(Received for publication, October 23, 1995)
From the
The mammalian 5`-AMP-activated protein kinase (AMPK) is related
to a growing family of protein kinases in yeast and plants that are
regulated by nutritional stress. We find the most prominent expressed
form of the hepatic AMPK catalytic subunit () is
distinct from the previously cloned kinase subunit (
).
The
(548 residues) and
(552
residues) isoforms have 90% amino acid sequence identity within the
catalytic core but only 61% identity elsewhere. The tissue distribution
of the AMPK activity most closely parallels the low abundance
6-kilobase
mRNA distribution and the
immunoreactivity rather than
, with substantial
amounts in kidney, liver, lung, heart, and brain. Both
and
isoforms are stimulated by AMP and contain
noncatalytic
and
subunits. The liver
isoform accounts for approximately 94% of the enzyme activity
measured using the SAMS peptide substrate. The tissue distribution of
the
immunoreactivity parallels the
8.5-kilobase mRNA and is most prominent in skeletal muscle,
heart, and liver. Isoforms of the
and
subunits present in
the human genome sequence reveal that the AMPK consists of a family of
isoenzymes.
The 5`-AMP-activated protein kinase (AMPK) ()was
initially identified as a protein kinase regulating
hydroxymethylglutaryl-CoA reductase(1) . Subsequently, the AMPK
was shown to phosphorylate hepatic acetyl-CoA carboxylase (2) and adipose hormone-sensitive lipase(3) . The AMPK
appears to act as a metabolic stress-sensing protein kinase switching
off biosynthetic pathways when cellular ATP levels are depleted and
when 5`-AMP rises in response to fuel limitation and/or
hypoxia(4) . Partial amino acid sequencing of hepatic AMPK (5, 6) revealed that it consists of 3 subunits, the
catalytic subunit
(63 kDa), and two noncatalytic subunits,
(40 kDa) and
(38 kDa).
The AMPK is a member of the yeast SNF1
protein kinase subfamily that includes protein kinases present in
plants, nematodes, and humans (5, 6, 7, 8, 9) . The AMPK
catalytic subunit, , has strong sequence identity to the catalytic
domain of the yeast protein kinase SNF1, which is involved in the
induction of invertase (SUC2) under conditions of nutritional stress
due to glucose starvation(10) . Both Snf1p and the AMPK require
phosphorylation by an activating protein kinase for full
activity(11) . The sequence relationship between Snf1p and AMPK
led to the finding that these enzymes share functional similarities,
both phosphorylating and inactivating yeast acetyl-CoA
carboxylase(5, 11, 12) . Nevertheless, the
AMPK does not complement SNF1 in yeast(11) , indicating that
their full range of functions are not identical. The noncatalytic
and
subunits of AMPK are also related to proteins that interact
with Snf1p; the
subunit is related to the SIP1/SIP2/GAL83 family
of transcription regulators and the
subunit to SNF4 (also called
CAT3)(6, 13) .
In the course of sequencing the porcine AMPK, we found that
the amino acid sequence of some peptides derived from the pig liver
AMPK subunit did not match those deduced from the rat liver cDNA
sequence(7, 8) . Therefore, the rat liver AMPK
catalytic subunit,
, was purified, and peptides accounting for 40%
of the protein were sequenced (222/548 residues, Fig. 1). Eight
of the sixteen peptides contained mismatched residues with the reported
AMPK cDNA sequence, but did match the pig liver enzyme sequence. Using
reverse transcription-polymerase chain reaction and cDNA library
screening, we obtained cDNA sequence of the rat hypothalamus enzyme
that accounted for all of the peptide sequences of the purified rat
liver AMPK catalytic subunit containing mismatches (Fig. 1). The
cDNA sequence of this AMPK catalytic subunit has been named
, since it corresponds to the purified enzyme and is
clearly derived from a gene different from the previously cloned
sequence (now referred to as
). The
isoform of the AMPK catalytic subunit accounts for approximately
94% or more of the SAMS peptide phosphotransferase activity of rat
liver and is therefore the predominant expressed hepatic isoform.
Despite sequencing multiple preparations of the AMPK catalytic subunit
from both pig and rat liver, no peptides were obtained that matched the
isoform sequence.
Figure 1:
Comparison of AMPK ,
, and peptide sequence. The alignment of rat AMPK
cDNA-derived amino acid sequence (rAlpha 1)
to human AMPK
cDNA-derived amino acid sequence (hAlpha 1), rat
cDNA-derived amino acid
sequence (rAlpha 2)(8) , and rat and porcine
peptide sequences (rat aa and porcine
aa) is shown. Amino acids that could not be identified confidently
are represented with an X. The sequences, aligned with the
Pileup program (GCG, University of Wisconsin and (26) ), were
formatted with the residues identical with the AMPK
cDNA-derived sequence being shaded.
