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

AMP-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 αβγ heterotrimer comprising a catalytic α and two non-catalytic subunits, β and γ. The β 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 β subunit, β1-(186–270), is sufficient to form an active AMP-dependent heterotrimer α1β1-(186–270)-γ1, whereas the 25-residue β1 C-terminal (246–270) sequence is sufficient to bind γ1, γ2, or γ3 but not the α subunit. Deletion of the β C-terminal Ile-270 precludes βγ association in the absence of the α subunit, but the presence of the α subunit or substitution of Ile-270 with Ala or Glu restores βγ binding. Truncation of the α subunit reveals that β1 binding requires the α1-(313–473) sequence. The conserved C-terminal 85-residue sequence of the β subunit (90% between β1 and β2) is the primary αγ binding sequence responsible for the formation of the AMPK αβγ heterotrimer.