From the Medical Research Council Clinical Sciences Centre, The AMP-activated protein kinase (AMPK) is a
member of a growing family of related kinases, including the SNF1
complex in yeast, which respond to nutritional stress. AMPK is a
heterotrimeric complex of a catalytic subunit ( In mammals, the AMP-activated protein kinase
(AMPK)1 plays a major role in
the response to metabolic stress (1-3). AMP activates AMPK via a
number of independent mechanisms, including activation of an upstream
kinase (AMPKK), which in turn phosphorylates and activates AMPK (4, 5).
The effects of AMP are antagonized by high concentrations of ATP so
that it appears that the kinase responds to the AMP/ATP ratio, rather
than AMP itself (4). Once activated, AMPK phosphorylates a number of
enzymes involved in biosynthetic pathways causing their inactivation
and preventing further ATP utilization. These findings have led to the
hypothesis that the AMPK system has evolved to monitor the energy
status, or fuel supply, within the cell (2-4).
Molecular characterization of AMPK has revealed that it is composed of
three distinct subunits: a catalytic subunit, A second In an attempt to identify novel downstream targets of AMPK, we screened
a two-hybrid library with AMPK Materials--
A human skeletal muscle library in pGAD10 was
obtained from CLONTECH. pYTH9 used to make a fusion
of AMPK Plasmid Construction--
In order to remove an internal
XbaI site, AMPK Two-hybrid Library Screening--
The vector pYTH9 Analysis of Positive Clones--
cDNA inserts in pGAD10 were
amplified from yeast extracts by PCR using primers GADF1
(GATAAGATACCCCACCAAACCC) and GADR1 (CTTGCGGGGTTTTTCAGTATCTACG) flanking the cloning sites. Following an extended hot start of 10 min
at 95 °C, 35 cycles of 95 °C for 1 min, 56 °C for 1 min, and
72 °C for 1 min were used. Products were analyzed by electrophoresis either before or after digestion with either RsaI,
Sau3A, and HaeIII. Clones were grouped according
to their restriction digest pattern, and two separate clones from each
group were isolated from the original yeast colonies and transformed
into E. coli. The inserts were sequenced by the dideoxy
chain termination method (19) using Sequenase (version 2.0 from
Amersham Pharmacia Biotech) in accordance with the manufacturer's
instructions.
Identification of Human AMPK Northern Blot Analysis--
A human multiple tissue Northern
blot (CLONTECH) was probed with either
random-primed human AMPK Antibody Production--
Peptides based on the amino acid
sequence of rat AMPK Immunoprecipitations--
Affinity-purified isoform-specific
antibodies were bound to either protein A-Sepharose (rabbit antibodies)
or protein G-Sepharose (sheep antibodies) and used to immunoprecipitate
AMPK from rat tissue extracts. For comparison of AMPK in liver and
skeletal muscle, AMPK was partially purified by chromatography on
DEAE-Sepharose (21). A mouse monoclonal antibody (clone 9E10; Ref. 22)
was bound to protein A-Sepharose and used to immunoprecipitate AMPK (in
which both the Western Blots--
Samples were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride membrane. The membrane was
blocked by incubation in 10 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 0.5% Tween 20, 5% low fat milk powder overnight
at 4 °C. The membrane was then incubated with primary antibody in
the same buffer for 2-4 h at room temperature. After extensive washing
with 10 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 0.5%
Tween 20, the membrane was incubated for 1 h at room temperature with either protein A conjugated with horseradish peroxidase (for primary antibodies raised in rabbits) or protein G conjugated with
horseradish peroxidase (for sheep antibodies). After further extensive
washing, the membrane was developed using enhanced chemiluminescence (Boehringer Mannheim).
In Vitro Translations--
cDNAs encoding AMPK Mammalian Cell Transfections--
For mammalian expression all
AMPK cDNAs were constructed in pCDNA3. cDNAs encoding AMPK Activity--
Activity was measured by phosphorylation of
the SAMS peptide as described previously (24).
