(Received for publication, December 20, 1995)
From the
The mammalian 5`-AMP-activated protein kinase (AMPK) is a
heterotrimeric protein consisting of -,
-, and
-subunits. The
-subunit is the catalytic subunit and is
related to the yeast Snf1p kinase. In this study, we report the cloning
of full-length cDNAs for the non-catalytic
- and
-subunits.
The rat liver AMPK
-subunit clone predicts a protein of 30,464 Da,
which is related to the Sip1p, Sip2p, and Gal83p subfamily of yeast
proteins that interact with Snf1p and are involved in glucose
regulation of gene expression. The AMPK
-subunit, when expressed
in bacteria and in mammalian cells, migrates anomalously on SDS gels at
an apparent molecular mass of 40 kDa. Rat and human liver AMPK
-subunit clones predict a protein of 37,577 Da
(AMPK-
), which is related to the yeast Snf4p protein
that copurifies with Snf1p and to a larger family of other human AMPK
-isoforms. The mRNAs for both AMPK-
and AMPK-
are widely expressed in rat tissues, consistent with a broad role
for AMPK in cellular regulation. These data reveal a mammalian
multisubunit protein kinase strikingly similar to the multisubunit
glucose-sensing Snf1 kinase complex. The identification of isoform
families for the AMPK subunits indicates the potential diversity of the
roles of this highly conserved signaling system in nutrient regulation
and utilization in mammalian cells.
The mammalian 5`-AMP-activated protein kinase (AMPK) ()and its homologs are expressed in plants, yeast, and
mammals(1, 2, 3, 4, 5) .
AMPK, a member of the SNF1 (sucrose non-fermentor) kinase
family(4, 5, 6, 7) , was first
recognized as a regulator of fatty acid and sterol synthesis through
its phosphorylation of acetyl-CoA carboxylase and
hydroxymethylglutaryl-CoA reductase, respectively(8) . In
particular, AMPK mediates responses of these pathways to several
metabolic or other cellular stresses, including glucose depletion, heat
shock, and ATP
depletion(9, 10, 11, 12) .
Purification of pig and rat liver AMPKs has revealed a
heterotrimeric kinase structure consisting of a 63-kDa catalytic
-subunit and non-catalytic
(40 kDa)- and
(38
kDa)-subunits(4, 6) . The AMPK
-subunit is 64%
identical in its catalytic core to the Saccharomyces cerevisiae Snf1p (
)protein kinase, which is responsible for the
glucose derepression response of the SUC1 gene(4, 5, 7, 13) . In contrast
to AMPK, Snf1p occurs as a heterodimer with Snf4p and does not purify
with other identified interacting proteins(4, 6) . We
have recently found that multiple isoforms of AMPK-
(
and
), which are products of distinct genes, are
present in liver and other tissues(14) . The AMPK
-isoform accounts for
90% of total AMPK activity
in liver extracts, yet its corresponding mRNA level is low relative to
that of the AMPK
-isoform. Preliminary peptide
sequencing and limited PCR product analysis of the non-catalytic
subunits have indicated that the AMPK
-subunit is related to the S. cerevisiae protein Snf4p (CAT3), (
)whereas AMPK-
is related to the S. cerevisiae Sip1p/Sip2p/Gal83p family of proteins. These are known to
associate with the Snf1p kinase and to participate in glucose-regulated
gene expression(6) .
In this study, we report the molecular
cloning of full-length cDNAs for the mammalian AMPK - and
-subunits. These clones have been used to characterize the tissue
distribution of subunit mRNA and to express subunit protein in both
bacteria and mammalian cells. Knowledge of their complete sequences has
also led to the identification of protein isoform families for each of
these non-catalytic units.
The
non-degenerate 23-mer cDNA was then used in conjugation with degenerate
primers constructed from two other peptide sequences to generate a
larger AMPK- cDNA by PCR. Both sense and antisense degenerate
oligonucleotide primers corresponding to the peptide sequences EELQIG
and FPKPEFM were used together with the sense MOPAC-derived
non-degenerate sequence to generate all possible PCR products, using
rat liver cDNA as template. The largest product (192 bp) obtained was
subcloned in pCR-Script (Stratagene) and sequenced. This sequence (see Fig. 4), which actually predicted amino acid sequence
corresponding to all three AMPK-
peptides used in the PCR
strategy, was then used for library screening as described above.
