©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of AMP-activated Protein Kinase and Subunits
ASSEMBLY OF THE HETEROTRIMERIC COMPLEX IN VITRO(*)

(Received for publication, December 8, 1995; and in revised form, January 12, 1996)

Angela Woods (§) Peter C. F. Cheung (¶) Fiona C. Smith Matthew D. Davison (1) James Scott Raj K. Beri (1) David Carling (**)

From the Medical Research Council Clinical Sciences Centre, Department of Molecular Medicine, Royal Postgraduate Medical School, DuCane Road, London W12 0NN, United Kingdom andCardiovascular and Infection Departments, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

There is growing evidence that mammalian AMP-activated protein kinase (AMPK) plays a role in protecting cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. The active form of AMPK from rat liver exists as a heterotrimeric complex and we have previously shown that the catalytic subunit is structurally and functionally related to the SNF1 protein kinase from Saccharomyces cerevisiae. Here we describe the isolation and characterization of the two other polypeptides, termed AMPKbeta and AMPK, that together with the catalytic subunit (AMPKalpha) form the active kinase complex in mammalian liver. Sequence analysis of cDNA clones encoding these subunits reveals that they are related to yeast proteins that interact with SNF1, providing further evidence that the regulation and function of AMPK and SNF1 have been conserved throughout evolution. The amino acid sequence of the beta subunit is most closely related to SIP2 (35% identity), while the amino acid sequence of the subunit is 35% identical with SNF4. We show that both AMPKbeta and AMPK mRNA and protein are expressed widely in rat tissues. We show that AMPKbeta interacts with both AMPKalpha and AMPK in vitro, whereas AMPKalpha does not interact with AMPK under the same conditions. These results suggest that AMPKbeta mediates the association of the heterotrimeric AMPK complex in vitro, and will facilitate future studies aimed at investigating the regulation of AMPK in vivo.


INTRODUCTION

A number of recent studies have led to the proposal that in mammals an AMP-activated protein kinase (AMPK) (^1)plays a major role in the response to metabolic stress (Corton et al., 1994; Hardie, 1994; Hardie et al., 1994). AMPK was first identified through its role in the phosphorylation and inactivation of a number of enzymes involved in lipid metabolism (Carling et al., 1987; Hardie et al., 1989; Hardie, 1992), and subsequently was shown to phosphorylate enzymes in other metabolic pathways (Carling and Hardie, 1989). AMPK has been purified from a number of species, including human, rat, and pig, and in each case is activated allosterically by micromolar concentrations of AMP (Carling et al., 1989; Mitchelhill et al., 1994; Sullivan et al., 1994). The kinase is itself regulated by reversible phosphorylation, being phosphorylated and activated by a distinct AMPK kinase (AMPKK), thereby forming a protein kinase cascade (Carling et al., 1987; Weekes et al., 1994). The phosphorylation and activation of AMPK is markedly stimulated by AMP (Moore et al., 1991; Hawley et al., 1995), making AMPK extremely sensitive to changes in the intracellular concentration of AMP. These findings have led to the proposal that one of the primary roles of AMPK is to conserve ATP during periods of excessive ATP utilization, when AMP levels are elevated (Corton et al., 1994; Hardie et al., 1994).

We recently reported that the deduced amino acid sequence of the catalytic subunit of rat liver AMPK is remarkably similar to the sequence of the yeast protein kinase SNF1 (Carling et al., 1994). In a further study we went on to show that SNF1 is functionally related to mammalian AMPK (Woods et al., 1994). In vitro, SNF1 phosphorylates a specific peptide substrate for AMPK, and there is good evidence that SNF1 phosphorylates and inactivates acetyl-CoA carboxylase in vivo. Furthermore, like AMPK, SNF1 is inactivated by protein phosphatases and can be reactivated by a partially purified preparation of mammalian AMPKK, suggesting functional conservation of the upstream kinases (Woods et al., 1994). The SNF1 protein kinase from Saccharomyces cerevisiae is required for the expression of glucose repressed genes in response to glucose starvation (Celenza and Carlson, 1986; Estruch et al., 1992; Gancedo, 1992), e.g. the SUC2 gene, which encodes invertase (Carlson and Botstein, 1982). snf1 mutants are unable to utilize a wide range of non-glucose sugars (Carlson et al., 1981; Estruch et al., 1992). In addition, snf1 mutants have been shown to be defective in other aspects of cell growth, e.g. glycogen synthesis and sensitivity to heat stress (Thompson-Jaeger et al., 1991). SNF1 is physically associated with a 36-kDa polypeptide, termed SNF4 (Celenza et al., 1989), which is itself required for expression of many glucose-repressible genes. SNF4 is thought to function as an activator of SNF1 (Celenza and Carlson, 1989; Celenza et al., 1989), although the mechanism by which SNF4 activates SNF1 is not known.

A number of yeast proteins, which interact with SNF1 in vivo, termed SNF1 interacting proteins or SIPs, have been identified using the two-hybrid system (Yang et al., 1992). Two of these proteins, SIP1 and SIP2, share significant amino acid sequence identity, particularly at their C termini (Yang et al., 1992). Furthermore, the amino acid sequence of SIP2 is 52% identical to GAL83 (Erickson and Johnston, 1993; Yang et al., 1994). GAL83 is involved in the glucose repression of GAL genes, and genetic evidence suggests that GAL83 is involved in the SNF1 pathway (Matsumoto et al., 1981; Erickson and Johnston, 1993). SIP1, SIP2, and GAL83 have been shown to co-immunoprecipitate with SNF1, and all three proteins are phosphorylated in an immune complex SNF1 kinase assay (Yang et al., 1994). In the same study it was shown that the C-terminal 80 amino acids of SIP2, termed the ASC domain, were sufficient to mediate interaction with SNF1. The functions of SIP1, SIP2, and GAL83 remain unknown, although it has been proposed that they may act as modulators of SNF1, targeting the kinase to specific intracellular locations and/or substrates (Yang et al., 1994).