Identification of a Novel AMPK Enzyme Pharmacology Unit,
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
) and two regulatory
subunits (
and
), and proteins related to all three subunits have
been identified in the SNF1 complex. We have used the two-hybrid system in order to identify proteins interacting with the catalytic subunit (
2). Using this approach, we have isolated a novel AMPK
isoform, which we designate AMPK
2. The N-terminal region of
2 differs significantly from that of the previously characterized isoform (
1),
suggesting that this region could play a role in isoform-specific AMPK
activity. Comparison of the C-terminal sequences of
1 and
2 with
their related proteins in yeast identifies two highly conserved regions
predicted to be involved in binding of the
and
subunits. The
expression of
1 and
2 was examined in a number of tissues,
revealing that the
1 isoform is highly expressed in liver with low
expression in skeletal muscle, whereas the opposite pattern is observed
for the
2 isoform. These results suggest that the
isoforms have
tissue-specific roles, which may involve altered responses to upstream
signaling and/or downstream targeting of the AMPK complex.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(molecular mass
approximately 63 kDa); and two regulatory subunits,
(30 kDa) and
(36 kDa) (6-10). In vitro binding studies indicate that
the
and
subunits and the
and
subunits interact
directly, whereas the
and
subunits do not form a stable
interaction (10). The formation of the heterotrimeric complex may
therefore be mediated, at least in part, by the
subunit. Proteins
related to all three subunits have been identified in the SNF1 kinase complex in Saccharomyces cerevisiae, which is involved in
the derepression of glucose-repressible genes (11). AMPK
is 47% identical to Snf1p (we refer to the individual subunits as Snf1p, etc.,
and the complex as SNF1) (6), AMPK
is 35% identical to Snf4p (10,
12), and AMPK
is related to the Sip1p/Sip2p/Gal83p family of
proteins (10, 12). In addition to their primary sequence similarities,
the AMPK and SNF1 complexes are functionally related since SNF1, like
AMPK, phosphorylates and inactivates acetyl-CoA carboxylase (13).
Indeed, the similarities between the two complexes extend even further
as it has recently been shown that active SNF1, e.g. under
glucose derepressing conditions, most likely exists as a heterotrimeric
complex of Snf1p, Snf4p, and one of the Sip1p/Sip2p/Gal83p proteins
(14, 15). Furthermore, analogous to AMPK, formation of the complex is
mediated by one of the Sip1p/Sip2p/Gal83p family of proteins (14,
15).
isoform has been isolated recently, which was termed,
somewhat unconventionally,
1 (the isoform that was first identified
becoming
2) (16). It was reported that the
1 isoform accounted
for virtually all of the AMPK activity measurable in rat liver extracts
and that
2 was virtually inactive (16), but a subsequent study has
challenged this finding (17). Immunoprecipitation of specific isoforms
from rat liver show that both
1 and
2 contribute equally to AMPK
activity and expression of recombinant enzyme demonstrate that both
isoforms have comparable specific activities (17). The only difference
that could be detected between the two isoforms was in their
specificity for peptide substrates (17). Although subtle, this does
raise the possibility that there could be differences in the downstream
targets in vivo leading to different physiological roles for
1 and
2.
2 as bait. Here, we report the
identification and characterization of a second isoform for AMPK
,
which is highly expressed in skeletal muscle. Comparison of the amino
acid sequences of the two
isoforms with Sip2p and Gal83p reveal two
highly conserved regions, which are predicted to interact with the
and
subunits. The sequences of the
isoforms diverge at their N
termini, suggesting that this region may play an important role in
conferring isoform specificity, either by targeting to different
substrates and/or intracellular locations or by responding to different
stimuli.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
2 with the DNA-binding domain of Gal4 was a gift from Dr.
Julia White, GlaxoWellcome Research Group. Yeast strain Y190 was used
for the two-hybrid screening, and standard methods were used for
manipulation and growth of yeast (18). Oligonucleotides were obtained
from Genosys. CCL13 cells were obtained from the American Type Culture
Collection and grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum.