Within the catalytic cores of
the and
isoforms, there is 90%
amino acid identity but only 61% identity outside the catalytic core (Fig. 1). Strong homology between the
and
sequences in the vicinity of the substrate binding
groove, inferred from the protein kinase crystal structure for
positions P
to P
(17) ,
suggest that the substrate specificities will be related. The substrate
anchoring loop (also called the lip or activation loop) contains an
insert Phe-Leu
for
,
,
and Snf1p that may provide a hydrophobic anchor for a P
or P
hydrophobic residue in the peptide
substrate. There is also Glu
(Glu
in
cAMP-dependent protein kinase) and Asp
available for a
P
basic residue specificity determinant for the
,
, and Snf1p. Both isoforms contain
a Thr
residue equivalent to Thr
in the
cAMP-dependent protein kinase, which is likely to be phosphorylated and
necessary for optimal activity. Since the major differences in the
and
sequences occur in their
COOH-terminal tails, they may interact with different proteins within
this region.
The 8.5-kb mRNA is most abundant in
skeletal muscle with lower levels in liver, heart, and kidney as
reported recently(8, 18) . In contrast, there were
very low levels of the
6-kb mRNA in all tissues
examined except testis, where a low level of 2.4-kb mRNA was observed (Fig. 2A). A testis-specific kinase related to Snf1p
has been reported(19) , but the corresponding transcript is 1.6
kb and may not be related to the 2.4-kb transcript seen here. The low
levels of
mRNA explains why
was
more difficult to clone than the
isoform (Fig. 2B). Northern blot analysis of the
and
subunits revealed a complex pattern of expression. The
subunit mRNA was least abundant with similar levels across a range of
tissues except brain, whereas the
subunit mRNA was abundant in
heart, lung, skeletal muscle, liver, and kidney. (
)An
earlier report on the tissue distribution of the AMPK activity had
claimed that it was predominantly a liver enzyme(15) . In view
of the mRNA distribution of the
and
subunits,
we reassessed the tissue distribution of the AMPK activity. The kidney
contained the highest specific activity with similar levels in the
liver, lung, and heart (Fig. 3) and little, if any, activity in
skeletal muscle. It is clear that the AMPK activity has a wider tissue
distribution than appreciated heretofore(15) , and this closely
parallels the distribution of
mRNA and not that of
mRNA. Using peptide-specific antisera to
(residues 339-358) and
(residues
352-366), we found that the
immunoreactivity
was predominant in the heart, liver, and skeletal muscle (Fig. 2E) where there is also the highest
concentrations of
mRNA. In contrast, the
immunoreactivity is widely distributed (Fig. 2D)
as is the less abundant
mRNA. The antibody to
recognized a minor component in the purified
preparation (Fig. 2E, lane
1), but sufficient amounts of this have not been obtained to
determine whether it represents weak cross-reactivity with a form of
, an additional isoform of the AMPK or a low level
contaminant of the
preparation by the
isoform. The antibody to
does not
immunoprecipitate
activity from affinity-purified
AMPK. Both
and
migrate on SDS-PAGE at approximately 63 kDa (Fig. 2, D and E). Unexpectedly, we found that the liver
immunoreactivity was not bound by the peptide substrate affinity
column. This column specifically binds the
isoform.
Using immune precipitation of the effluent from the peptide substrate
affinity column with
specific antibody, we found that
the
isoenzyme contained
and
subunits (Fig. 4) and catalyzed the phosphorylation of the SAMS peptide.
Immune precipitates of
and
showed
variable activation by 5`-AMP ranging from 2-3- and
3-4-fold, respectively. There was also an approximate 60-kDa band
recognized by the
-specific antibody in tissue
extracts from heart and lung (Fig. 2D). This band is
not present in the purified liver enzyme, and its relationship to the
isoform is not yet known.
Figure 2:
Distribution of rat AMPK isoforms:
mRNA and protein. A rat multiple tissue Northern (MTN) blot (Clontech)
containing 2 µg of poly(A)
RNA of individual
tissues was successively probed and is shown in A-C. A,
MTN hybridized with a 2.3-kb rat
cDNA.The blot was
washed with 2
SSC, 0.5% SDS at 42 °C for 3
20 min. B, MTN hybridized with a 1.6-kb rat
cDNA.
The blot was washed with 2
SSC, 0.5% SDS at 42 °C and 0.1
SSC, 0.5% SDS at 65 °C for 30 min. C, MTN
hybridized with a 2-kb
-actin cDNA, the blot was washed with 2
SSC, 0.5% SDS at 42 °C for 2
20 min. RNA markers
are shown in kilobases. A rat multiple tissue Western blot containing
100 µg of protein/lane is shown in D and E. D was
probed with affinity-purified
peptide serum, and E was probed with affinity-purified
peptide
serum. The
control lane represents 0.1 µg of
purified rat liver AMPK(5) , and
represents
0.1 µg of expressed AMPK
(J. Dyck, unpublished
data). Protein markers are shown in kDa. Legend: H, heart; B, brain; Sp, spleen; Lu, lung; Li,
liver; Sk, skeletal muscle; K, kidney; and T, testis.