Recently, AMPK has been purified to apparent homogeneity from both rat and pig liver (Mitchelhill et al., 1994; Davies et al., 1994). Two other polypeptides co-purified with the catalytic subunit (molecular mass 63 kDa), and biochemical analysis of the purified kinase complex indicated that AMPK isolated from rat liver exists as a heterotrimer (Davies et al., 1994). We subsequently reported that the catalytic subunit of AMPK isolated from rat skeletal muscle did not appear to be associated with any other polypeptides and that this observation might account for the low activity of AMPK detectable in skeletal muscle (Verhoeven et al., 1995). In this paper we report the isolation and cDNA cloning of two AMPK subunits from rat liver which we refer to as AMPKbeta and AMPK (the catalytic subunit is designated AMPKalpha) following the terminology of Kemp and colleagues (Stapleton et al., 1994). The beta subunit is most closely related to SIP2 and contains a region at its C terminus, which is 50% identical with the ASC domain of SIP1/SIP2/GAL83 (Yang et al., 1994). The subunit has a high degree of amino acid sequence identity with SNF4, and this conservation of sequence suggests that, like SNF4, it is necessary for the catalytic activity of AMPK. We show here that AMPKbeta interacts with both AMPKalpha and AMPK and that this mediates the assembly of the ternary complex in vitro. The similarity between the mammalian AMPK complex and the SNF1 complex from yeast emphasizes the likelihood that the role of these kinases have been highly conserved throughout evolution.


MATERIALS AND METHODS

Purification and Amino Acid Sequencing of AMPK Subunits

AMPK was partially purified from rat liver up to and including the DEAE-Sepharose ion-exchange step, as described previously (Carling et al., 1989). AMPK was further purified by immunoaffinity chromatography using affinity-purified antibodies raised against a synthetic peptide based on the deduced sequence of AMPKalpha (Carling et al., 1994). Approximately 200 mg of partially purified AMPK was incubated overnight at 4 °C with 5 mg of affinity-purified antibody that had been cross-linked to protein A-Sepharose (Harlow and Lane, 1988). Following extensive washing of the resin with 50 mM Tris/HCl, pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol (buffer A), protein was eluted with 5 bed volumes of 0.1 M glycine, pH 2.5. The eluate was immediately neutralized by the addition of 0.1 volumes of 1 M Tris/HCl, pH 8, and concentrated in a Centricon-30 microconcentrator (Amicon). Proteins were resolved by SDS-PAGE on a 10% polyacrylamide gel and visualized by staining with Coomassie Blue. The polypeptides migrating at apparent molecular masses of 38 kDa (AMPKbeta) and 36 kDa (AMPK) were excised from the gel and the proteins cleaved in the gel slice by overnight incubation with CNBr in 90% formic acid at room temperature. The supernatant was removed and dried in a Speed-Vac. The residue was washed twice with water, dried, and resuspended in SDS-gel loading buffer. Peptides were resolved by SDS-PAGE using a Tricine buffer system (Schagger and von Jagow, 1987) and transferred onto a Problot membrane (Applied Biosystems) for sequencing using an Applied Biosystems model 475 sequencer.

Autophosphorylated AMPK was prepared by incubating the immune complex with 0.2 mM [-P]ATP and 0.2 mM AMP at 30 °C for 30 min. Unincorporated ATP was removed by extensive washing with buffer A. Protein was eluted from the resin as above. Following SDS-PAGE the gel was dried and subjected to autoradiography at -70 °C.

Amplification of cDNAs Encoding the beta and Subunits

Degenerate oligonucleotide primers based on the peptide sequences obtained from either AMPKbeta or AMPK (shown in Table 1) were synthesized as follows: beta forward primer 1, CCNGARAARGARGARTT; forward primer 2, AARGARGARTTYYTNGC; reverse primer 1, ACRTAYTTYTTYTGRTA; reverse primer 2, TGRTANC(GT)NACNGTNGC (primers corresponding to the reverse and complemented sequence of these oligonucleotides were also synthesized but did not yield a distinct product after amplification, indicating that peptide 38CB3 is N-terminal to peptide 38CB1); forward primer 1, GAYTTYAT(ACT)AAYAT(ACT)YT; forward primer 2, YTNCAY(AC)GNTAYTAYAA; reverse primer 1, TGYTGNACRAA(AGT)ATNCC; reverse primer 2, CCNARNGCNACRTANAC. For both subunits, rat liver cDNA (Clontech) was amplified using forward primer 1 and reverse primer 1 for 30 cycles of 94 °C, 1 min; 50 °C, 1 min; and 72 °C, 1 min. In each case, an aliquot (0.1 µl) of the products of the reaction were used for a second round of amplification using forward primer 2 and reverse primer 2 (same cycles as before). The products from the reaction were purified by agarose gel electrophoresis, cloned into a pGEM-T vector (Promega), and sequenced to confirm their identity.



