AMP-activated protein kinase β subunit tethers α and γ Subunits via its C-terminal sequence (186-270)

dAMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable alpha beta gamma heterotrimer comprising a catalytic alpha and two non-catalytic subunits, beta and gamma. The beta subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the beta subunit, beta 1-(186-270), is sufficient to form an active AMP-dependent heterotrimer alpha 1 beta 1-(186-270)-gamma 1, whereas the 25-residue beta 1 C-terminal (246 270) sequence is sufficient to bind gamma 1, gamma 2, or gamma 3 but not the alpha subunit. Deletion of the beta C-terminal Ile-270 precludes beta gamma association in the absence of the alpha subunit, but the presence of the alpha subunit or substitution of Ile-270 with Ala or Glu restores beta gamma binding. Truncation of the alpha subunit reveals that beta 1 binding requires the alpha 1-(313-473) sequence. The conserved C-terminal 85-residue sequence of the beta subunit (90% between beta 1 and beta 2) is the primary alpha gamma binding sequence responsible for the formation of the AMPK alpha beta gamma heterotrimer.