are antago­
nized by high concentrations of ATP21,23. Therefore, the
kinase acts as an energy sensor.
Because of the adenylate kinase reaction (FIG. 1), the
AMP:ATP ratio is a more sensitive indicator of cellular
energy status than the ADP:ATP ratio. The finding that
AMPK activation is greatly reduced during contraction
of muscles from mice that are deficient in adenylate
kinase24 supports the important role of that enzyme in
the generation of the activating signal. One puzzling
finding is that the S. cerevisiae SNF1 complex is not
activated by AMP in cell-free assays, despite the fact that
there are large increases in the AMP:ATP ratio in vivo
under conditions where it is activated6,25. Although the
yeast kinase contains a residue equivalent to Thr172
(Thr210) that must be phosphorylated for activity26,
exactly how the SNF1 complex is activated in response
to glucose starvation remains unclear.
An important recent development was the determina­
tion of a crystal structure for the core of the αβγ complex
from Schizosaccharomyces pombe27 (FIG. 4). This structure
contains the whole γ-subunit but only the C‑terminal
domains of the α- and β-subunits. Unfortunately, we
know almost nothing about the properties of the enzyme
from S. pombe, but this structure does define the core
interactions between the three subunits, which are likely
to be conserved between different eukaryotes.
The α-subunits: catalytic subunits. Two α-subunit iso­
forms (α1/α2) are encoded by distinct genes in mammals
(PRKAA1 and PRKAA2) whereas in budding yeast, only
one isoform exists, encoded by the single gene SNF1.
All have conventional Ser/Thr kinase domains at the
N terminus, with the conserved Thr residue that must be
phosphorylated for activity in the activation loop18, which
is a common feature in many protein kinases (FIG. 3). The
structures of the kinase domains from S. cerevisiae Snf1
(Ref. 28) (Protein Data Bank (PDB) ID 2FH9) and human
α2 (PDB ID 2H6D) have recently been determined. They
contain the canonical two-lobed structure of eukaryotic
kinase domains, but in both structures the protein was
in an inactive, dephosphorylated state and the activation
loops were not fully resolved.
volume 8 | october 2007 | 775
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Adipocyte
Thiazolidinediones
PPARγ
Cannabinoids Low glucose
Leptin
Ghrelin
Resistin
Leptin
Adiponectin
IL-6
CNTF
Berberine
Resveratrol
EGCG
Muscle, liver, other cells
AMPK
Hypothalamus
Food intake
AMPK
Glucose
uptake ↑
Glycolysis ↑
AS160
EF2K
PFK2
ACC2 PGC1α
Fatty acid
oxidation ↑ Mitochondrial
biogenesis ↑
ACC1
HMGR
Fatty acid
synthesis ↓
GS
mTOR
Sterol
synthesis ↓
Protein
synthesis ↓
Glycogen
synthesis ↓
Figure 2 | Activation of AMPK by cytokines, drugs and polyphenols, and key downstream events. In the hypothalamus,
Nature
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| Molecular
Cell Biology
AMP-activated protein kinase (AMPK) is inhibited by agents that inhibit food intake (leptin)
and
is stimulated
by agents
that
stimulate food intake (cannabinoids, low glucose, ghrelin). In other cells such as liver and muscle, AMPK is inhibited by the
adipokine resistin, activated by the adipokines leptin and adiponectin, activated by other cytokines such as interleukin‑6
(IL‑6) and ciliary neurotrophic factor (CNTF), and by plant products such as berberine, resveratrol and (–)epigallocatechin‑3gallate (EGCG). The thiazolidinediones (for example, rosiglitazone and pioglitazone) may activate AMPK in a similar manner
to metformin (FIG. 1) at high concentrations, but their main effect appears to be to promote the release of adiponectin from
adipocytes by activation of peroxisome proliferator-activated receptor‑γ (PPARγ). Some of the major targets downstream
of AMPK, and their effects on energy metabolism, are also shown; not all of these are phosphorylated directly. Overall,
AMPK switches off ATP-consuming processes (such as protein synthesis, glycogen synthesis, sterol synthesis) and
upregulates processes that increase ATP (such as glycolysis, mitochondrial biogenesis and glucose uptake). ACC1, acetylCoA carboxylase‑1; ACC2, acetyl-CoA carboxylase‑2; AS160, AKT substrate of 160 kDa; EF2K, elongation factor‑2 kinase;
GS, glycogen synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; mTOR, mammalian target of rapamycin;
PGC1a, PPARγ co-activator‑1α; PFK2, 6-phosphofructo‑2-kinase.
Ubiquitin-associated
domain
A type of protein domain.
The role of some, but not all,
of these domains is to cause
association with ubiquitylated
or polyubiquitylated proteins.
α1–6-linked branch point
A branch point in α1–4-linked
glucans (such as starch or
glycogen) that is formed by a
linkage between carbon-1 (in
the α‑anomeric configuration)
of the glucose at one end of
the side chain, and carbon-6 of
a glucose unit on the main
chain to which the side chain is
attached.
α1–4-linked glucan
A polymer of glucose (for
example, amylose) that is
formed by linkages between
carbon-1 of one glucose unit
and carbon-4 of the next, with
all glucose units in the
α‑anomeric configuration.
A region that is C‑terminal to the kinase domain
appears to act as an autoinhibitory sequence (AIS) that
represses kinase activity, because bacterially expressed
constructs that contain the kinase domain are >10-fold less
active when they also contain the AIS29,30. The AIS shows
some sequence similarity with the ubiquitin-associated
domains that are found in the same position in several
members of the AMPK-related kinase family31.