Isolation of cDNA Clones Encoding the beta and Subunits

Standard molecular biology techniques were used (Sambrook et al., 1989). The products isolated from amplification of rat liver cDNA were used separately to screen a rat liver cDNA library ( UniZAP, Stratagene). Hybridization conditions were: 50 ng of probe (10^9 cpm/µg; 10^6 cpm/ml) in 5 times SSPE, 100 µg/ml sonicated and denatured salmon sperm DNA, 2 times Denhardt's, 0.1% SDS at 65 °C for 12 h. Filters were washed with 2 times SSC, 0.1% SDS at room temperature for 1 h, followed by 2 times SSC, 0.1% SDS at 65 °C for 30 min and autoradiographed at -70 °C for 18 h, with intensifying screens. Twelve positive clones encoding AMPKbeta were isolated from approximately 4 million plaques and one positive clone for AMPK was isolated from approximately 1 million plaques. Plasmids were recovered by in vivo excision from the phage (Short et al., 1988) and the inserts sequenced manually by dideoxy chain termination (Sanger et al., 1977) using vector and cDNA specific primers.

In order to obtain the 5` end of the cDNA encoding the subunit, antisense oligonucleotide primers were synthesized based on cDNA sequence and used to perform 5` RACE-PCR (Frohman et al., 1988). (AMPK: RACE primer 1, TGGCGTAGGTGCCAATCTG; RACE primer 2, ATCTGTAGCTCTTCCAGAG). Rat liver RACE-ready cDNA (Clontech) was used as a template for amplification using primer 1 and the anchor primer for 30 cycles of 94 °C, 1 min; 58 °C, 1 min; 72 °C, 1 min. A second round of amplification on an aliquot (0.1 µl) of the reaction products was performed using primer 2 and the anchor primer under the same conditions as before. Products from the second round of amplification were isolated by agarose gel electrophoresis, cloned into pGEM-T vector, and sequenced. In order to construct a cDNA encoding the entire amino acid sequence of the subunit oligonucleotide primers spanning the initiating methionine (GCCAAGGTCGACGGCCGGGTGCTAGCAATG) and downstream of the stop codon (GGCCACTAGTCGACTCCGTTCTCTCAGG) and containing a SalI restriction site (underlined) were synthesized and used to amplify rat liver cDNA. The product (1.1 kb) was cloned into pGEM-T vector to yield pGEM-. The inserts from each of three independent clones were sequenced to confirm their authenticity.

Northern Analysis

A rat multiple tissue Northern (Clontech) was probed with either a random primed (Feinberg and Vogelstein, 1983) 1.9-kb fragment encoding the beta subunit or a random primed 1.1-kb cDNA fragment encoding the subunit according to the manufacturer's instructions. Following hybridization the blot was washed with 2 times SSC, 0.5% SDS at room temperature for 1 h, followed by 0.2 times SSC, 0.5% SDS at 65 °C for 2 times 20 min and autoradiographed at -70 °C for 2-5 days.

Antibody Production

The entire coding sequence of AMPK (330 residues) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. A polypeptide containing the C-terminal 217 residues of the beta subunit was expressed in E. coli as a fusion protein with glutathione S-transferase. The fusion proteins were purified on a glutathione-agarose column (Pharmacia) and used to immunize male New Zealand White rabbits. Following three rounds of immunization, antiserum was collected and used for Western blot analysis and immunoprecipitations. Antiserum against the catalytic subunit of AMPK was obtained as described previously (Carling et al., 1994).

Western Blotting of Tissue Lysates

Female Wistar rats (250-300 g body weight) were killed by stunning and cervical dislocation, tissues removed, and immediately frozen in liquid nitrogen. Approximately 0.5 g of frozen tissue was ground to a fine powder using a pestle and mortar and homogenized in 10 ml of buffer (50 mM Tris/HCl, pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.25 M sucrose, 0.1 mM phenylmethylsulfonyl fluoride) using a Polytron homogenizer. After homogenization, SDS was added to a final concentration of 0.5% and the homogenate was boiled for 15 min. The homogenate was centrifuged at 14,000 times g for 15 min and the supernatant removed. The protein concentration of the supernatant was determined using the Lowry assay. 100 µg of protein for each tissue was resolved by SDS-PAGE and transferred to a polyvinylidene membrane. The membrane was blocked by incubation in 10 mM Tris/HCl, pH 7.5, 1 M NaCl, 0.5% Tween, 5% nonfat milk powder (w/v) for 1 h at room temperature. Primary antibody was added to this buffer and the blot incubated for another 2 h. The blot was washed extensively in 10 mM Tris/HCl, pH 7.5, 1 M NaCl, 0.5% Tween and then incubated with an anti-rabbit antibody conjugated to horseradish peroxidase. Antibodies were detected using enhanced chemiluminesence (Boehringer Mannheim).

Interactions Using the Two-hybrid System

Vectors expressing the GAL4 DNA binding domain (pGBT9) and the GAL4 activation domain (pGAD) from the ADH1 promoter (Clontech) were used to construct fusion proteins with each of the AMPK subunits. Plasmids containing the entire coding sequence of the catalytic subunit were constructed by insertion of a 1.9-kb EcoRI fragment of p63 cDNA (Carling et al., 1994) into the EcoRI site of pGBT9 or pGAD, producing G-alpha or G-alpha. Plasmids containing the entire coding sequence of the subunit were made by insertion of the 1.1-kb SalI fragment from pGEM- into the SalI site in pGBT9 or pGAD to produce G- and G-. In order to construct vectors expressing the beta subunit, the following oligonucleotide, GACTGCGAATTCCGGTCGACGGTGAATGAGAAAGCC, was used with the T7 promoter primer to amplify AMPKbeta cDNA from a clone contained in pBluescript. The product was digested with EcoRI (underlined sequence in the oligonucleotide) and XhoI (contained within the polylinker of Bluescript) and ligated into either pGBT9 or pGAD that had been digested with EcoRI and SalI. This creates an in-frame fusion between either the GAL4 DNA binding domain or GAL4 activation domain and the C-terminal 201 amino acids of the beta subunit. Vectors expressing fusions of the GAL4 activation domain with SNF1, SNF4, SIP1, and SIP2 (Yang et al., 1994) were generously provided by Dr. Marian Carlson, Columbia University.