Screening of 2
10
plaques with this larger PCR
product yielded several positive clones, which were further
characterized (see below); however, none of the rat cDNAs (1-1.3
kilobases) isolated corresponded to a full-length open reading frame.
In an effort to extend the sequence to the 5`-end of the open reading
frame, a primer extension library was constructed using a
AMPK-
-specific antisense primer (Stratagene;
ZAPII).
Additional screening of this library, while yielding some 5`-extended
sequence, did not yield the start Met codon. The application of a
5`-RACE strategy with rat liver cDNA was also unsuccessful in attempts
at sequence extension, although a 5`-RACE product from porcine liver
was obtained (data not shown). The most 5` rat cDNA sequence (520 bp)
was then used to screen a human fetal liver library, which yielded a
full-length AMPK-
cDNA (see below).
Figure 4:
Nucleotide and deduced amino acid
sequences of AMPK-. Shown are the nucleotide and deduced amino
acid sequences of a human fetal liver AMPK-
cDNA. The start codon
is assigned, as indicated under ``Results.'' Peptide
sequences corresponding to those determined by analysis of peptides
derived from the rat and porcine purified proteins are underlined. Those sequences, which extend information
previously reported(6) , include newly determined amino acids
60-64, 111-113, 225-232, 255-259,
261-264, and 280-291. The original PCR-derived 192-bp rat
cDNA, used in screening of the rat liver cDNA library, extends from
nucleotides 650 to 841.
Figure 5:
Homology of human liver AMPK-, rat
liver AMPK-
, and yeast Snf4p. Shown are alignments of deduced
amino acid sequences of human (h) fetal liver AMPK-
, a
partial-length rat (r) liver AMPK-
, and S. cerevisiae Snf4p as configured by the Pileup program. Residues identical to
human fetal liver AMPK-
are boxed.
Figure 1:
Nucleotide and deduced amino acid
sequences of rat liver AMPK-. Shown is the nucleotide and deduced
amino acid sequences of the 1107-bp rat liver AMPK-
clone. The underlined peptide sequences correspond to those determined by
direct peptide sequencing of the isolated rat liver AMPK-
protein,
as previously reported (6) and as completed for this study (new
sequences include amino acids 55-57, 108-125,
161-181, and 183-199). Assignment of the start codon is
explained under ``Results.''
This clone contains an open reading frame
encoding for a 270-amino acid peptide, which contains all of the 15
independent (some overlapping) peptide sequences obtained from
extensive sequence analysis of the purified protein. The translational
start methionine codon is assigned from the typical Kozak sequence
present for a initiation codon (19) and the lack of any other
upstream in-frame methionine codons. While no in-frame stop codon is
present in this 5`-upstream sequence, a human expressed sequence tag
cDNA (GenBank accession number T78033; see below) in the
data base contains such a stop codon preceding the same assigned start
methionine codon. This reading frame, however, predicts a protein of
30,464 Da, well below the estimated molecular mass of 40 kDa evident on
SDS gel electrophoresis(4, 6) .
To clarify the size
of the protein product that could be synthesized from this cDNA, the
AMPK- clone was expressed both in bacteria and mammalian cells. As
shown in Fig. 2, in both expression systems, the protein product
migrates at a higher than predicted molecular mass. When purified as a
His
-tagged fusion protein from E. coli, the
isolated protein migrates on SDS gels with an apparent molecular mass
of
43,000 Da (the same as the ovalbumin standard). This
corresponds to a AMPK-
polypeptide product of 40 kDa with an
additional 3 kDa of fusion tag sequence derived from the pET vector.
When expressed in mammalian cells from an HA-tagged expression vector,
two polypeptides are evident, with the major product corresponding to a
40-kDa species (after correction for the size of the HA epitope tag). A
second product of 42-43 kDa is also evident using this expression
system. Taken together, these data demonstrate that the protein product
of this AMPK-
migrates on SDS-polyacrylamide gel electrophoresis
with an anomalously high molecular mass.
Figure 2:
Bacterial and cellular expression of
AMPK- and AMPK-
. In the upper panel is shown a
Coomassie Blue-stained SDS-polyacrylamide gel (9% acrylamide) of
fractions from a nickel affinity column obtained on elution of the
bacterially expressed His
-tagged AMPK-
fusion protein.