The C‑terminal region of the α-subunit is required for
formation of the complex with the β- and γ-subunits29,32
and, in the crystal structure of the S. pombe core complex,
forms a compact domain around which the C‑terminal
region of the β-subunit (which is required for the forma­
tion of the mammalian complex33) is entwined (FIG. 4).
The extreme C terminus of the β-subunit in the S. pombe
structure forms two strands of a β-sheet with the γ-subunit
providing the third strand, whereas the contacts between
the α and γ subunits are more limited. The S. pombe
structure is consistent with previous models that have
been proposed for the mammalian enzyme, in which the
β- and γ-subunits interact directly with each other34,35.
An alternative model36, which predicts that the α- and
γ-subunits can interact in the absence of the β-subunit,
is not supported by the S. pombe structure.
The β-subunits: scaffolds with glycogen-binding domains.
Two β-subunit isoforms (β1 and β2) are encoded by
distinct genes in mammals (PRKAB1 and PRKAB2),
whereas there are three β-subunit genes (SIP1, SIP2
776 | october 2007 | volume 8
(Snf1-interacting protein‑1 and ‑2) and GAL83 (galactose
metabolism‑83)) in budding yeast. Because their
C‑terminal domains appear to bridge the α- and γ-subunits,
they can be regarded as protein ‘scaffolds’ on which
the AMPK complex assembles. The β-subunits also
contain a central carbohydrate-binding domain (FIG. 3)
that causes the mammalian αβγ complex to associate
with glycogen in intact cells33,37. The domain is related
to non-catalytic domains found in enzymes that meta­
bolize the α1–6-linked branch points in starch and glyco­
gen. The glycogen-binding domain of mammalian β1
has been crystallized in the presence of β‑cyclodextrin
(a circular α1–4-linked glucan of seven glucose units),
thus defining the carbohydrate-binding site38. However,
cyclodextrins do not occur naturally in mammals, and
it seems likely that the real physiological ligands might
be the branch points in glycogen. Although the physio­
logical function of glycogen binding is unknown, in
muscle cells it may function to localize the kinase
close to glycogen synthase — a substrate of AMPK.
Alternatively, AMPK may sense some aspect of glycogen
structure, and therefore respond to the medium-term
reserves of cellular energy in the form of glycogen,
as well as to the immediately available energy in the
form of AMP and ATP. Another interesting finding is
that this domain appears to be responsible for an inter­
action between AMPK and the glycogen debranching
enzyme39, although the physiological significance of
this remains unclear.
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α-subunits:
Upstream kinases
β-binding
P
N
Kinase
Glycogen
binding
β-subunits:
α-CTD
AIS
N
C
α-binding
β-CTD
GBD
C
γ-binding
Bateman domains
AMP/ATP binding
AMP/ATP binding
γ 1:
N
γ 2 (long): N
γ 3 (long):
N
CBS1
CBS2
CBS3
CBS4
C
γ2-NTD
CBS1
CBS2
CBS3
CBS4
C
γ3-NTD
CBS1
CBS2
CBS3
CBS4
C
CBS2
CBS3
CBS4
C
β-binding
Starch
binding?
Plant βγ subunits:
N
SBD
CBS1
Figure 3 | Domain structure of AMPK subunits. The figure is drawn approximately to
scale, and domains shown in the same colour are related
in sequence. The mammalian
Nature Reviews | Molecular Cell Biology
α1/α2 and β1/β2 isoforms are very similar and a generalized example is shown for each.
The α subunits have a C‑terminal domain (α-CTD) that is required for binding the βγ
subunits and an autoinhibitory sequence (AIS) that inhibits the activity of the kinase
domain. The kinase domain also contains the Thr residue that must be phosphorylated (P)
for activity. The β-subunits contain central glycogen-binding domains (GBD) and
C‑terminal domains (β-CTD) that are required for binding the α- and γ-subunits. The
three γ-subunit isoforms have variable N‑terminal domains (NTDs), a short region that is
required for binding to the β-subunit, and four conserved cystathionine β‑synthase
motifs (CBS1–4). The CBS motifs act in pairs to form two Bateman domains that bind AMP
or ATP. Some plants express unusual βγ subunits (bottom) that have a domain that is
related to the mammalian GBD (a postulated starch-binding domain (SBD)), which
appears to have fused with a γ-subunit. Plants also express β-subunits that contain just
the β‑CTD, as well as more conventional β- and γ-subunits.
Ventricular pre-excitation
A clinical condition in which the
delay between the excitation of
the atria (small chambers) and
ventricles (large chambers) of
the heart is reduced.
The γ-subunits: the AMP/ATP binding subunits. Three
γ-subunit isoforms (γ1, γ2, γ3) are encoded by distinct
genes in mammals (PRKAG1, PRKAG2 and PRKAG3),
whereas in budding yeast, only one isoform exists,
encoded by the single gene SNF4. The mammalian γ2
and γ3 isoforms contain unrelated N‑terminal exten­
sions (FIG. 3), which are subject to truncation by RNA
splicing and whose functions are currently unknown.
C‑terminal to these are short conserved regions that are
involved in the interaction with the β-subunit, as shown
by truncation experiments 35. Consistent with this,
in the S. pombe structure this region of the γ-subunit
forms the αB helix and the β1 strand, which interact
with the β2-β3 loop and the β3 strand, respectively, on
the β-subunit (FIG. 4). This region is followed by four
tandem repeats of a sequence of ~60 residues (FIGS 3,4),
which were first recognized by Bateman in the enzyme
cystathionine β‑synthase (CBS) and other proteins40,
and termed a CBS motif. These motifs act in pairs to
form two domains now referred to as Bateman domains.
nature reviews | molecular cell biology
When the Bateman domains (that is, CBS1–CBS2 and
CBS3–CBS4) from human γ2 were expressed separately,
they each bound one molecule of AMP and, as expected
from the antagonistic effects of ATP on kinase activation,
they also bound ATP in a mutually exclusive manner,
although with an affinity that was fivefold lower41.
