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 t he 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 subuni t, 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 g amma 2 is widely expressed in human tissues, whereas significant expression of gamma 3 mRNA was only detected in skeletal muscle, Using isoform-specif ic antibodies, we determined the AMPK activity associated with the differen t 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, w ith the remaining activity being accounted for mainly by the gamma 2 isofor m, Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no eviden ce for any selective association between the eel and alpha 2 isoforms and t he various gamma isoforms. However, the AMP-dependence of the kinase comple x is markedly affected by the identity of the gamma isoform present, with g amma 2-containing complexes having the greatest AMP-dependence: gamma 3 the lowest, and gamma 1 having an intermediate effect. Labelling studies, usin g the reactive AMP analogue 8-azido-[P-32]AMP, indicate that the gamma subu nit may participate directly in the binding of AMP within the complex.