Yeast (strain SFY526 harboring a GAL1-lacZ reporter gene) were transformed with various combinations of the vectors and grown on selective media. To test for interactions, several colonies from each transformation were patched onto selective plates and grown for 2 days at 30 °C. Colonies were transferred to nitrocellulose filters and cells were permeabilized by freeze-thawing in liquid nitrogen. The filters were incubated in the presence of X-Gal at 30 °C for 1-2 h in order to determine any blue coloration. For quantitative analysis, transformants were grown to mid-log phase in selective liquid culture and beta-galactosidase activity was determined in permeabilized cells. In every case, activities were measured in at least two transformants and assays were performed in triplicate. Values are expressed in Miller units (Miller, 1972) with a standard error of less than 20% of the mean.

In Vitro Translation and Immunoprecipitation of AMPK Subunits

cDNA containing the entire coding sequence of AMPKalpha was inserted into pET-21a (Novagen), while cDNAs containing the entire coding sequence of AMPKbeta, AMPK and the C-terminal 204 amino acids of AMPKbeta were inserted into pET-14b. The full-length AMPKbeta construct does not have any additional protein sequence, whereas the other constructs contain additional protein sequence encoded by the vector (including a polyhistidine sequence). 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 [S]methionine. Total labeled products of translation were analyzed by SDS-PAGE and fluorography. For immunoprecipitations, the volume of the lysate was adjusted to 1 ml with phosphate-buffered saline containing 1% Triton X-100 and incubated with preimmune serum and protein A-Sepharose for 2 h at 4 °C. The precleared reticulocyte lysate was incubated with antibodies against the alpha, beta, or subunits and protein A-Sepharose for 2 h at 4 °C. The immune complex was collected by centrifugation and washed extensively with with phosphate-buffered saline containing 1% Triton X-100 and either 0.2 M NaCl (alpha antibody), 1 M NaCl (beta antibody), or 0.5 M NaCl ( antibody) and analyzed by SDS-PAGE and fluorography.


RESULTS

Purification of AMPK Subunits from Rat Liver

Affinity-purified antibodies raised against the catalytic subunit of AMPK (Carling et al., 1994) were covalently cross-linked to protein A-Sepharose and used to purify AMPK from rat liver. Fig. 1shows that in addition to the 63 kDa catalytic subunit two other polypeptides, with apparent molecular masses of 38 and 36 kDa, as judged by SDS-PAGE, were identified (a faint band corresponding to IgG heavy chains could also be detected). We refer to these polypeptides as the beta subunit (38 kDa) and subunit (36 kDa) of AMPK, with the catalytic subunit being designated the alpha subunit. As can be seen from Fig. 1, both the alpha and beta subunits undergo autophosphorylation, whereas there is no detectable incorporation of phosphate into the subunit. In order to characterize the beta and subunits further, the purified polypeptides were transferred to a Problot membrane and subjected to N-terminal amino acid sequence analysis. On two separate occasions, no sequence was derived from the N terminus of either AMPKbeta or AMPK. However, cleavage of the gel-purified beta and subunits with cyanogen bromide yielded several peptides from which amino acid sequence was obtained (Table 1). Analysis of the Swiss-Prot data base with the peptide sequences from the subunit revealed that they were most closely related to sequences within yeast SNF4. No significant identity with any sequences in the data base was found with the peptides from the beta subunit.


Figure 1: Co-purification of two polypeptides with the catalytic subunit of rat liver AMPK. Partially purified AMPK from rat liver was purified using protein A-Sepharose cross-linked to affinity-purified antibodies raised against AMPKalpha. Following extensive washing, the antibody-protein A-Sepharose resin was incubated with 0.2 mM [-P]ATP and 0.2 mM AMP at 30 °C for 30 min. Proteins were eluted from the antibody-protein A-Sepharose resin, resolved by SDS-PAGE, and visualized by staining with Coomassie Blue (lane A). An autoradiograph of the same gel is shown in lane B. The migration of molecular mass standards is shown on the right of the figure.



Isolation of AMPKbeta cDNA

A partial cDNA sequence encoding AMPKbeta was obtained by PCR using rat liver cDNA and degenerate oligonucleotide primers corresponding to potential sequences encoding the peptide sequences 38CB1 and 38CB3 shown in Table 1(see ``Materials and Methods''). A product of approximately 600 bp was isolated and sequenced, revealing that it encoded peptide sequences corresponding to 38CB2 and 38CB4. The residue predicted to be immediately N-terminal to peptide 38CB4 was found to be an aspartic acid, rather than a methionine as expected following CNBr cleavage, indicating that peptide 38CB4 was produced by acid cleavage of an Asp-Pro bond. The PCR product was used to screen a rat liver cDNA library in order to isolate full-length clones. Twelve positive hybridizing clones for AMPKbeta were isolated from approximately 4 million plaques. Sequence analysis of the longest clone showed that the insert contained an in-frame methionine, which was preceded by a stop codon in the same frame, followed by an open reading frame of 269 amino acids that included all of the peptides obtained by amino acid sequencing of the purified protein from rat liver. The open reading frame was followed by approximately 1 kb of untranslated sequence, including a polyadenylation site consensus sequence and a poly(A) tail (Fig. 2A). The predicted molecular mass of AMPKbeta from the deduced protein sequence is 30 kDa, which is significantly lower than the apparent mass observed by SDS-PAGE analysis (38 kDa). However, in vitro translation of RNA synthesized from AMPKbeta cDNA (beginning at the first methionine) produced a protein that exactly co-migrated on SDS-PAGE with the beta subunit immunoprecipitated from rat liver (Fig. 3). This result confirms that there is no additional coding sequence upstream of the first methionine in the cDNA we isolated. At present we do not know the reason for the anomalous migration of the beta subunit on SDS-PAGE.