When the tandem Bateman domains were expressed
together (CBS1–CBS2–CBS3–CBS4), they bound two
molecules of AMP or ATP in a highly cooperative
manner, suggesting that the second binding site only
becomes available when nucleotide has bound to the
first. Binding of ATP did not require Mg2+, which agrees
with the finding that no divalent metals were present
in the crystals of the S. pombe complex grown in the
presence of ATP, even when they were included in
the medium27. Because free ATP is present in cells at
concentrations that are almost two orders of magnitude
lower than the Mg2+–ATP complex, this helps to explain
how AMP in the micromolar range can effectively com­
pete with ATP for binding to the γ subunit, even though
the total cellular concentration of ATP is usually in the
millimolar range.
How does binding of AMP to the Bateman domains
cause allosteric activation of the αβγ complex? Many
protein kinases are inhibited by internal autoinhibitory
sequences that resemble the sequences at target sites for
the kinase (pseudosubstrate sequences), the effects of
which are relieved by binding of the activating ligand42.
Recently, myself and co-workers identified pseudo­
substrate sequences within CBS2 in the N‑terminal
Bateman domain of all eukaryotic γ-subunits43. These
sequences closely resemble the consensus recognition
motif for AMPK substrates 44, except that they have
non-phosphorylatable residues in place of Ser or Thr.
The pseudosubstrate sequence contains basic residues
that we propose are involved both in the interaction
between the pseudosubstrate sequence and the kinase
domain, and in the binding of AMP to the N‑terminal
Bateman domain. Because these two interactions
would be mutually exclusive, this suggests an elegant
mechanism for allosteric activation. In the absence of
AMP, the pseudosubstrate sequence would bind to the
substrate-binding groove on the kinase domain, which
inhibits kinase activity. AMP binding probably occurs
initially at the C‑terminal Bateman domain (as in the
S. pombe enzyme; FIG. 4). This then promotes binding
of the nucleotide at the N‑terminal domain, which pre­
vents the interaction of the pseudosubstrate sequence
with the kinase domain and therefore causes activa­
tion43. How this mechanism interfaces with the effect
of the AIS on the α-subunit (see above) remains unclear
at present.
Mutations in the γ-subunit cause heart disease. Several
point mutations in the human γ2 subunit isoform are
associated with heart disease41,45–47. Most cause a heredi­
tary, autosomal dominant form with an adult onset.
The main clinical feature of this disorder is ventricular
pre-excitation (Wolff–Parkinson–White syndrome) — a
premature excitation of the muscle of the large chambers
(ventricles) of the heart caused by abnormal electrical
volume 8 | october 2007 | 777
© 2007 Nature Publishing Group
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Glycogen-binding
domain
N
β-CTD
α-CTD
C
C
β2-β3 loop
β1 strand
αB helix
CBS1
CBS2
γ-NTD
N
CBS3
C
CBS4
AMP
Figure 4 | Structure of the core of the
Nature
Reviews
| Molecular
Cell Biology
Schizosaccharomyces
pombe
αβγ complex.
The
structures that have been solved27 contain the C‑terminal
domains (CTDs) of the α-subunit (yellow) and β-subunit
(red), and the entire γ-subunit. The crystals contained
dimers of heterotrimers, but only a monomer is shown. The
N‑terminal end of the α‑C-terminal domain (α-CTD), to
which the autoinhibitory and kinase domains would
be linked, lies in the centre to the rear in this view.
The N‑terminal domain of γ (γ-NTD) is shown in white and
its four cystathionine β‑synthase motifs (CBS1–4) are
shown in pale blue, magenta, green and orange,
respectively. A single molecule of AMP lies in the cleft
between CBS3 and CBS4 with the phosphate group at the
top in this view (C atoms in green, N in blue, O in red, P in
magenta; H atoms are omitted). The equivalent site in the
cleft between CBS1 and CBS2 is unoccupied. The main
interaction between the β- and γ-subunits is via the central
three-stranded β‑sheet with two strands (β2/β3, red) from
β and one strand (β1, white) from γ, but there are also
interactions between the αB helix on γ and the β2‑β3 loop
on β. Image created using the coordinates from Protein
Data Bank ID 2OOX in MacPyMOL100.
connections with the small chambers (atria). There
are also more severe forms of the disease that are not
inherited because they result in death from heart failure
or respiratory distress in early infancy. The common
feature of both types of disease is elevated storage of
glycogen in cardiac myocytes45–48, which, in the milder
forms, appears to affect the development of the electri­
cally insulating layer between the atria and ventricles49
and, in the severe forms, also appears to compromise
contractile function. When these mutations were made
in bacterially expressed Bateman domains, or in AMPK
heterotrimers expressed in mammalian cells, they inter­
fered with AMP binding and also reduced or abolished
AMP activation41,45,46,50. This provided the crucial evi­
dence that the Bateman domains represent the regulatory
AMP-binding sites.
778 | october 2007 | volume 8
At least eight different point mutations in the γ2 sub­
unit have been found to be associated with heart disease,
and five of them neutralize positively charged side chains
on conserved Arg or His residues in similar positions
in CBS motifs 1, 2 and 4. Modelling suggests that these
residues bind the negatively charged α-phosphate group
of AMP41,51. In agreement with this, the Arg residue in
CBS4 is conserved in the S. pombe γ-subunit (R290) and
in human γ1 (R299), and in recent crystal structures of
these domains the side chains of these Arg residues form
ionic interactions with the phosphate groups of AMP
or ATP27,52.
