Characterization of AMP-activated protein kinase γ-subunit isoforms and their role in AMP binding

PCF CHEUNG, IP SALT, SP DAVIES… - Biochemical …, 2000 - portlandpress.com
PCF CHEUNG, IP SALT, SP DAVIES, DG HARDIE, D CARLING
Biochemical Journal, 2000portlandpress.com
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 (α) and two regulatory subunits (β and γ). We have isolated and characterized two
isoforms of the γ subunit, termed γ2 and γ3. Both γ2 (569 amino acids) and γ3 (492 amino
acids) have a long N-terminal domain which is not present in the previously characterized
isoform, γ1. As with γ1, mRNA encoding γ2 is widely expressed in human tissues, whereas …
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 (α) and two regulatory subunits (β and γ). We have isolated and characterized two isoforms of the γ subunit, termed γ2 and γ3. Both γ2 (569 amino acids) and γ3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, γ1. As with γ1, mRNA encoding γ2 is widely expressed in human tissues, whereas significant expression of γ3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different γ isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the γ1 isoform, with the remaining activity being accounted for mainly by the γ2 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 α1 and α2isoforms and the various γ isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the γ isoform present, with γ2-containing complexes having the greatest AMP-dependence, γ3 the lowest, and γ1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[32P]AMP, indicate that the γ subunit may participate directly in the binding of AMP within the complex.
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