Biochem. J. (2000) 346
(659–669) (Printed in Great Britain)
Characterization of AMP-activated protein kinase g-subunit isoforms and their role in AMP binding
Peter C. F. CHEUNG*1, Ian P. SALT†‡, Stephen P. DAVIES†§, D. Grahame HARDIE† and David CARLING*2
*Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K., †Biochemistry Department, Dundee University, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, Scotland, U.K., ‡Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K., and §Division of Signal Transduction Therapy, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, Scotland, U.K.
The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of a catalytic subunit (a) and two regulatory subunits (b and g). We have isolated and characterized two isoforms of the g subunit, termed g2 and g3. Both g2 (569 amino acids) and g3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, g1. As with g1, mRNA encoding g2 is widely expressed in human tissues, whereas significant expression of g3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different g isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the g1 isoform, with the remaining activity being accounted for mainly by the g2 isoform. Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no evidence for any selective association between the a1 and a2isoforms and the various g isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the g isoform present, with g2-containing complexes having the greatest AMP-dependence, g3 the lowest, and g1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[32P]AMP, indicate that the g subunit may participate directly in the binding of AMP within the complex.
Abbreviations used: AMPK, AMP-activated protein kinase; AMPKK, AMP-activated protein kinase kinase; ASC, association with SNF1 complex; BLAST, Basic Local Alignment Search Tool; CBS, cystathionine
b-synthase; DTT, dithiothreitol; EST, expressed sequence tag; FSBA,
p-fluorosulphonylbenzoyl adenosine; KIS, kinase interacting sequence.
1 Present address: MRC Protein Phosphorylation Unit, Biochemistry Department, Dundee University, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, Scotland, U.K.
2 To whom correspondence should be addressed (e-mail dcarling@csc.mrc.ac.uk).
The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AJ249976 (AMPK-g2) and AJ249977 (AMPK-g3).
Key words: allosteric activation, cell signalling, metabolic regulation.
Received 15 October 1999/3 December 1999; accepted 14 January 2000
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