The effect of the γ2 mutations on AMPK activity are
complex because, as well as reducing binding of the activ­
ating nucleotide AMP (a loss-of-function effect), they
also reduce binding of the inhibitory nucleotide ATP
(a gain-of-function effect). Therefore, although they pre­
vent activation by AMP, they also appear to increase the
basal activity45,48, which explains why these mutations
are dominant in effect. A recent study of transgenic mice
expressing in cardiac muscle the N488I mutation (which
had been previously identified in a family affected by
pre-excitation syndrome and cardiac hypertrophy)
suggested that this causes increases in basal cardiac
glucose uptake, glucose‑6-phosphate content and glyco­
gen synthesis53. Although increased basal AMPK would
be expected to inactivate glycogen synthase owing to its
increased phosphorylation54, this inhibitory effect would
be overridden by the high levels of glucose‑6-phosphate,
an allosteric activator of glycogen synthase.
Some γ2 mutations have been reported to cause
elevated glycogen in skeletal muscle (where γ2 is also
expressed) as well as cardiac muscle, although this does
not seem to cause any clinical problems. Interestingly,
a mutation in the skeletal muscle-specific γ3 isoform
(R200Q, equivalent to R302Q in human γ2, a mutation
that causes heart disease) is associated with elevated
skeletal muscle glycogen content in pigs55. It is worth
pointing out that the γ1 isoform provides the majority
of AMPK activity in most cell types, including skeletal
and cardiac muscle. As yet, no mutations in γ1 have
been identified, possibly because they would be more
deleterious than γ2 or γ3 mutations. However, musclespecific expression of an R70Q mutant γ1 transgene in
mice (equivalent to R302Q in human γ2) also caused
elevated muscle glycogen56.
Because mutations in either Bateman domain reduce
or abolish activation by AMP, it seems likely that both
sites must be occupied for AMP activation to occur. It
was therefore surprising that in the structure of the core
complex from S. pombe, which had been crystallized in
the presence of high concentrations of AMP, only one
of the potential binding sites was occupied (FIG. 4) .
However, whether the S. pombe kinase is activated by
AMP like the human enzyme, or is insensitive to AMP
like the S. cerevisiae enzyme, is a question that remains to
be resolved. It seems most likely that the S. pombe enzyme
will also be AMP-insensitive because basic residues
that are required for AMP binding to the N‑terminal
Bateman domain of human γ2 (R302 and H383), which
are conserved in all γ-subunit sequences in species where
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AMP activation has been demonstrated (mammals,
Drosophila melanogaster and Caenorhabditis elegans),
are not conserved in S. pombe or S. cerevisiae.
Calmodulin
A small protein that binds
Ca2+, causing a conformational
change that causes the
complex to bind to and
activate many downstream
target proteins.
MAP kinase kinase kinase
A Ser/Thr protein kinase at the
head of a cascade of three
protein kinases, the final one
being a mitogen-activated
protein kinase such as ERK1 or
ERK2.
GLUT4
A member of the plasmamembrane glucose transporter
(GLUT) family expressed in
insulin-sensitive tissues such as
muscle and adipose tissue.
GLUT4 translocates to the
membrane in response to
insulin.
GTPase-activating protein
A protein that activates the
intrinsic GTP-hydrolysing
activity of small GTP-binding
proteins of the Ras/Rab family.
Rab protein
A member of the family of
small GTP-binding proteins
related to Ras, most of which
are thought to be involved in
the regulation of membrane
traffic.
Identification of upstream kinases
For many years, the identities of the upstream kinase(s)
responsible for phosphorylation of the critical Thr172
phosphorylation site on the α-subunit18 were elusive.
The initial breakthrough came from the yeast system,
where Sak1 (Snf1-activating kinase‑1, formerly known
as Pak1), Elm1 (elongated morphology‑1) and Tos3
(target of Sbf3) were identified as kinases that func­
tion upstream of the SNF1 complex by various wholegenome screening strategies26,57,58. There is sufficient
redundancy in the function of these three kinases such
that all three must be knocked out to generate the same
phenotype as a snf1 mutant57,58. Although there are no
clear orthologues of SAK1, TOS3 or ELM1 in the human
genome, the protein kinases with kinase domains closest
in sequence are LKB1 and the two calmodulin-dependent
protein kinase kinases, CaMKKα and CaMKKβ.
Subsequent studies revealed that both LKB1 (Refs 20,59)
and CaMKKs60–62, especially CaMKKβ, can act upstream
of AMPK in mammalian cells. A recent screen for
mammalian kinases that activate the SNF1 complex
when expressed in yeast has yielded a further candidate,
transforming growth factor‑β (TGFβ)-activated kinase‑1
(TAK1)63. TAK1 was originally identified as a MAP kinase
kinase kinase that acts upstream of members of the stressactivated MAP kinase family; whether activation of
AMPK by TAK1 is physiologically relevant remains
uncertain at present.
The discovery that LKB1 was an upstream kinase
for AMPK was particularly interesting because LKB1
was originally discovered as a tumour suppressor that is
mutated in an inherited susceptibility to human cancer,
Peutz–Jeghers syndrome64. LKB1 also functions upstream
of 12 other kinases (AMPK-related kinases) that fall on
the same branch as AMPK by phylogenetic analysis of
kinase domain sequences64,65. The roles of AMPK to
inhibit cell growth and proliferation and promote cell
polarity (see below) suggest that it could be responsi­
ble for the tumour-suppressor role of LKB1, although
the involvement of the other AMPK-related kinases
cannot be excluded at present. Although LKB1 must
be bound to two accessory subunits (STRAD (sterile‑
20-related adaptor) and MO25 (mouse protein‑25)) to
be functional, the protein kinase activity appears to be
constitutively active65,66. The trigger for increased phos­
phorylation of AMPK appears instead to be the binding
of AMP to AMPK, which inhibits dephosphorylation of
Thr172 (Refs 21,22).
