The mammalian 5′-AMP-activated protein kinase is a heterotrimer consisting of an α catalytic subunit and β and γ 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 α subunit isoforms, α-1 and α-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 α subunits. Co-expression of α subunits with the β or γ subunits modestly increases kinase activity accompanied by the formation of α/β or α/γ 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 α subunit, the β and γ 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 α subunit expression. Co-expression of a catalytically defective α subunit or the β/γ-binding COOH-terminal domain of the α subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic β and γ 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.

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.

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 (alpha 1) is distinct from the previously cloned kinase subunit (alpha 2). The alpha 1 (548 residues) and alpha 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 alpha 1 mRNA distribution and the alpha 1 immunoreactivity rather than alpha 2, with substantial amounts in kidney, liver, lung, heart, and brain. Both alpha 1 and alpha 2 isoforms are stimulated by AMP and contain noncatalytic beta and gamma subunits. The liver alpha 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the alpha 2 immunoreactivity parallels the alpha 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the beta and gamma subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.

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 (AMPK63(cat)) 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 AMPK63(cat), 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 AMPK63(cat) (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 AMPK63(cat) 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.

The 5'-AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation and inactivation of acetyl-CoA carboxylase. The porcine liver 5'-AMP-activated protein kinase 63-kDa catalytic subunit co-purifies 14,000-fold with a 38- and 40-kDa protein (Mitchelhill, K.I. et al. (1994) J. Biol. Chem. 269, 2361-2364). The 63-kDa subunit is homologous to the Saccharomyces cerevisiae Snf1 protein kinase, which regulates gene expression during glucose derepression. Peptide amino acid and polymerase chain reaction-derived partial cDNA sequences of both the pig and rat liver enzymes show that the 38-kDa protein is homologous to Snf4p (CAT3) and that the 40-kDa protein is homologous to the Sip1p/Spm/GAL83 family of Snf1p interacting proteins. Sucrose density gradient and cross-linking experiments with purified 5'-AMP-activated protein kinase suggest that both the 38- and 40-kDa proteins associate tightly with the 63-kDa catalytic polypeptide in either a heterotrimeric complex or in dimeric complexes. The 40-kDa subunit is autophosphorylated within the 63-kDa subunit complex. The sequence relationships between the mammalian 5'-AMP-activated protein kinase and yeast Snf1p extend to the subunit proteins consistent with conservation of the functional roles of these polypeptides in cellular regulation by this family of metabolite-sensing protein kinases.

1. In the rat, acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid metabolism, exists as at least two different isozymes (Mr 265,000 and 280,000) that display distinct tissue-specific distribution and regulation. 2. Based on the study of human tissue and human-derived breast cancer cell lines by enzyme isolation and protein blotting techniques, we have now identified two human isoforms of Mr 265,000 (HACC 265) and 275,000 (HACC 275), each of which is homologous to one of the rat isozymes. 3. Human breast carcinoma cell lines show variable expression of these two isoforms, mirrored in the estimation of ACC acetyl-CoA kinetics.

Heterogeneous zonation of hepatic protein expression over the liver lobule has been recognized by using several analytical techniques, including microdissection, selective cell isolation, immunohistochemistry and hybridization of mRNA in situ. We previously employed the technique of dual-digitonin-pulse perfusion for the highly selective collection and analysis of periportal and perivenous soluble protein. In the present work we have now documented the feasibility of the application of this technique to the study of zonal distribution of mRNA. By using a split-stream design, both protein and RNA fractions can be simultaneously collected from hepatic zones. High-quality RNA (average yield approximately 9-33 micrograms of total RNA per mg of eluted protein) is obtained for analysis. As analysed by immunoblotting and Northern-blot analysis, the zonal distribution of several important cytosolic metabolic enzymes and their mRNAs can be documented. This technique is also applicable to the study of mRNAs for organelle- and membrane-associated proteins that are not recoverable with this digitonin-lysis technique. The application of this experimental technique should allow further molecular insight into the mechanisms underlying zonation of hepatic function.