Regulation of 5′-AMP-activated Protein Kinase Activity by the Noncatalytic β and γ Subunits

Abstract

The mammalian 5-AMP-activated protein kinase is a heterotrimer consisting of an ␣ catalytic subunit and ␤ and ␥ noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast. In this study, we have used mammalian cell transfection to compare the activities of the two ␣ subunit isoforms, ␣-1 and ␣-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity. Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic ␣ subunits. Co-expression of ␣ subunits with the ␤ or ␥ subunits modestly increases kinase activity accompanied by the formation of ␣/␤ or ␣/␥ heterodimers. Co-expression of all three subunits, however, is accompanied by a 50 -110-fold increase in kinase activity with the formation of a heterotrimeric complex. In addition to binding of each noncatalytic subunit to the ␣ subunit, the ␤ and ␥ subunits bind to each other, likely resulting in a more stable heterotrimeric complex. The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced ␣ subunit expression. Co-expression of a catalytically defective ␣ subunit or the ␤/␥-binding COOH-terminal domain of the ␣ subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic ␤ and ␥ subunits of 5-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.

AMP-activated Protein Kinase β Subunit Tethers α and γ Subunits via Its C-terminal Sequence (186–270)

“…Maximum activity requires all three subunits (3). The catalytic AMPK ␣1-(1-312) fragment is constitutively active whereas the ␣1-(1-392) fragment is autoinhibited, and neither bind ␤␥ subunits (2).…”

β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit

“…In mammals, the formation of the heterotrimer is necessary for AMPKa activity (Dyck et al, 1996;Woods et al, 1996). In yeast, SNF4 (the g-subunit) deletion or simultaneous deletions of the three b-subunits totally inactivates the SNF1 activity in vivo (Carlson et al, 1981;Schmidt and McCartney, 2000), indicating that they all play important roles within the complex.…”

A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell

“…A constitutively active AMPK mutant containing only the a-subunit kinase domain, CcaAMPK, provided an alternative, but the kinase domain alone possesses weak kinase activity in mammalian cells, and maximum AMPK activity requires all three subunits. 6,14 We therefore constructed a replication-defective adenovirus expressing NcaAMPK, which has higher kinase activity and suppresses anoxia-induced apoptotic cell death more efficiently than CcaAMPK. Although higher dose, 100 MOI, CcaAMPK transduction in the cells increased SAMS peptide phosphorylation rate by B35% compared with 50 MOI, the dose we used in this study, we did not find more potent inhibition of cell death in CcaAMPK-overexpressing cells with 100 MOI (data not shown).…”

“…Several kinds of constitutively active AMPK mutants have been investigated, including AMPKa1 (amino acids (aa) 1-312, Thr172-Asp: T172D), 14,15 AMPKg2 (Arg302-Gln), 16 and AMPKg1 (His150-Arg). 17 Although it has been reported that maximum activity of AMPK requires all three subunits, 6 AMPKa1 (1-312, T172D) lacks both the binding domain for interactions with the b-subunit and the autoinhibitory domain, which inhibits the self-kinase activity. 14 The b-subunit plays a role in modulating subcellular localization through its phosphorylation and myristoylation, 18,19 so that the ability to bind the b-subunit might be critical for the catalytic a-subunit to select its appropriate substrates.…”

“…4 AMPK was identified as a homolog of yeast sucrose non-fermenting 1 and is known as a metabolite-sensing protein kinase. 5 AMPK is a heterotrimeric serine/threonine protein kinase consisting of a catalytic a-subunit and two regulatory subunits, b and g. 6 There are multiple isoforms of each AMPK subunit, with a1, a2, b1, b2, g1, g2, and g3 forming heterotrimers 7 that differ in tissue and subcellular localization. In mammalian cells, AMPK is activated by increases in the AMP/ATP ratio, 4 which occur under conditions of hypoxia or anoxia.…”