The capability of CaMKKβ to act as an alternate
upstream kinase means that, in cells in which it is
expressed, signals that increase cytosolic Ca2+ would
be able to activate AMPK in the absence of an increase
in AMP. Increasing cytosolic Ca2+ often triggers ATPconsuming processes, such as activation of motor pro­
teins or membrane traffic, and Ca2+ must also be pumped
back out of the cytosol by ATP-driven membrane
pumps. Activation of AMPK could therefore be viewed
nature reviews | molecular cell biology
as a mechanism to anticipate the large increased demand
for ATP that usually accompanies Ca2+ release. Unlike
LKB1, which appears to be ubiquitous, the expression of
CaMKKs is more restricted. Both isoforms are expressed
predominantly in neural tissue, although CaMKKβ is
also found in cells of the endothelial or haematopoietic
lineage. Recently, AMPK has been shown to be activated
via the CaMKK pathway in response to K+-induced
depolarization in rat brain slices60, thrombin activa­
tion of endothelial cells67, and stimulation of the T-cell
receptor in T cells68: all of these are situations in which
Ca2+ is released into the cytosol and in which cellular
energy turnover is likely to increase rapidly following
stimulation.
Downstream functions of AMPK/SNF1
In general, AMPK activation stimulates ATP-producing,
catabolic pathways and inhibits ATP-consuming anabolic
pathways, both by rapid effects via direct phosphoryla­
tion of metabolic enzymes, and by longer-term effects
via regulation of transcription. Some of the metabolic
targets are shown in FIG. 2, and a more complete list of
AMPK targets is shown in TABLE 1. A more exhaustive
account of these targets and effects can be found in
recent reviews elsewhere7,15–17.
Regulation of glucose uptake. One key effect of AMPK
is its capability to stimulate glucose uptake in muscle by
increasing the translocation of the glucose transporter
GLUT4 to the plasma membrane69. Insulin is thought
to produce the same effect, at least in part, by phos­
phorylation of the AKT substrate of 160 kDa (AS160)
by the insulin-activated protein kinase, AKT/PKB
(protein kinase B)70. AS160 contains a GTPase-activating
protein (GAP) domain for small G proteins of the Rab
family, and insulin treatment appears to cause AS160
to dissociate from intracellular GLUT4 storage vesicles.
A current model is that AS160 dissociation allows for
activation of a Rab protein by GTP, which triggers the
docking and/or fusion of GLUT4 storage vesicles with
the plasma membrane (FIG. 5a). Intriguingly, activation
of AMPK causes phosphorylation of AS160 at some of
the same sites as insulin71,72, indicating that insulin and
AMPK might trigger GLUT4 translocation by intersecting
mechanisms.
Regulation of transcription. A paradigm for the mecha­
nism by which AMPK regulates gene expression came
from studies of glucose-repressed genes in S. cerevisiae,
involving phosphorylation of the transcription factor
Mig1 (FIG. 5b). Mig1 binds to and inhibits the promoters
of glucose-repressed genes, including the SUC2 gene that
encodes a secreted invertase enzyme, which is required
to metabolize sucrose. The removal of glucose from the
medium activates the SNF1 complex, which phospho­
rylates Mig1 at four sites73. Phosphorylation of Mig1
abolishes its interaction with the co-repressor complex,
Cyc8–Tup1, relieving repression74. At the same time,
phosphorylation triggers the interaction of Mig1 with
the nuclear export factor Msn5, causing its translocation
from the nucleus to the cytoplasm75 (FIG. 5b).
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Table 1 | Protein targets for which there is good evidence for direct phosphorylation by AMPK*
Protein target
Effect on protein function
Pathway
Tissue
Effect on pathway
ACC1
↓ activity
Fatty acid synthesis
All cells?
↓ fatty acid synthesis
ACC2
Lipid metabolism
↓ activity
Fatty acid oxidation
Muscle, liver
↑ fatty acid oxidation
HMGR
↓ activity
Isoprenoid synthesis
Liver
↓ cholesterol synthesis
HSL
↓ activity
Lipolysis
Adipose tissue
↓ lipolysis
AS160
↓ Rab-GAP?
GLUT4 trafficking
Muscle
↑ glucose uptake
Glycogen synthase
Carbohydrate metabolism
↓ activity
Glycogen synthesis
Muscle
↓ glycogen synthesis
PFK2 (cardiac isoform)
↑ activity
Glycolysis
Heart
↑ glycolysis
PFK2 (inducible isoform)
↑ activity
Glycolysis
Monocytes, macrophages
↑ glycolysis
Protein metabolism (translation)
EF2K
↑ activity
Protein synthesis
All cells?
↓ translation elongation
TSC2 (tuberin)
↑ Rheb-GAP
Regulation of TOR
All cells?
↓ translation initiation,
↓ cell growth,
↓ protein synthesis
eNOS
↑ activity
Nitric oxide production
Endothelial cells
↑ nitric oxide,
↑ increased blood flow?
IRS1
↑ PI 3‑kinase
Insulin signalling
All cells?
↑ insulin signalling?
p300
↓ interaction
Gene expression
All cells?
