AMP-activated protein kinase kinase: detection with recombinant AMPK α1 subunit

Abstract

The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic α subunit and two non-catalytic subunits, β and γ, and is regulated both by the allosteric action of AMP and by phosphorylation of the α and β subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the α subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK α1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in α subunit regulation. Recombinant α1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6× His fusion proteins and purified to homogeneity by Ni 2+ chromatography. Both wild-type α1(1–312) and α1(1–312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKβ. The corresponding AMPKα1(1–392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant α1(1–312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active.