Figure 2: Nucleotide sequence and predicted amino acid sequence of AMPKbeta and AMPK. A, AMPKbeta. The initiating codon and stop codon are shown in bold, and peptide sequences derived from purified AMPKbeta are underlined. An in-frame stop codon upstream of the initiating methionine is shown in bold, and a potential polyadenylation signal sequence is underlined. Nucleotides are numbered on the right and amino acids on the left. B, AMPK.




Figure 3: In vitro translation of AMPKbeta. [S]Methionine-labeled AMPKbeta was translated in vitro in rabbit reticulocyte lysate programmed with RNA synthesized from AMPKbeta cDNA cloned into pET-14b. The lysate was immunoprecipitated using anti-beta antibodies attached to protein A-Sepharose and the immune complex analyzed by SDS-PAGE. AMPK was immunoprecipitated from rat liver with anti-beta antibodies and resolved by SDS-PAGE on the same gel in order to compare the electrophoretic mobility of the native and recombinant beta subunits. Proteins were visualized by staining with Coomassie Blue, and labeled products were detected by fluorography. Lane 1, total lysate in the absence of RNA; lane 2, total lysate programmed with AMPKbeta RNA; lane 3, immunoprecipitation of AMPK from rat liver; lane 4, immunoprecipitation of lysate programmed with AMPKbeta RNA. Note that in lane 3 all three AMPK subunits are precipitated and that AMPKbeta is the upper polypeptide in the 38/36-kDa doublet. The migration of molecular mass markers is shown on the left.



Isolation of AMPK cDNA

A partial cDNA sequence encoding AMPK was obtained by amplifying rat liver cDNA using degenerate oligonucleotide primers corresponding to potential sequences encoding the peptide sequences 36CB1 and 36CB2 shown in Table 1(see ``Materials and Methods''). A single product of approximately 400 bp was isolated, and sequence analysis confirmed that it encoded amino acid sequence within the peptides derived from the subunit. This cDNA product was used to screen a rat liver cDNA library. One positive hybridizing clone was isolated from approximately 1 million plaques. Sequence analysis of the clone revealed that it contained a 900-bp insert with an open reading frame of 200 amino acids, which encoded the sequence of peptide 36CB2. However, this clone did not encode the N-terminal region of including peptide 36CB1. In order to isolate the 5` region of cDNA encoding the subunit, we performed 5` RACE (Frohman et al., 1988) with rat liver cDNA. A unique product was isolated, and sequence analysis showed that it contained an in-frame methionine residue that was followed downstream by sequence corresponding to peptide 36CB1. A composite of the overlapping nucleotide sequences, together with the deduced protein sequence, is shown in Fig. 2B. The deduced mass of the protein encoded by the cDNA is 37 kDa, which is in reasonable agreement with the apparent mass observed from SDS-PAGE analysis of the purified protein. The open reading frame is followed by approximately 300 bp, but there is no obvious polyadenylation signal or poly(A) tail.

The beta and Subunits of AMPK Share Significant Amino Acid Sequence Identity with Yeast Proteins That Interact with SNF1

A search of the SwissProt data base with the amino acid sequence of the beta subunit showed that it is most closely related to SIP2 from S. cerevisiae, sharing 35% sequence identity overall. The highest conservation of sequence occurs at the C termini of the polypeptides, where there is 50% identity over a stretch of 68 amino acids (Fig. 4A, sequence beginning PPILPP to end). A similar, but slightly lower, degree of sequence identity was found with two other yeast proteins, SIP1 and GAL83, which have been shown previously to be related to SIP2 (Erickson and Johnston, 1993). A search of the SwissProt data base with the deduced protein sequence of the subunit identified the yeast protein SNF4 as having the highest degree of identity. The amino acid sequences of AMPK and SNF4 are 35% identical overall (Fig. 4B). No other significant similarities were identified with either of the subunits.


Figure 4: AMPKbeta and AMPK are related to yeast proteins that interact with SNF1. A, the deduced amino acid sequences of AMPKbeta (top) and SIP2 (bottom) were aligned using the GAP program in the University of Wisconsin package with a gap weight of 3.0 and a length weight of 0.1. Dots indicate gaps introduced to maximize the alignment. Identities between the two sequences are shown in shaded boxes. B, the deduced amino acid sequences of AMPK (top) and SNF4 (bottom) were aligned as above.



Tissue Distribution of AMPKbeta and AMPK

Poly(A)-rich RNA isolated from a number of rat tissues was probed with cDNA encoding the beta and subunits. Fig. 5shows the results of the Northern analysis and compares the expression of the beta and mRNA with the expression of AMPKalpha, which we have reported previously (Verhoeven et al., 1995). A strongly hybridizing band of approximately 2.4 kb was detected in all of the tissues tested following labeling with a cDNA probe specific for AMPKbeta. A weakly hybridizing signal at approximately 4.5 kb, present in all tissues, could also be detected with the AMPKbeta probe. When a subunit-specific probe was used, a single hybridizing band at approximately 1.8 kb was detected in all tissues, although only a faint signal was detected in testis. A band at approximately 2.4 kb was also detected with mRNA isolated from brain. These results indicate that the messages for both subunits are expressed in a wide number of rat tissues. In contrast to the beta and subunits, the mRNA expression of the alpha subunit shows marked differences in tissue distribution and is most highly expressed in skeletal and cardiac muscle (see Verhoeven et al.(1995)).


