Abstract
Abstract The 5′-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic α-subunit, an AMP-binding γ-subunit, and a scaffolding β-subunit thought to bind directly both the α- and γ-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the α2-subunit binds directly not only to the β-subunit, confirming previous work, but also to the γ1-subunit. Deletion analysis of the α2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the β-subunit. The γ1-subunit binds directly to the α2-subunit at two interaction sites, one within the catalytic domain consisting of α2 amino acids 1-312 and a second within residues 386-552. Binding of the α2 and the γ1-subunits was not affected by 400 μm AMP or ATP. Furthermore, we show that the β-subunit C terminus is essential for binding to the α2-subunit but, in contrast to previous work, the β-subunit does not bind directly to the γ1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the β-subunit binds to the α-subunit that, in turn, binds to the γ-subunit. There is no direct interaction between the β- and γ-subunits.
Highlights
The 5Ј-AMP-activated protein kinase (AMPK)[2] is a master metabolic regulator; it is activated by high intracellular AMP/ ATP ratios to respond to diminishing cellular energy (1, 2) and by the adipokines leptin (3) and adiponectin (4)
This study presents a new model for AMPK heterotrimer structure where through its C terminus the -subunit binds to the ␣-subunit that, in turn, binds to the ␥-subunit
AMPK is functionally related to the yeast Snf[1] protein kinase complex which plays a major role in regulating the nutritional stress response in Saccharomyces cerevisiae (2, 15)
Summary
THE ␣-SUBUNIT BINDS TO BOTH THE - AND ␥-SUBUNITS THERE IS NO DIRECT BINDING BETWEEN THE - AND ␥-SUBUNITS*. The KIS and ASC domains present in the three yeast -subunits (Sip1/Sip2/Gal83) were applied to the mammalian AMPK--subunits by sequence homology, and similar functional conservation of binding the ␣- and ␥-subunits was inferred. It was subsequently shown that the C-terminal 186 –270 residues of AMPK-1 were sufficient to tether the ␣- and ␥-subunits (19) Another layer of complexity was uncovered by elucidation of the crystal structure of the yeast Snf[1] catalytic domain. FLAG and HA epitope-tagged versions of full length Snf[1] co-immunoprecipitated together and many residues that mediate the dimer interaction are conserved across species These data suggest that AMPK may exist in a stoichiometry other than 1:1:1 (␣::␥) with additional subunit interactions potentially involved in activation or repression of the kinase complex. This study provides evidence for a new model of AMPK heterotrimer structure where the -subunit binds to the ␣-subunit which, in turn, binds the ␥-subunit but with no direct interaction between the - and ␥-subunits (Fig. 1)
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