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.

• PMID: 7718624
• DOI full text
• PubMed

Reference Type

Comparative Study | Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Gao G, Widmer J, Stapleton D, Teh T, Cox T, Kemp BE, Witters LA

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