Figure 5: Northern blot analysis of AMPKalpha, beta, and mRNA. Approximately 2 µg of poly(A)-rich RNAs isolated from the indicated tissues were separated on a 1.2% agarose gel under denaturing conditions, transferred to a charged-modified nylon membrane, and probed separately with either a 1.9-kb fragment of AMPKbeta cDNA or a 1.1-kb fragment of AMPK cDNA. In each case the blot was washed under stringent conditions (0.2 times SSC, 0.5% SDS at 65 °C) and exposed for either 2 days (AMPKbeta) or 5 days (AMPK) at -70 °C. For comparison a Northern blot of the alpha subunit is shown (Verhoeven et al., 1995). The migration of RNA markers are indicated.



Antibodies raised against fusion proteins of AMPKbeta or AMPK with glutathione S-transferase were used to determine the expression of the polypeptides in various rat tissues. We also examined the expression of AMPKalpha (the catalytic subunit) using antibodies raised against alpha specific peptides (Carling et al., 1994). Fig. 6shows the expression of the polypeptides in a number of tissue lysates. All three polypeptides were detected in every tissue tested, although there appeared to be some variation in the relative amounts of the three subunits present in different tissues (for instance compare the expression of the alpha and beta subunits in brain and skeletal muscle). As we have noted previously, there is a small but detectable shift in the mobility of the alpha subunit between different tissues, which we believe may reflect differences in the phosphorylation state of the enzyme (Verhoeven et al., 1995).


Figure 6: Western blot analysis of AMPKalpha, beta, and . Approximately 100 µg of tissue lysate (14,000 times g supernatant) from the indicated tissues were separated by SDS-PAGE and transferred to a polyvinylidene membrane. Separate blots were probed with polyclonal antibody to AMPKalpha, AMPKbeta, or AMPK. Primary antibody was detected using a goat anti-rabbit antibody conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence.



Interaction of the alpha, beta and Subunits in the Two-hybrid System

We employed the yeast two-hybrid system (Fields and Song, 1989) in order to examine the interaction of the three AMPK subunits in more detail. The entire coding sequence of the alpha, , or the C-terminal 201 amino acids of beta were expressed as fusion proteins with either the GAL4 DNA binding domain (G) or the GAL4 transcriptional activation domain (G). Table 2shows the activity of beta-galactosidase in yeast transformed with various combinations of the fusion proteins. Transformation of yeast with G-alpha and G-beta or G-beta and G- resulted in significant beta-galactosidase activity, and this was confirmed by the appearance of blue colonies in the presence of X-Gal (Table 2). Similar results with these combinations were obtained when the G and G fusions were switched. The activity detected in transformants with G-alpha and G- or G-alpha and G- was very low in comparison with the other combinations, which could indicate that the interaction between AMPKalpha and AMPK is weaker than the other interactions. Very low levels of beta-galactosidase activity were detected when yeast were transformed with the same subunit expressed with both G and G, and these transformants remained white in the presence of X-Gal, indicating that the subunits do not form homodimers.



Since the two-hybrid system is carried out in yeast, it was important to determine any interactions between the mammalian subunits and their yeast counterparts. We therefore extended the study to determine interactions between AMPK subunits and SNF1, SNF4, SIP1, and SIP2. Table 3shows the results of the various combinations of rat and yeast proteins in the two-hybrid system. We were unable to detect any interaction of AMPKalpha with any of the yeast proteins. However, AMPKbeta gave a signal with both SNF1 and SNF4 and AMPK interacted with SNF1, SIP1, and SIP2. Furthermore, AMPK interacted with all of the SIP1 and SIP2 fusions tested, including a fusion expressing the C-terminal 120 amino acids of SIP2 (G-SIP2; Yang et al. (1994)).



Association of the AMPK Complex in Vitro

In order to confirm the results of the interactions determined by the two-hybrid system, we looked at the association of the subunits in vitro. The alpha, beta, and subunits were translated in rabbit reticulocyte lysates either alone or with one, or both, of the other subunits in the presence of [S]methionine. Translated products were immunoprecipitated with anti-alpha, anti-beta, or anti- antibodies bound to protein A-Sepharose, and the [S]methionine-labeled subunits in the immune complexes were resolved by SDS-PAGE and detected by fluorography. The products of the translations before immunoprecipitation (panel A) or following immunoprecipitation with AMPK subunit specific antibodies (panel B) are shown in Fig. 7. When alpha or were co-translated with beta, they could be co-precipitated with anti-beta antibodies. In contrast, however, the subunit was not detected in anti-alpha immunoprecipitates, nor was the alpha subunit detected in anti- immunoprecipitates, from lysates programmed with both the alpha and subunits. All three subunits were immunoprecipitated from lysates programmed with alpha, beta, and using anti-alpha, anti-beta, or anti- antibodies. These results indicate that when all three subunits are co-translated, a ternary complex between alpha, beta, and is formed, rather than a mixture of alphabeta and beta dimers. For clarity we have shown the results using a truncated form of AMPKbeta since full-length beta has a similar mobility to on SDS-gels. These results were consistent with those obtained using the full-length form of AMPKbeta (data not shown).