↓ transcription by nuclear
receptors
HNF4‑α
↓ DNA binding,
↑ degradation
Gene expression
Liver, others
↓ transcription
ChREBP
↓ DNA binding
Gene expression
Liver
↓ transcription of lipogenic
genes
TORC2
↑ cytoplasmic translocation
Gene expression,
localization
Liver
↓ transcription of
gluconeogenic genes
↓ channel opening
Ion transport, fluid
secretion
Airway, gut epithelium
↓ ion transport,
↓ fluid secretion
Cell signalling
Transcription
Ion transport/ion balance
CFTR
ACC1, acetyl-CoA carboxylase‑1; ACC2, acetyl-CoA carboxylase‑2; AS160, AKT substrate of 160 kDa; CFTR, cystic fibrosis transmembrane conductance regulator;
ChREBP, carbohydrate-response element-binding protein; EF2K, elongation factor‑2 kinase; eNOS, endothelial nitric oxide synthase; GLUT4, glucose transporter
type-4; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; HNF4-α, hepatocyte nuclear factor-4α; HSL, hormone-sensitive lipase; IRS1, insulin receptor
substrate‑1; PFK2, 6-phosphofructo‑2-kinase; PI, phosphatidylinositol; Rab-GAP, Rab GTPase-activating protein; Rheb-GAP, Ras homologue enriched in brain
GTPase-activating protein; TOR, target of rapamycin; TORC2, transducer of regulated CREB (cyclic AMP-responsive element binding) activity; TSC2, tuberous
sclerosis-2. *Detailed references can be found elsewhere16.
Cyclin
A protein that controls
progress through the cell
division cycle by binding to and
activating a cyclin-dependent
protein kinase.
G1–S boundary
An event in the cell division
cycle: the boundary between
the first phase (Gap 1) and the
second (S phase, when DNA
replication occurs). Quiescent
(non-dividing) cells are usually
arrested just before the G1–S
boundary.
In mammals, AMPK activation downregulates
expression of biosynthetic genes, such as those involved
in gluconeogenesis and lipogenesis in the liver, and
upregulates genes involved in catabolism, such as
GLUT4 and mitochondrial genes in muscle. These
effects are achieved by phosphorylation of numerous
targets, including transcription factors and co-activators
(TABLE 1). The expression of other transcriptional regula­
tors are upregulated (for example, the key co-activator for
mitochondrial biogenesis, PPARγ (peroxisome prolifera­
tor-activated receptor‑γ) co-activator‑1α (PGC1α)76,77)
or downregulated (for example, the lipogenic tran­
scription factor, sterol response element-binding
protein-1c (SREBP-1c)10), although the mechanisms for
these effects remain unclear.
780 | october 2007 | volume 8
Regulation of cell growth and proliferation. Because cell
growth and proliferation are energy-intensive processes,
it is not surprising that AMPK activation should inhibit
them. Pharmacological activation of AMPK inhibits the
growth of cancer cells, and causes phosphorylation of
p53 on Ser15 and accumulation of p53 and the cyclindependent kinase inhibitors, p21 and p27 (Refs 78,79).
Mouse embryo fibroblasts (MEFs) that are deprived
of glucose arrest at the G1–S boundary, and this effect
required phosphorylation of Ser18 on p53 (equivalent
to human Ser15) by AMPK, suggesting that the kinase
forms an ‘energy checkpoint’ that delays progress
through the cell cycle if insufficient energy is available80.
Recently, p27 was reported to become phosphorylated
at the C‑terminal residue Thr198 in response to AMPK
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a
b
Plasma
membrane
GLUT4
GLUT4
storage
vesicle
Plasma
membrane
P
P
SNF1
Rab–GTP
P
P
AS160
Rab–GDP
Low
glucose
P
PKB
P
Mig1
Cytoplasm
Mig1
Cyc8
SNF1
AMPK
Mig1
SUC2 promoter
Insulin
Metabolic
stress
Msn5
Cyc8
Tup1
Tup1
SUC2 coding sequence
Transcription of SUC2
Nucleus
Figure 5 | Models for regulation by AMPK/SNF1 of glucose uptake in muscle and gene expression in yeast.
Nature storage
Reviews vesicles.
| Molecular
Cell Biology
a | In resting muscle, the glucose transporter GLUT4 is mainly located in intracellular GLUT4
Activation
of
AKT/protein kinase B (PKB) by the insulin signalling pathway, or activation of the AMP-activated protein kinase (AMPK)
pathway by metabolic stress, causes phosphorylation of overlapping sets of sites on AKT substrate of 160 kDa (AS160). This
triggers dissociation of AS160 from the GLUT4 storage vesicle, preventing AS160 from converting the Rab protein to its
inactive GDP form. The activated Rab–GTP complex then promotes docking and/or fusion of the GLUT4 storage vesicle
with the plasma membrane. b | Regulation of expression of the SUC2 gene (which encodes a sucrose-hydrolyzing enzyme)
by the SNF1 complex in Saccharomyces cerevisiae. In high glucose conditions, the repressor protein Mig1 is bound to the
promoter of the SUC2 gene, to which it recruits the co-repressor Cyc8–Tup1 complex, thus repressing transcription.
Removal of glucose from the medium activates the SNF1 complex, which translocates to the nucleus and phosphorylates
Mig1 at multiple sites. This disrupts the interaction of Mig1 with Cyc8–Tup1, and also causes binding of Mig1 to the
nuclear export factor, Msn5. Mig1 is then exported from the nucleus and the SUC2 gene is expressed.
AU-rich element
A region in mRNA that is rich in
the bases adenine and uracil.
It is often a binding site for
proteins that control mRNA
degradation.
Autophagy
A process that occurs inside
cells in which cytoplasmic
components are engulfed by
membrane vesicles and
degraded. It is thought to be
used to recycle amino acids
and other components.
Anterior–posterior axis
The line between the head and
tail of an organism.