2 cDNA was amplified by the polymerase
chain reaction (PCR) with the following oligonucleotides:
AGATCGGACACTACGTG (forward primer) and CCTCATCATCGATGCTTTTAAAGTCCAGAAG (reverse primer). The amplified product was digested with
ClaI and BglII and ligated with AMPK
2
(ClaI/BglII). A second round of amplification was
carried out on the modified AMPK
2 cDNA using the following
primers: GAGCTCAGGTCGACGCCCATGGCTGAGAAGC and
TCTGTCAGGAATTCAGAAAGACAGAGACCG. The product was digested
with SalI and EcoRI (restriction sites shown
underlined in the primers) and cloned into the
SalI-EcoRI sites of plasmid pYTH9 to yield
pYTH9
2. For mammalian cell expression, AMPK subunit cDNAs were
cloned into pCDNA3 expression vector (Invitrogen). Plasmid DNA was
prepared using a QIAGEN maxiprep kit according to the manufacturer's
instructions.
2 was
linearized with XbaI and integrated into the genome of yeast
strain Y190. Competent cells were transformed with a human skeletal
muscle library in pGAD10 and plated on selective medium lacking
histidine, leucine, and tryptophan. After incubation for 10 days at
30 °C, filter lifts of the colonies were taken using Whatman No. 3MM
paper and replicated onto fresh plates before being permeabilized by
immersing twice in liquid nitrogen for 5 s. Filters were incubated
with 5-bromo-4-chloro-3-indoyl
-D-galactoside at room
temperature for 1 h, and positive clones were identified by the
appearance of blue colonies. The position of the positive colonies was
marked on the replica plates, and these were incubated overnight at
30 °C. Positive clones were individually transferred to plates
lacking leucine and tryptophan in order to isolate single colonies.
Where appropriate, plasmid DNA was extracted from 1.5-ml yeast cultures
and used to transform Escherichia coli.
1 cDNA--
The nucleotide
sequence of rat AMPK
(10) was used to search the dbEST (Expressed
Sequence Tag) data base using the BLAST (Basic Local Alignment Search
Tool) program. Two clones (accession numbers H06094 and R20494) were
obtained from the IMAGE consortium. Plasmid DNA from the clones was
prepared and the inserts sequenced.
1 cDNA or AMPK
2 cDNA. Following hybridization the blots were washed with 2 × SSC, 0.5% SDS at room temperature for 1 h, followed by 0.2 × SSC, 0.5% SDS
at 60 °C for 2 × 1 h, and autoradiographed for 1-5 days
at
70 °C.
1 (residues 20-33, PRRDSSGGTKDGDR) and human
AMPK
2 (residues 44-57, SVFSLPDSKLPGDK) were synthesized and coupled
to keyhole-limpet hemocyanin via a cysteine residue added at the N
terminus of the peptide. The conjugated peptides were used to immunize
sheep (AMPK
1-specific peptide) or rabbits (AMPK
2-specific
peptide). In some cases, antibodies were affinity-purified from serum
using the appropriate peptide conjugated to thiol-Sepharose (20).
1 and
2 subunits have the myc peptide EQKLISEEDL added at the N terminus) from transiently transfected CCL13 cells. Extracts were precleared by incubation with rabbit preimmune serum bound to protein A-Sepharose for 2 h at 4 °C and then incubated with an excess of the appropriate antibody for 2-16 h at 4 °C. Immune complexes were collected by centrifugation at 6000 × g for 5 min, washed extensively with buffer A (50 mM Tris-HCl, pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 1% (v/v) Triton
X-100), and then analyzed for AMPK activity and by Western
blotting.
isoforms
were constructed in pCDNA3 (Invitrogen). RNA transcripts were
synthesized using T7 polymerase and translated in reticulocyte lysates
using a coupled transcription/translation system (TNT system, Promega)
in the presence of [35S]methionine. Total labeled
products of translation were analyzed by SDS-PAGE and fluorography.