Figure 3:
Distribution of AMPK activity in rat
tissues. AMPK activity, measured as described under ``Experimental
Procedures,'' is expressed as nanomoles of P
transferred to SAMS peptide substrate per min per mg of protein.
Legend:
, -AMP;
,
+AMP.
Figure 4:
SDS-PAGE analysis of purified rat AMPK
and
. Samples were analyzed by
SDS-PAGE (13%, w/v, acrylamide). Shown is the unbound fraction from the
substrate affinity column (flow-through), enzyme eluted from the
substrate affinity column (AMPK
), and enzyme eluted
from the
specific antibody column (AMPK
) as described under ``Experimental
Procedures.'' Molecular masses of the AMPK subunits are shown in
kDa.
The proportion of SAMS
peptide phosphotransferase activity bound to the peptide affinity
column with a single pass varied (ranged 90-92%, n = 7, and 74-86%, n = 6 rat liver
preparations). With recycling, approximately 94% of the activity was
bound to the column. The residual activity was attributable to
isoform activity based on immunoprecipitation with
the
-specific antibody. However, the amount of protein
immunoprecipitated based on Coomassie Blue staining (Fig. 4)
indicated that there was substantially more
protein
than was expected from only 6% of the total SAMS peptide activity. The
specific activity of the
isoform is not yet known in
the absence of bound antibody. Based on the
cDNA
sequence, Carling et al.(7) reported that a peptide
specific antibody immunoprecipitated virtually all of the partially
purified AMPK activity from liver. The peptide used in their
experiments, PGLKPHPERMPPLI, contains 8/15 residues that are identical
(underlined) between
and
so it
seems reasonable that their polyclonal antisera may recognize both
isoforms. In this event, their immunoprecipitation data are consistent
with our results.
The present work makes plain that there is an
isoenzyme family of AMPK catalytic subunits, increasing the
complexity of activity analysis. This also raises the question of what
function the
isoform has and whether
associates with a specific subset of
and
subunits. A
significant fraction of the
isoform mRNA has a
142-base pair out-of-frame deletion within its catalytic domain that
would encode a truncated, nonfunctional
protein(8, 18) . The close sequence relationship
between the
isoforms from pig, rat, and human (Fig. 1) means that there is strong conservation across species.
Previously, it was reported that human liver does not contain AMPK
mRNA(20) ; however, it is now clear that
mRNA
was being probed for and not the dominant
isoform
mRNA. The gene encoding the human liver AMPK catalytic subunit reported
on chromosome 1 (20, 21) is therefore the gene for the
isoform, whereas the gene for the
isoform is located on chromosome 5.
Recent genome
sequencing has revealed multiple isoforms of the noncatalytic and
subunits of the AMPK. There appear to be at least three isoforms
of the
subunit in brain with
and
present, distinct from the rat liver
isoform.
Human brain also contains multiple
subunit isoforms distinct from
the rat liver
isoform. The accession numbers for
putative AMPK
and
subunit isoforms are:
,
M78939;
, R35524;
, R20494;
, R14746. Thus, a potentially large subfamily of
heterotrimeric AMPKs, based on various combinations of all three AMPK
subunits, may be present.
The structural relationships between the
AMPK and SNF1 kinase, as well as the presence of multiple isoforms,
brings into focus a vista of questions concerning the diverse
physiological roles of this new subfamily of protein kinases. Whereas
the AMPK regulates lipid metabolism in hepatocytes under conditions of
metabolic stress, its role in other tissues, including the heart and
kidney, are unknown. Recent studies by Kudo et al. (22) have shown that the AMPK is activated during cardiac
ischemia, and the activation persists during reperfusion, possibly
contributing to the ischemia-driven decoupling of metabolism and
cardiac mechanical function. Regulation of cardiac acetyl-CoA
carboxylase by AMPK plays an important role in the switching of cardiac
metabolism between the use of glucose and fatty acids as oxidative
fuel(23) . In the cell of the pancreas, where AMPK
subunits are highly expressed in islet cells, (
)glucose
availability rapidly regulates acetyl-CoA carboxylase through changes
in AMPK-directed phosphorylation, suggesting strongly a role for AMPK
in stimulus-secretion coupling for insulin release(24) . In
addition to these metabolic roles, members of the SNF1 protein kinase
subfamily appear to play important roles in development, with the par-1 gene of Caenorhabditis elegans playing an
essential role in embryogenesis(25) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40819[GenBank].