Apical–basal polarity
In epithelial cells, which
separate the interior of an
organism from the exterior or
the gut, the term apical–basal
polarity refers to the unequal
distribution of proteins and
other materials between the
apical side (facing the exterior
or the gut) and the basal side
(facing the interior).
activation, although, similar to the phosphorylation
of Ser15/18 on p53, it is not yet clear whether this is a
direct phosphorylation. However, phosphorylation of
p27 appears to stabilize the protein, which contributes
to cell-cycle arrest81.
Another mechanism by which AMPK may cause
cell-cycle arrest is by preventing nuclear export of the
RNA-binding protein human antigen R (HuR). This
reduces the binding of HuR to AU‑rich elements, which is
required to stabilize mRNAs that encode vital cell-cycle
regulators such as cyclins A and B1 (Ref. 82). Both the
p53 and the HuR pathways appear to be involved in
the ability of AMPK to promote senescence in fibro­
blasts, which is associated with an elevated cellular
AMP:ATP ratio80,83.
As well as causing cell-cycle arrest, AMPK activation
also inhibits cell growth. It can achieve this, in part, by
its classical effects that inhibit lipid synthesis, but also
by switching off protein synthesis by two pathways
(FIG. 2). These are the activation of elongation factor‑2
kinase, which causes inhibition of the elongation step of
translation84, and inhibition of the target-of-rapamycin
(TOR) pathway, which stimulates the initiation step
of protein synthesis by phosphorylation of multiple
targets85. TOR is activated by an upstream pathway
involving the TSC1–TSC2 (tuberous sclerosis complex)
heterodimer and the small Ras-related G protein Rheb
(Ras homologue enriched in brain). The TSC2 subunit
has a GTPase-activating protein (GAP) domain that
inhibits the ability of Rheb to activate TOR. AMPK
phosphorylates the TSC1–TSC2 complex and appears
to enhance its ability to switch off TOR86. By contrast,
AKT/PKB phosphorylates TSC1–TSC2 at different
nature reviews | molecular cell biology
sites in response to activation of the insulin/insulin-like
growth factor‑1 (IGF1) signalling pathway, and inhibits
its ability to switch off Rheb87. Therefore, insulin and
IGF1, which signify the availability of nutrients, and
AMPK, which signifies a lack of energy or nutrients,
have opposing effects on cell growth (FIG. 6).
Inhibition of the TOR pathway using rapamycin (the
antibiotic that led to the initial discovery of the pathway)
also stimulates autophagy. Therefore, it is not surprising
that autophagy is stimulated by activation of the SNF1
complex in S. cerevisiae88 and by AMPK in mamma­
lian cells89. Phosphorylation of p27 may be involved in
this effect81.
Establishment and maintenance of cell polarity. An
unexpected role for AMPK that has recently emerged is
in the regulation of cell polarity, especially in epithelial
cells. In D. melanogaster, mutations in the gene encoding
the upstream kinase LKB1 are embryonic lethal. LKB1
is required for polarization of the oocyte cytoskeleton
that defines the embryonic anterior–posterior axis, as well
as for apical–basal polarity in epithelial cells90. Consistent
with this finding, activation of LKB1 in mammalian
intestinal epithelial cell lines by inducing expression
of its accessory subunit, STRAD, results in cell polari­
zation, with reorganization of the actin cytoskeleton
and the formation of tight junctions and brush border
membranes91. Because some of the other protein kinases
downstream of LKB1 were known to have roles in the
establishment of cell polarity (especially MARKs,
the mammalian homologues of C. elegans PAR‑1), it had
been assumed that the effects of LKB1 on cell polarity
were probably not mediated by AMPK. However, this
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Insulin
IRS1
PtdIns(4,5)P2
Pi
PI3K
PTEN
PtdIns(3,4,5)P3
PDK1
Rheb–GDP
PKB/AKT
TSC2
TSC1
ATP
AMP
Stress
AMPK
Pi
TOR
Protein synthesis,
cell growth
Rheb–GTP
LKB1
Figure 6 | The opposing effects of insulin and AMPK activation on the target-of-rapamycin (TOR) pathway. Insulin
Nature Reviews | Molecular Cell Biology
activates phosphatidylinositol 3‑kinase (PI3K) by phosphorylation of insulin receptor substrate‑1 (IRS1) by the insulin
receptor. PI3K catalyses the conversion of phosphatidylinositol‑4,5-bisphosphate (PtdIns(4,5)P2) to phosphatidylinositol‑
3,4,5-trisphosphate (PtdIns(3,4,5)P3), an effect that is reversed by PTEN (phosphatase and tensin homologue deleted on
chromosome ten). PtdIns(3,4,5)P3 triggers the phosphorylation of protein kinase B (PKB/AKT) by its upstream kinase
phosphoinositide-dependent kinase‑1 (PDK1). Activated PKB then phosphorylates tuberous sclerosis‑2 (TSC2), which
inhibits its Ras homologue enriched in brain (Rheb)-GTPase activating protein (GAP) activity and promotes the activation
of TOR by Rheb–GTP. TOR, in turn, promotes protein synthesis and cell growth. Conversely, AMP-activated protein kinase
(AMPK) is activated by metabolic stresses that increase the AMP:ATP ratio, causing binding of AMP to AMPK, promoting
net phosphorylation of Thr172 by the protein kinase LKB1 (this effect involves inhibition of dephosphorylation, see main
text). AMPK phosphorylates TSC2 at different sites from PKB, stimulating the Rheb-GAP activity of the TSC1–TSC2
complex and inhibiting activation of TOR. Proteins shown in blue are tumour suppressors, for which loss-of-function
mutations cause TOR activation.
view may have to be modified. Intriguingly, activation
of AMPK appears to be required for repolarization of
Madin–Darby canine kidney cells in response to changes
of extracellular calcium92,93. Activation of AMPK in an
intestinal epithelial cell line by depletion of ATP with
deoxyglucose also induces polarization94. Because the
maintenance of cell polarity requires energy, it may
seem counter-intuitive that a protein kinase that is
switched on during negative energy balance should
promote it. However, the maintenance of the perme­
ability barrier provided by epithelia is clearly crucial,
and this may be one case where the AMPK system is
diverting what limited energy is available to a critical
survival function.
