The mammalian 5-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (␣ 1 ) is distinct from the previously cloned kinase subunit (␣ 2 ). The ␣ 1 (548 residues) and ␣ 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase ␣ 1 mRNA distribution and the ␣ 1 immunoreactivity rather than ␣ 2 , with substantial amounts in kidney, liver, lung, heart, and brain. Both ␣ 1 and ␣ 2 isoforms are stimulated by AMP and contain noncatalytic ␤ and ␥ subunits. The liver ␣ 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the ␣ 2 immunoreactivity parallels the ␣ 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the ␤ and ␥ subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.The 5Ј-AMP-activated protein kinase (AMPK) 1 was initially identified as a protein kinase regulating hydroxymethylglutaryl-CoA reductase (1). Subsequently, the AMPK was shown to phosphorylate hepatic acetyl-CoA carboxylase (2) and adipose hormone-sensitive lipase (3). The AMPK appears to act as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are depleted and when 5Ј-AMP rises in response to fuel limitation and/or hypoxia (4). Partial amino acid sequencing of hepatic AMPK (5, 6) revealed that it consists of 3 subunits, the catalytic subunit ␣ (63 kDa), and two noncatalytic subunits, ␤ (40 kDa) and ␥ (38 kDa).The AMPK is a member of the yeast SNF1 protein kinase subfamily that includes protein kinases present in plants, nematodes, and humans (5-9). The AMPK catalytic subunit, ␣, has strong sequence identity to the catalytic domain of the yeast protein kinase SNF1, which is involved in the induction of invertase (SUC2) under conditions of nutritional stress due to glucose starvation (10). Both Snf1p and the AMPK require phosphorylation by an activating protein kinase for full activity (11). The sequence relationship between Snf1p and AMPK led to the finding that these enzymes share functional similarities, both phosphorylating and inactivating yeast acetyl-CoA carboxylase (5,11,12). Nevertheless, the AMPK does not complement SNF1 in yeast (11), indicating that their full range of functions are not identical. The noncatalytic ␤ and ␥ subunits of AMPK are also related to proteins that interact with Snf1p; the ␤ subunit is related to the SIP1/SIP2/GAL83 family of transcription regulators and the ␥ subunit to SNF4 (also called CAT3) (6, 13). EXPERIMENTAL PROCEDURESPeptide Sequencing-Peptides were derived from rat and porcine ␣ 1 subunit of the AMPK, by in situ proteolysis (5), and sequenced on either an Ap...

The mammalian 5-AMP-activated protein kinase is a heterotrimer consisting of an ␣ catalytic subunit and ␤ and ␥ noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast. In this study, we have used mammalian cell transfection to compare the activities of the two ␣ subunit isoforms, ␣-1 and ␣-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity. Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic ␣ subunits. Co-expression of ␣ subunits with the ␤ or ␥ subunits modestly increases kinase activity accompanied by the formation of ␣/␤ or ␣/␥ heterodimers. Co-expression of all three subunits, however, is accompanied by a 50 -110-fold increase in kinase activity with the formation of a heterotrimeric complex. In addition to binding of each noncatalytic subunit to the ␣ subunit, the ␤ and ␥ subunits bind to each other, likely resulting in a more stable heterotrimeric complex. The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced ␣ subunit expression. Co-expression of a catalytically defective ␣ subunit or the ␤/␥-binding COOH-terminal domain of the ␣ subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic ␤ and ␥ subunits of 5-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.

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-␥ 1 ), 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-␥ 1 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 5'-AMP-activated protein kinase (AMPK) regulates the fatty acid and sterol synthesizing pathways via phosphorylation of acetyl-CoA carboxylase and HMG-CoA reductase, respectively. Highly purified kinase from porcine liver contains three apparent subunits of molecular mass 63 kDa, 40 kDa and 38 kDa. Peptide sequencing of the 63 kDa protein (AMPK63cat) revealed that this polypeptide is the catalytic subunit of the kinase. Porcine peptide sequences were used to clone by RT-PCR partial length cDNAs for the catalytic domains of the porcine AMPK63cat, and its rat homolog, which were virtually identical in deduced amino acid sequence. Screening of a rat liver cDNA library with these partial length cDNAs and with degenerate oligonucleotides yielded several unique clones, some of which had a 142 bp deletion in the catalytic domain of the kinase. A consensus full-length sequence with a 1.7 kb open reading frame has been constructed from overlapping library and PCR-derived clones. A large mRNA for rat AMPK63cat (8.5 kb) is expressed in nearly all rat tissues, with highest levels detectable in heart and skeletal muscle. Using PCR, the presence of two mRNA species with or without the 142 bp deletion in the catalytic domain was noted in all rat tissues examined. Comparison of the deduced protein sequence of AMPK63cat reveals highly conserved homologies in both the catalytic and non-catalytic domains to several members of the SNF1 kinase family, including kinases from Arabidopsis, barley, rye, and S. cerevesiae, as well as to other mammalian kinases and to a C. elegans kinase. The high evolutionary conservation of both kinase structure and function (metabolite sensing) coupled with their pattern of tissue/organism expression suggest that the mammalian members of this kinase family likely play wider roles than the regulation of cellular lipid metabolism.

Acetyl-CoA carboxylase (ACC), an important enzyme in fatty acid biosynthesis and a regulator of fatty acid oxidation, is present in at least two isoenzymic forms in rat and human tissues. Previous work has established the existence of a 265,000 Da enzyme in both the rat and human (RACC265; HACC265) and a higher-molecular-mass species (275,000-280,000 Da) in the same species (RACC280; HACC275). An HACC265 gene has previously been localized to chromosome 17. In the present study, we report cloning of a partial-length human cDNA sequence which appears to correspond to HACC275 and its rat homologue, RACC280, as judged by mRNA tissue distribution and cell-specific regulation of mRNA/protein expression. The gene encoding this isoenzymic form of ACC has been localized to the long arm of human chromosome 12. Thus, ACC is represented in a multigene family in both rodents and humans. The newly discovered human gene and its rat homologue appear to be under different regulatory control to the HACC265 gene, as judged by tissue-specific expression in vivo and by independent modulation in cultured cells in vitro.