Figure 7: Association of AMPK subunits in vitro. A, [S]methionine-labeled AMPK subunits were generated in rabbit reticulocyte lysates programmed with the indicated RNAs and an aliquot of the products was analyzed by SDS-PAGE and fluorography. The AMPKbeta RNA used for these studies lacked sequence encoding the N terminus. AMPKbeta, therefore, migrates with a lower apparent molecular mass compared to AMPK, allowing the subunits to be clearly resolved. The migration of molecular mass markers is shown on the left. B, interaction of the subunits was determined by co-immunoprecipitation from the lysates (2 times volume used in A). Following incubation with preimmune serum and protein A-Sepharose, lysates were immunoprecipitated with subunit-specific antibodies as indicated. The immune complexes were washed extensively (as described under ``Materials and Methods''), boiled with SDS-sample buffer, and resolved by SDS-PAGE. Labeled products were detected by fluorography.



The results of the in vitro translation studies demonstrate that the alpha and beta subunits and the beta and subunits interact with each other forming relatively stable complexes, whereas there is no evidence for a stable complex between the alpha and subunits. If the alpha and subunits do interact, then their association must be either transient or weak, or both, and does not survive immunoprecipitation. The translations and immunoprecipitations are carried out in buffers lacking protein phosphatase inhibitors and under conditions that would not be expected to cause activation of endogenous AMPKK, which may be present in the reticulocyte lysate. It is likely that AMPKalpha is in the dephosphorylated form following translation, suggesting that phosphorylation by AMPKK is not necessary for formation of the ternary complex. This may also explain why we have been unable to detect AMPK activity in any of the translations.


DISCUSSION

Full-length cDNA clones encoding AMPKbeta were isolated by conventional library screening, and a composite cDNA clone encoding the full-length sequence of AMPK was constructed from overlapping clones isolated by a combination of library screening and 5` RACE. The deduced amino acid sequence of AMPKbeta predicts a protein with a molecular mass of 30 kDa. This is considerably lower than the apparent mass of the beta subunit isolated from rat liver, as judged by SDS-PAGE. In vitro translation of RNA synthesized from AMPKbeta cDNA produced a major product, which exactly co-migrated with rat liver AMPKbeta following SDS-PAGE. This finding, coupled with the fact that an in-frame stop codon is present upstream of the first methionine, confirms that the AMPKbeta cDNA reported here is full-length. The reason for the anomalous electrophoretic mobility of the beta subunit in denaturing gels is unclear, but if it is due to post-translational modification of the polypeptide the reticulocyte lysate system must be competent in carrying out the modification.

The finding that AMPKbeta and AMPK share sequence identity with yeast proteins that interact with SNF1 strengthens the proposal that the functions of the two kinases have been highly conserved throughout evolution (Woods et al., 1994). Although the function of SNF4 is not known, it is necessary for the protein kinase activity of SNF1 and it seems likely that AMPK will have a similar role in the activity of AMPK. Despite the obvious similarity between the amino acid sequences of AMPK and SNF4, we have not been able to complement snf4 mutants by expression of AMPK. (^2)In a previous study, we reported that we were unable to complement snf1 mutants by expression of the catalytic subunit of AMPK, which shares 47% amino acid sequence identity with SNF1 (Carling et al., 1994; Woods et al., 1994). Taken together these results indicate that, although the AMPK and SNF1 complexes are highly related, significant differences between the two complexes must exist. One notable difference that is already known is that, while the mammalian kinase is markedly activated by AMP (Carling et al., 1989), no measurable effect of AMP on SNF1 activity has been demonstrated (Mitchelhill et al., 1994; Woods et al., 1994). Whether this difference alone is sufficient to explain the inability of the catalytic subunit to rescue snf1 mutants, and AMPK to rescue snf4 mutants, is not yet clear. In order to address this question, a detailed comparison of the structures of the mammalian and yeast kinase complexes, e.g. from crystallographic studies, and the elucidation of the regulation of the kinases are required.

Western blot analysis of rat tissue lysates shows that AMPKbeta and AMPK subunits are expressed in every tissue examined. Although the blots are not quantitative, there does appear to be some variation in the relative expression of the three subunits in different tissues (Fig. 6). AMPK purified from rat liver appears to exist entirely as a heterotrimeric complex of AMPKalpha, beta, and (see Fig. 1and Davies et al.(1994)). Immunoprecipitation of alpha from skeletal muscle, however, suggests that it exists predominantly as a monomer, with no kinase activity (Verhoeven et al., 1995), even though AMPKbeta and AMPK are present in this tissue. These findings raise the interesting possibility that the association of the catalytic subunit with AMPKbeta and AMPK is regulated and that the structure of the complex may vary between different tissues and/or different conditions. At present the mechanism that leads to the association of the subunits is not known, although our results suggest phosphorylation is not required. Dephosphorylation of the active AMPK complex to an inactive form in vitro does not result in dissociation of the subunits (data not shown), and it is interesting to note that in yeast the association of SIP1 or SIP2 with SNF1 does not require SNF1 protein kinase activity or the presence of SNF4 (Yang et al., 1994).

The results from the two-hybrid experiments indicate that AMPKbeta interacts with both AMPKalpha and AMPK, but that the interaction of AMPKalpha with AMPK is very weak. We also tested the interaction of the mammalian subunits with their yeast counterparts in the two-hybrid system. In this case we found evidence for the interaction of AMPKbeta with both SNF1 and SNF4 and AMPK with SNF1 and SIP1 and SIP2. However, there was no detectable interaction between AMPKalpha and any of the yeast proteins. These results do not rule out the possibility that the AMPK-SNF1 interaction is indirect and could be mediated by an AMPKbeta homologue in yeast, e.g. SIP1/SIP2/GAL83, which could act as a bridging protein in a ternary complex.