1
and
2 were constructed with a sequence encoding a 10-amino acid
epitope tag derived from c-Myc (EQKLISEEDL; Ref. 22) immediately
following the initiating methionine. CCL13 cells were transfected with
plasmid (10 µg of each plasmid) by calcium phosphate precipitation
(23). The precipitate was incubated with the cells overnight, followed
by a 2-min incubation with phosphate-buffered saline containing 10%
(v/v) dimethyl sulfoxide. Cells were harvested 60 h after
transfection and lysed in buffer A. Insoluble material was removed by
centrifugation and the supernatant analyzed by Western blotting or used
for immunoprecipitation using anti-AMPK antibodies or an anti-Myc
antibody (22).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
Subunit Using the Two-hybrid
System--
In order to identify proteins interacting with the
catalytic subunit of AMPK, we screened a human skeletal muscle
two-hybrid library with AMPK
2 as bait. A total of 2.3 million clones
were screened, and 136 positive clones were identified by blue/white selection from the lacZ reporter gene. Grouping of the
clones by restriction digest mapping and partial sequencing of the
cDNA inserts revealed that 50 clones, which we designate AMPK
2,
shared considerable sequence identity with the cDNA encoding the
rat AMPK
1 subunit. A representative clone was sequenced in its
entirety, and the nucleotide and predicted amino acid sequences are
shown in Fig. 1A. The
nucleotide sequence of
2 is 66.7% identical to rat
1, whereas
the predicted amino acid sequence is 70.6% identical to the rat
sequence (10). In order to determine whether the cDNA we had
isolated was the human homologue of rat
1, we searched the human EST
data base with the rat
1 and human
2 sequences. A number of
clones were identified with sequences, which, although highly related
to rat
1, did not match
2. Two of these clones (with accession
numbers H06094 and R20494) encoded the sequence corresponding to the
N-terminal region of rat
1 (including the initiating methionine) and
were obtained from the IMAGE consortium through the UK Human Genome
Mapping Resource Center, Cambridge. Sequence analysis of these clones
revealed that they were identical, except that clone H06094 had a
343-base pair deletion compared with clone R20494 (see Fig.
1B). The nucleotide and predicted amino acid sequence of
clone R20494, which we designate human
1, is shown in Fig.
1B. At the nucleotide level, human
1 is 89% identical to
rat
1 (97% amino acid sequence identity) and 66% identical to
human
2. These results indicate that clone R20494 is the human
homologue of rat
1 and that
2, therefore, is a novel
subunit
isoform.
View larger version (36K):
[in a new window]
Fig. 1.
Nucleotide sequence and predicted amino acid
sequence of human AMPK 1 and AMPK
2. A,
2. The
initiating and stop codons are shown in bold. Nucleotides
are numbered on the right and amino acids on the
left. B,
1. Arrows mark the limits
of the 343-base pair deletion found in clone H06094.
|
Tissue Distribution of AMPK1 and AMPK
2--
Poly(A)-rich RNA
isolated from a number of human tissues was probed with cDNA
encoding
1 or
2 and the results are shown in Fig.
3.
1 mRNA is detected as a single
band of approximately 3 kilobase pairs and is expressed in all tissues
examined at approximately equal levels. A single band of approximately
7.5 kilobase pairs is detected with the
2 probe, although in this
case there is clearly a difference in the pattern of expression.
Relatively high levels of expression are detected in skeletal and
cardiac muscle with low levels in the kidney and lung.
|
|
Expression of Subunits in Mammalian Cells--
cDNAs encoding
1/
1/
1,
1/
2/
1,
2/
1/
1, or
2/
2/
1 were
co-transfected into CCL13 cells. AMPK was immunoprecipitated from
transfected cells with an anti-Myc antibody (a Myc epitope tag is
present on both
1 and
2) and kinase activity in the immune complexes determined. No significant differences in AMPK activity between
1-containing complexes compared with
2-containing
complexes were detected (Fig.
5A). As has been reported
previously, complexes containing
1 were expressed at higher levels
than those containing
2 and yield correspondingly higher activities
(7). The immune complexes were analyzed by Western blotting using an
antibody that recognizes both
isoforms (10) (Fig. 5B).
Transfections with
1 yield a single band migrating with an apparent
molecular mass of approximately 38 kDa, whereas transfections with
2
reveal a single band migrating with an apparent molecular mass of 34 kDa. No products could be detected in untransfected cells. There is no
significant difference in the Western blots from immunoprecipitates of
1- or
2-transfected cells, indicating that
1 and
2 can form
complexes with either
1 or
2, consistent with the results from
liver and skeletal muscle. As we have reported previously, co-transfection of
,
, and
is required in order to detect expression of recombinant protein or activity in this system (10) (data
not shown).