Shown left to right are molecular weight standards (MWS), the
inclusion body extract applied to the column (Load), a sample
of the column wash just prior to elution (Wash), and six
elution fractions obtained after increasing the imidazole concentration
to 300 mM. In the lower panel is shown a composite
anti-HA immunoblot following SDS-polyacrylamide gel electrophoresis of
lysates obtained from COS-7 cells after transfection with pMT2-HA
vector alone (lane 1), pMT2-HA-rat AMPK-
(lane
2; truncated cDNA), pMT2-HA-AMPK-
(lane 3),
pMT2-HA-human AMPK-
(lane 4; full-length cDNA), and both
pMT2-HA-AMPK-
and pMT2-HA-human AMPK-
(lane 5). Each
lane was loaded with lysate equivalent to 4% of total cells/well after
transfection. The migration positions of prestained molecular mass
standards are indicated to the right.
Comparison of the rat liver
AMPK- sequence to the data base reveals that it is highly
homologous to three yeast proteins (Sip1p, Sip2p, and Gal83p) and to
two recently cloned human expressed sequence tag cDNA sequences (Fig. 3). This alignment, as gapped according to the sequence of
the S. cerevisiae protein Sip1p (20) , is most
striking at the C terminus of AMPK-
and these yeast proteins.
Figure 3:
Yeast and human homologs of rat liver
AMPK-. Shown are alignments of the rat liver AMPK-
deduced
amino acid sequence with S. cerevisiae proteins Sip1p, Sip2p,
and Gal83p and with two partial-length human cDNAs in the data base
(GenBank
accession numbers T78033 and F11147), as matched
by BLAST searching. Sequences were aligned with the Pileup program of
the Genetics Computer Group software package and are gapped with
reference to the Sip1p sequence. Residues identical to AMPK-
are boxed. The NCBI data base also contains four other human
sequences that are either very similar or identical to the two human
sequences indicated (data not shown); these sequences are accessible as
R14746, H06094, R20494, and R25722.
A typical Kozak
translation initiation sequence surrounds the assigned methionine start
codon; this start is also in frame with a 5`-upstream stop codon. The
assigned start methionine codon is followed by an open reading frame
predicting a protein of 331 amino acids and of 37,546 Da, which
corresponds to the molecular mass observed on SDS gel electrophoresis
of the protein as purified from rat and porcine liver (4, 6) . Expression of a truncated rat AMPK- cDNA
(amino acids 33-331) and the full-length human AMPK-
(331
amino acids) in COS-7 cells yields products consistent with the
molecular mass predicted for each cDNA (34,081 and 37,577 Da,
respectively) (Fig. 2). The rat liver AMPK-
product
expressed in bacteria also displayed the molecular mass predicted by
the cDNA (data not shown). Thus, unlike AMPK-
, there is no
anomalous migration of the protein product of AMPK-
cDNA.
Comparison of the human and rat liver AMPK- amino acid
sequences to the data base yields a significant alignment of this
protein with S. cerevisiae Snf4p (Fig. 5). In addition,
our human (and rat; data not shown) full-length cDNA also aligns with
several other human partial-length expressed sequence tag cDNA
sequences from brain, breast, placenta, liver, and heart, recently
reported in the data base (Fig. 6). Inspection of these
sequences reveals that there are multiple isoforms of the human
AMPK-
protein. There are likely also similar AMPK
-isoform
families expressed in rat and pig. This latter expectation is based on
sequence analysis of 14 other MOPAC-derived partial AMPK-
cDNA
sequences, as identified on colony hybridization of the AMPK-
MOPAC products with
P-labeled degenerate oligonucleotides
(see ``Experimental Procedures''). These products showed at
least two reproducible patterns of nucleotide heterogeneity within the
non-degenerate core (data not shown).
Figure 6:
Identification of an AMPK -isoform
family. Shown are alignments of deduced amino acid sequences of human (h) fetal liver AMPK-
with 11 partial-length human cDNAs
identified in the GenBank
and EMBL Data Banks by BLAST
searching. These latter sequences are indicated by accession numbers
and by the human tissue of origin. Residues identical to human fetal
liver AMPK-
are boxed.