The results from the in vitro study show that AMPKbeta interacts with both AMPKalpha and AMPK forming stable complexes. However, under the same conditions, we could not detect stable complexes between AMPKalpha and AMPK. These results suggest that the formation of the ternary complex between alpha, beta, and is mediated by the beta subunit. We have not been able to detect AMPK activity in lysates programmed with all three subunits, or in immune complexes from these lysates. This may be due to the dephosphorylated form of the kinase and/or the lack of sufficient protein in the translation system to allow detection of kinase activity.

Two different models for the association of the three subunits can be predicted based on the results of this study. The first model is one in which the beta subunit links the alpha and subunits (Fig. 8A). The second model involves a conformational change in either the alpha or subunit, or both, upon binding of the beta subunit, which would then allow direct interaction between the alpha and subunits (Fig. 8B). It is interesting to note the similarities between the AMPK complex and the heterotrimeric complex formed between CDK-activating kinase, cyclin H, and p36/MAT1 (RING finger protein) (Fisher et al., 1995; Devault et al., 1995). Although there is no significant amino acid sequence identity between the AMPK subunits and the CDK-activating kinase subunits, the regulation and association of the two kinase complexes bear some obvious resemblances.


Figure 8: Model for the association of an active AMPK complex. Our results demonstrate that AMPKbeta interacts with both AMPKalpha and AMPK and that this mediates the formation of a stable trimeric complex. AMPKbeta could act as a bridge linking the other two subunits (A), or alternatively AMPKbeta could cause a conformational change in AMPKalpha, AMPK, or both (depicted by the different labeling of the subunits) allowing the alpha and subunits to interact (B). AMPKalpha is phosphorylated by an upstream kinase (AMPKK), leading to activation of the complex (Weekes et al., 1994; Hawley et al., 1995). Our preliminary results indicate that AMPKK can only phosphorylate AMPKalpha when it is in the heterotrimeric form, implying that association of the complex is a prerequisite for phosphorylation. AMP activates the complex both allosterically and by promoting the phosphorylation of AMPKalpha by AMPKK, although it is not known whether AMP has any direct effect on the association of the complex.



Although the functions of AMPKbeta and AMPK remain unclear, a possible insight can be gained by comparison with a proposed model for the SNF1 complex in yeast. SNF4 is necessary for SNF1 kinase activity in vitro (Woods et al., 1994) and may therefore fulfill a similar function to cyclins in the activation of cyclin dependent kinases (Jeffrey et al., 1995). AMPK by analogy would play a similar role in the activation of AMPK. Biochemical evidence suggests that SNF1 forms a relatively stable complex with SNF4, and that this complex has protein kinase activity in vitro (Mitchelhill et al., 1994). The SNF1bulletSNF4 complex interacts with one of a number of related proteins, which include SIP1, SIP2, and GAL83 (Yang et al., 1994). It has been proposed that these proteins act as adaptors or targeting subunits, directing the kinase to specific intracellular substrates (Yang et al., 1994). In this model, the formation of different SNF1bulletSNF4bulletadaptor complexes would allow selective phosphorylation of the downstream targets of SNF1, if the adaptor proteins recognize different substrates. It is not clear whether the assembly of the SNF1bulletSNF4bulletadaptor complex is regulated, or how the adaptor proteins act to promote phosphorylation of target substrates. However, there is clear evidence from other systems that one mechanism for regulating the phosphorylation of a protein is to regulate the distribution of the kinase, or phosphatase, acting on that protein via specific targeting subunits (Hubbard and Cohen, 1993; Coghlan et al., 1995). Could AMPKbeta act as a targeting subunit for the AMPK complex? It seems unlikely that the function of AMPKbeta is merely to bring together the alpha and subunits, especially given the fact that in yeast there appears to be a family of proteins related to AMPKbeta. It will be interesting to determine whether or not there is a family of proteins related to AMPKbeta in mammalian cells, and whether these proteins act as adaptors for AMPK.

Finally, it is interesting to note that, although the interaction of SNF1 and SNF4 is often used as a model for the two-hybrid system, our results imply that this interaction could in fact be indirect. Given the similarities between the mammalian AMPK and yeast SNF1 complexes it is possible that the interaction between SNF1 and SNF4 is mediated by a member of the SIP1/SIP2/GAL83 family of proteins. In addition to SNF4, a number of other polypeptides were found to co-purify with SNF1 (Stapleton et al., 1994). Although SIP1 was not identified, it is possible that some of these co-purifying polypeptides are members of the SIP1/SIP2/GAL83 family, which could mediate the association of SNF1 and SNF4 in a ternary complex, analogous to AMPKbeta in the mammalian complex.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) X95577 [GenBank]and X95578[GenBank].

§
Supported by an intermediate research fellowship from the British Heart Foundation.

Supported by a Medical Research Council (United Kingdom) studentship.

**
To whom correspondence should be addressed. Tel: 44-181-740-3985; Fax: 44-181-749-8341; dcarling{at}rpms.ac.uk.

(^1)
The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKK, AMPK kinase; PAGE, polyacrylamide gel electrophoresis; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s); X-Gal, 5-bromo-4-chloro3-indoyl beta-D-galactoside; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^2)
P. C. F. Cheung and D. Carling, unpublished results.


ACKNOWLEDGEMENTS

We thank Robert Forder (Zeneca Pharmaceuticals) for the production of antisera against AMPKalpha.


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