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DISCUSSION |
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We report here the identification of a second isoform of AMPK
(termed
2), which we isolated from a two-hybrid screen for proteins
interacting with the catalytic subunit of AMPK (
2). cDNA
encoding the human homologue of rat
1 was identified from a search
of the EST data base, and a clone (accession number R20494) containing
the entire coding sequence was obtained from the IMAGE consortium. In a
recent paper, it was reported that at least three distinct gene
products for AMPK
were represented in the EST data base (16). Our
results do not support this claim, indicating that all of the
sequences in the EST data base correspond to either
1 or
2. It is
worth noting, however, that most of the entries in the data base do not
match exactly the sequences of
1 and
2 reported here, differing
by a number of single base changes. We believe that these differences
probably represent errors in the automated DNA sequence analysis used
to generate the EST sequences. The majority of the discrepancies occur
at the beginning and end of the EST sequences, which are more prone to
reading errors. At the present time, therefore, we believe that only
two
isoforms can be identified, although we do not exclude the
possibility that additional isoforms exist (see also "Addendum").
In addition to the
subunit isoforms, two isoforms of the
subunit have been characterized (16). Searching of the EST data base,
combined with preliminary results obtained from two-hybrid screening
using the
1 subunit as bait, demonstrate that there are at least
three genes coding for isoforms of the
subunit.2 The existence of
different isoforms for each of the AMPK subunits suggests that a large
subfamily of AMPK complexes is present in mammals (see below).
The amino acid sequences of 1 and
2 share considerable identity
with a family of related proteins in yeast, which interact with the
SNF1 protein kinase complex (10, 11, 16, 27, 28). Amino acid sequence
alignment of
1,
2, Sip2p, and Gal83p reveals two highly conserved
regions (Fig. 2B), which have been shown recently to be
important in mediating the interaction of the yeast proteins with Snf1p
and Snf4p (15). The conserved C-terminal region, termed the ASC domain,
interacts with Snf4p, while the conserved internal region, termed the
KIS domain, interacts with Snf1p (15). We have shown previously that
the
subunit plays an important role in the formation of the ternary
AMPK complex in vitro since it interacts with both
and
, whereas
and
do not appear to interact directly (10). The
simplest interpretation of these results is that the
and
subunits bind independently to the
subunit, probably through the
two regions related to the KIS and ASC domains in the yeast proteins.
It remains to be determined whether binding of the
and
subunits
to
then allows them to interact directly with one another, although
there is evidence that this may be the case with the homologous
subunits in the yeast complex (15). Studies in our laboratory are
currently under way to examine these interactions in more detail. It is unlikely that the sole purpose of the
subunit is to mediate the
formation of the AMPK complex, since both isoforms appear equally
competent to perform this function in vitro and in
vivo. Most of the amino acid sequence variation between
1 and
2 occurs within the N-terminal region, and this is also the case for
Sip2p and Gal83p (data not shown). It seems likely, therefore, that functional differences between the
isoforms will be attributable to
the divergent N-terminal regions and will not entail assembly of the
heterotrimeric complex.
Both isoforms are expressed in a wide range of tissues, but there
is a marked difference in their expression patterns.
1 is most
highly expressed in liver with low level expression in skeletal muscle,
and this pattern is reversed for
2. These findings suggest that the
isoforms may play tissue-specific roles in regulating the activity
and/or function of AMPK. To begin to address these questions, we
examined the effect of expression of the different
isoforms on AMPK
activity in a recombinant system. Co-expression of the
and
subunits with either
1 or
2 in CCL13 cells did not reveal a
significant difference in AMPK activity between the two
isoforms,
although, as reported previously, expression with
1 led to
substantially higher activity than with
2 (7). Furthermore,
1 and
2 both interact with
1 and
2, and we did not detect any
obvious difference in the association of the different
and
isoforms in vitro. These results imply that the
isoforms
do not directly alter the activity of AMPK per se, but they
do not exclude a less direct role in the regulation of AMPK. We decided therefore to examine the relative expression and activity of
1- and
2-containing AMPK in vivo, and since there was an obvious difference in the expression of the isoforms between the liver and
skeletal muscle, we concentrated on these tissues. Complexes containing
all four possible combinations of the
and
isoforms were
detected in liver and skeletal muscle, indicating that there are no
constraints on isoform-specific subunit composition in vivo.