Figure 7:
Northern blot analysis of rat tissue mRNAs
with AMPK- and AMPK-
. Shown are autoradiographs of Northern
blots (upper two panels) of rat tissue RNAs probed with either
P-labeled rat liver AMPK-
(upper panel) or
P-labeled rat liver AMPK-
(middle panel).
The lower panel is the ethidium bromide (EB)-stained
gel prior to transfer of the RNA preparations probed indicating the
equivalence of loading and the relative positions of the 28 S and 18 S
ribosomal RNA. The first lane in all panels was loaded with 2
µg of poly(A)
RNA isolated from rat liver; the
other lanes were loaded with 20 µg of total RNA from the rat
tissues indicated. WAT, white adipose tissue; SM,
skeletal muscle; mammary, lactating mammary
gland.
Mammalian AMPK, as isolated from rat and porcine liver,
contains three polypeptide subunits termed AMPK-, AMPK-
, and
AMPK-
. The
-subunit contains the kinase catalytic domain
sequence and is highly homologous to several members of the SNF1 kinase
family(4, 6, 7, 13, 14) .
There are multiple isoforms of the
-subunit, with
being responsible for
90% of the AMPK activity detected in
liver extracts(14) . The present report, based on very
extensive peptide sequence and on predicted amino acid sequence from
cDNA clones, establishes that full-length AMPK
- and
-subunits are likewise homologous to two classes of proteins in S. cerevisiae. This extends information previously available
from limited peptide sequence analysis and from smaller PCR-derived
cDNAs(6) . The present work further demonstrates, both by cDNA
cloning and by direct peptide sequencing, which isoforms of AMPK
-
and
-subunits interact with the catalytic
-subunit in liver. This work also establishes that
these non-catalytic subunits, like the
-subunit isoforms, have a
wider tissue distribution, as evidenced by mRNA content of several rat
tissues, than expected from the AMPK activity distribution previously
reported(13, 21) .
The AMPK -subunit is a
mammalian homolog of a class of proteins in yeast, represented by
Sip1p/Sip2p/Gal83p. The GAL83 gene product is known to affect
glucose repression of the GAL genes(22) . All of these
proteins have been shown to interact with the Snf1p protein kinase
either in the two-hybrid system or by
immunoprecipitation(20, 23) . It has been proposed
that these proteins serve as adaptors that promote the activity of
Snf1p toward specific targets(23) . Based on analysis of yeast
mutants, it has been suggested that these proteins may facilitate
interaction of Snf1p with unique and different targets. Of interest is
the demonstration of a highly conserved domain of
80 amino acids
in the C terminus of Sip1p/Sip2p/Gal83p, termed the ASC domain
(association with Snf1p complex)(23) . As studied in the
two-hybrid system, the ASC domain of both Sip1p and Sip2p interacts
strongly with Snf1p(23) . However, the interaction of Sip2p
with Snf1p is not entirely lost on deletion of this domain, suggesting
that the ASC domain is not solely responsible for this protein-protein
interaction. A putative ASC domain is also highly conserved in the C
terminus of rat liver AMPK-
(amino acids 203-270),
suggesting that this region may be responsible, in part, for binding to
the AMPK
-subunit.
AMPK-, like Sip2p and Gal83p, is
phosphorylated in vitro when associated with a catalytic
subunit (AMPK-
or Snf1p,
respectively)(4, 6, 23) . Mutations of Gal83p
can abolish most of the Snf1p kinase activity detectable in immune
complexes, precipitated with anti-Snf1p antibody(23) . A Sip2p
gal83
mutant shows reduced Snf1 protein kinase
activity, which is restored following expression of either Sip2p- or
Gal83p-LexA fusion proteins in the mutant strain(23) . Taken
together, these data suggest the possibility that AMPK-
may also
serve as an adaptor molecule for the catalytic AMPK
-subunit and
will positively regulate AMPK activity. This possibility is being
tested experimentally.