AMPK orthologues in other eukaryotes
Although studies of mammalian cells and budding
yeast have provided the most detailed insights into the
AMPK/SNF1 systems, it is worth briefly considering
some interesting findings that have been made in other
eukaryotic systems.
Dauer larval form
An alternative developmental
stage in worms, which is
activated under stressful
conditions. Dauer larvae are
sterile and are adapted for
long-term survival.
Green plants. Genes encoding α-, β- and γ-subunit
isoforms of AMPK are found in all plant genomes,
although there are also intriguing gene products in
which the carbohydrate-binding domains (which, pre­
sumably, bind starch in plants) appear to have switched
from the β- to the γ-subunits95 (FIG. 3, bottom). When
the two catalytic subunit genes were knocked out in a
primitive green plant, the moss Physcomitrella patens,
the plants survived if maintained in 24 hour light, but
died if subjected to a normal alternate light/dark cycle96.
Darkness is, of course, the equivalent of starvation for a
photosynthetic organism, and these results indicate that
the primary role of the plant kinases is the response to
starvation of a carbon source, as in yeast.
782 | october 2007 | volume 8
Caenorhabditis elegans. C. elegans has two genes
(AMP-activated kinases (aak)‑1 and ‑2) that encode
homologues of the AMPK/SNF1 catalytic subunit, and
AMPK complexes from this organism are activated by
AMP97. Application of a sublethal stress during early
life, such as high temperature, starvation or treatment
with mitochondrial inhibitors, can extend subsequent
lifespan. Intriguingly, aak‑2 mutants have a 12% shorter
lifespan than the wild-type but, more dramatically, the
effects of heat stress to increase lifespan are abolished,
and its effects to decrease fertility are also reduced.
The aak‑2 mutants also do not display the extension of
lifespan that is induced by reduced function mutations
in the insulin-like receptor DAF‑2, which signals the
availability of nutrients97. Thus, AAK‑2 appears to be
required for the sensing of starvation and other stresses
in early life, which leads to subsequent extension
of lifespan.
More recently, the aak‑1/‑2 and the par‑4 genes (the
latter encoding LKB1) have been shown to be required
for the extended cell-cycle arrest of germ cells98 — char­
acteristic of the long-lived but sterile dauer larval form
— which is induced by environmental stress. These
results are compatible with the ‘disposable soma’ theory
of ageing, which proposes that lifespan in multicel­
lular organisms is a compromise between the energy
expenditure required to maintain integrity of somatic
cells and the necessity to live long enough to reproduce.
An extension of this is the proposal that AMPK delays
ageing in response to stresses during early life, in order
to allow reproduction at a later date when conditions
improve. These findings raise the possibility that AMPK
is involved in the extension of lifespan induced by caloric
restriction in mammals and, consistent with this, the
AMPK activator resveratrol increases lifespan in mice
that are fed a high-fat diet13.
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REVIEWS
Drosophila melanogaster. The D. melanogaster genome
encodes single isoforms of the α-, β- and γ-subunits
of AMPK. D. melanogaster AMPK is activated by AMP,
and all three subunits are essential for activity99. In cul­
tured cells, the α-subunit is phosphorylated at Thr184
(equivalent to Thr172 in the mammalian α-subunit)
and, consequently, is activated in response to inhibi­
tors of ATP synthesis99. Recently, the catalytic subunits
of AMPK have been knocked out in D. melanogaster
in vivo. As already discussed above, mutations in LKB1
caused defects in cell polarity in the early embryo92 and
the phenotype of the AMPK null mutant was essen­
tially identical94. In the same study, it was reported
that AMPK phosphorylates the regulatory light chain
of myosin (MRLC) at Ser22, which is known to trig­
ger movement of the myosin motor protein on actin
fibres. Intriguingly, the defects in embryonic cell
polarity of LKB1 and AMPK mutations were rescued
by expression of a phosphomimetic mutant (T21E/
S22E) of MRLC 94. This is remarkable given that it
is likely that AMPK has tens, if not hundreds, of dif­
ferent targets in D. melanogaster. It seems likely that
the phosphorylation of MRLC will also have a role in the
polarization of mammalian epithelial cells, as already
discussed above.
Conclusions and remaining questions
For a unicellular eukaryote such as S. cerevisiae, it can
be argued that the most important factor regulating cell
growth and proliferation is the availability of its favoured
carbon source, glucose. However, with the development
of multicellular eukaryotes, glucose deprivation became
less significant at the cellular level because of the sophis­
ticated systems that maintain glucose homeostasis in
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Acknowledgements
Recent studies in the author’s laboratory have been supported by Programme Grants from the Wellcome Trust and
by the EXGENESIS Integrated Project of the European
Commission.
Competing interests statement
The author declares no competing financial interests.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
PRKAA1 | PRKAA2 | PRKAB1 | PRKAB2 | PRKAG1 | PRKAG2 |
PRKAG3 | SNF1
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
Peutz–Jeghers syndrome | type 2 diabetes |
Wolff–Parkinson–White syndrome
Protein Data Bank: http://www.pdb.org/pdb/home/home.do
2FH9 | 2H6D | 2OOX
UniProtKB: http://ca.expasy.org/sprot
LKB1
FURTHER INFORMATION
D. Grahame Hardie’s homepage:
http://www.lifesci.dundee.ac.uk/people/grahame_hardie
All links are active in the online pdf.
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