In liver the
1-containing complexes account for virtually all of the
AMPK activity, and this may simply reflect the relative abundance of
the
1 complexes compared with
2. Consistent with this result is
the finding that purification of AMPK from rat liver yields a
preparation in which the
subunit is almost exclusively
1, as
judged by migration on SDS-PAGE and amino acid sequencing (9, 10, 26).
It is clear from our results, however, that a proportion of AMPK from
rat liver contains the novel
2 isoform. In skeletal muscle, although
there is more
2 complex compared with
1, AMPK activity associated
with the different
isoforms is approximately the same. These
results suggest that the
2 complex, or a proportion of it, is in a
relatively inactive state compared with the
1 complex. We reported
previously that AMPK isolated from skeletal muscle is in a relatively
inactive state and suggested that this could be due to lack of
association of the
subunit with the
and
subunits (29). The
results of our present study appear to rule out this possibility, and
we are currently investigating the basis for the low activity state of
AMPK in muscle.
We have detected the subunit in all of the complexes isolated
in vivo, although we have not been able to determine which particular isoform is present in the different complexes. AMPK isolated
from liver contains predominantly the
1 isoform, as judged by amino
acid sequencing (9, 10, 26), although the
2 and
3 isoforms are
also expressed in liver (25).2 It is likely that, at least
in liver, some complexes will contain the
2 and
3 isoforms.
We believe that our results provide valuable clues regarding the
possible role of the subunit in the regulation of AMPK. The marked
difference in the expression patterns of
1 and
2 in liver and
skeletal muscle strongly suggests a tissue-specific role for the
isoforms. It was reported recently that AMPK
2, but not AMPK
1, is
activated in response to increased contraction in rat skeletal muscle
(30). Although we have not assessed the relative amounts of the
isoforms in skeletal muscle, the predominant
isoform is
2.
Interestingly, however, both
1 and
2 account for approximately
equal AMPK activity in muscle. It is possible that association with the
different
isoforms could play a part in an altered response to the
upstream signaling pathways that lead to AMPK activation. This could
explain the differential activation of
2 in skeletal muscle during
contraction if there were a pool of inactive
2
2-containing AMPK.
Subtle differences in substrate recognition have been reported between
1 and
2, suggesting that the isoforms could phosphorylate
different proteins, at different rates, within the cell (17, 31). The
ability of the different isoform complexes to have a varying response
to specific stimuli, coupled with the differing substrate specificity
between
1 and
2, would allow an enormous degree of flexibility
within the AMPK cascade.
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ACKNOWLEDGEMENT |
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We are grateful to Julia White for help with the two-hybrid screening.
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Addendum |
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During the preparation of this manuscript, Stapleton
et al. reported the partial sequence of a mouse cDNA
identified in the EST data base (accession number W07176), which
corresponds to the mouse homologue of 2 (26).
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FOOTNOTES |
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* This work was supported by the Medical Research Council (United Kingdom) and by a studentship from GlaxoWellcome (to C. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ224515 and AJ224538.
§ To whom correspondence should be addressed: MRC Clinical Sciences Centre, Cellular Stress Group, Imperial College School of Medicine, Hammersmith Hospital, DuCane Rd., London W12 0NN, United Kingdom. Tel.: 44-181-383-4314; Fax: 44-181-383-2028; E-mail: dcarling{at}rpms.ac.uk.
1 The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKK, AMP-activated protein kinase kinase; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SAMS, the synthetic peptide substrate with the sequence HMRSAMSGLHLVKRR.
2 P. C. F. Cheung and D. Carling, manuscript in preparation.
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REFERENCES |
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