AMPK- appears to migrate anomalously on
SDS gels, with the polypeptide migrating at a molecular mass
10
kDa larger than the size predicted from the cDNA. This slower migration
is evident for both the bacterially expressed His
fusion
protein and the protein expressed in COS-7 cells. These observations
suggest that higher orders of structure are responsible for the
anomalous migration on SDS-polyacrylamide gel electrophoresis. The AMPK
-subunit is autophosphorylated in
vitro(4, 6) ; this suggests that the two
AMPK-
bands expressed on transfection of mammalian cells with
AMPK-
cDNA may result from a similar post-translational
modification giving rise to smaller mobility shifts. Interestingly,
this aberrant migratory behavior of AMPK-
is similar to that of
its yeast homolog, Gal83p. The LexA fusion protein(s) of Gal83p, as
expressed in yeast, also migrate at greater than the expected molecular
mass and display more than one band on SDS gels, consistent with the
known phosphorylation of Gal83p by Snf1p(23) .
Rat and human
liver AMPKs- are mammalian homologs of S. cerevisiae Snf4p (CAT3)(24, 25, 26) . Snf4p was
shown to interact with the Snf1p protein in the first reported use of
the two-hybrid system and also coimmunoprecipitates with
it(26) . Indeed, on isolation of the Snf1p kinase from yeast,
Snf4p, but not the other Snf1p-interacting proteins, copurifies in a
1:1 stoichiometry with the Snf1p polypeptide (4) . Analysis of SNF4 mutants in yeast suggests that Snf4p also positively
regulates the activity of its associated catalytic subunit,
Snf1p(24, 27) . By analogy, our prediction is that
AMPK-
will also have such a positive influence on the AMPK
-subunit.
Examination of the data base reveals that, in
addition to the homology of AMPK- to Snf4p, there are two or three
different human proteins highly homologous or identical to our human
and rat liver AMPK-
sequences. However, some of these data base
sequences, as predicted from expressed sequence tag cDNAs in brain,
heart, breast, and placenta, are distinct from each other and from our
clones; some, for example, have a C-terminal extension. This indicates
that there is a mammalian isoform family of potential AMPK
-subunits, each perhaps with different tissue expression and
regulatory roles. We propose that these different
-isoforms be
designated
,
,
. . .
.
, as their full-length sequences are delineated. We
have designated the rat liver/human liver AMPK-
sequence reported
herein as AMPK-
. The isoform diversity of both the
- and
-subunits of AMPK underscores the need for complete
characterization of the translation products of the enzyme, as isolated
from various sources, in order to properly identify the relevant
isoforms that make up the heterotrimeric complex. To what extent
various species of AMPK heterotrimers with varying composition of
individual subunits could exist in vivo is not yet known.
AMPK was first recognized as a protein kinase active on enzymes of
lipid metabolism (acetyl-CoA carboxylase, hydroxymethylglutaryl-CoA
reductase, and hormone-sensitive
lipase)(1, 2, 3) . However, as has been
observed for the AMPK
-subunit(5, 13, 14) , the AMPK
-
and
-subunits have wider tissue distribution than
might be expected for a protein active only in the regulation of lipid
metabolism. While mRNAs for each are detectable in
``classic'' lipogenic tissues like liver, white adipose
tissue, and lactating mammary gland, high concentrations of mRNA in
non-lipogenic tissues like heart, brain, spleen, and lung, for example,
suggest that these proteins have roles that extend beyond the
regulation of fatty acid and sterol metabolism. Of note are the
relatively low amounts of AMPK-
and AMPK-
mRNAs in skeletal
muscle; this observation is consistent with the relatively low levels
of AMPK activity reported by others in this tissue and with the failure
of skeletal muscle AMPK-
to immunoprecipitate with detectable
- and
-subunits(28) .
The wide tissue distribution of the mRNAs for all three subunits for AMPK raises the question of other potential roles for AMPK beyond lipogenic regulation. The striking homology of all three subunits to yeast proteins that are involved in nutrient (glucose) responses raises the possibility that the three mammalian proteins may be involved in glucose (or other nutrient) regulation of gene expression in mammalian tissues or in other adaptive responses to a changing nutrient environment. We (9) and others (12) have presented evidence that AMPK may be a important ``metabolic sensor'' linked to oxidative fuel choice in the heart and to glucose sensing in the pancreatic beta cell, perhaps being important for insulin secretion. There is every reason to believe that further study of the AMPK subunits may shed light on multiple aspects of cellular regulation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U42411[GenBank], U42412[GenBank], and U42413[GenBank].