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

The synergistic positive regulatory roles for both the noncatalytic beta and gamma subunits of 5'-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf 1p and Snf4p seem to be required for maximum kinase activity.

Abstract

The mammalian 5′-AMP-activated protein kinase is a heterotrimer consisting of an α catalytic subunit and β and γ 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 α subunit isoforms, α-1 and α-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 α subunits. Co-expression of α subunits with the β or γ subunits modestly increases kinase activity accompanied by the formation of α/β or α/γ 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 α subunit, the β and γ 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 α subunit expression. Co-expression of a catalytically defective α subunit or the β/γ-binding COOH-terminal domain of the α subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic β and γ 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. The mammalian 5′-AMP-activated protein kinase is a heterotrimer consisting of an α catalytic subunit and β and γ 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 α subunit isoforms, α-1 and α-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 α subunits. Co-expression of α subunits with the β or γ subunits modestly increases kinase activity accompanied by the formation of α/β or α/γ 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 α subunit, the β and γ 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 α subunit expression. Co-expression of a catalytically defective α subunit or the β/γ-binding COOH-terminal domain of the α subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic β and γ 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.