Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding.
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 (alpha) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma2 and gamma3. Both gamma2 (569 amino acids) and gamma3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma1. As with gamma1, mRNA encoding gamma2 is widely expressed in human tissues, whereas significant expression of gamma3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different gamma isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the gamma1 isoform, with the remaining activity being accounted for mainly by the gamma2 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 alpha1 and alpha2isoforms and the various gamma isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the gamma isoform present, with gamma2-containing complexes having the greatest AMP-dependence, gamma3 the lowest, and gamma1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[(32)P]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.
Cheung
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Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K.
Salt
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Davies
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Hardie
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Carling
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D055372:AMP-Activated Protein Kinases; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D020033:Protein Isoforms; D000249:Adenosine Monophosphate; D011494:Protein Kinases; C526222:PRKAG1 protein, human; C526225:PRKAG3 protein, human; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human