Characterization of AMP-activated protein kinase γ-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 (a) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma 2 and gamma 3. Both gamma 2 (569 amino acids) and gamma 3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma 1, As with gamma 1, mRNA encoding gamma 2 is widely expressed in human tissues, whereas significant expression of gamma 3 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 gamma 1 isoform, with the remaining activity being accounted for mainly by the gamma 2 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 eel and alpha 2 isoforms 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 gamma 2-containing complexes having the greatest AMP-dependence: gamma 3 the lowest, and gamma 1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[P-32]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.