Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
| The AMP-activated protein kinase (AMPK) is a conserved signaling molecule in a pathway that maintains adenosine triphosphate homeostasis. Recent studies have suggested that subunit of AMPK promote the formation of an active, phosphatase- resistant conformation. We propose an alternative model in which the kinase domain association with the heterotrimer core results in activation of the kinase catalytic activity, whereas low energy adenylate ligands bound in the kinase active site promote phosphatase resistance. Purified Snf1 subunit with a conservative, single amino acid substitution in the kinase domain is protected from dephosphorylation by adenosine subunits. Staurosporine, a compound known to bind to the active site of many protein kinases, mediates strong protection from dephosphorylation to yeast and mammalian AMPK enzymes. The analog- sensitive Snf1-I132G protein but not wild type Snf1 exhibits protection from dephosphorylation when bound by the adenosine analog 2NM-PP1 in vitro and in vivo. These data demonstrate that ligand binding to the Snf1 active site can mediate phosphatase resistance. Finally, Snf1 kinase with an amino acid substitution at the interface of the kinase domain and the heterotrimer core exhibits normal regulation of phosphorylation in vivo but greatly reduced Snf1 kinase activity, supporting a model in which kinase domain association with the heterotrimer core is needed for kinase activation. |
| Model for activation of Snf1 kinase and acquisition of phosphatase resistance. A, the Snf1 kinase domain (KD) is loosely tethered to the heterotrimer core (abg) via a disordered linker domain with the activation loop accessible to phosphorylation (reaction 1) and dephosphorylation (reaction 2). Once it is phosphorylated, the kinase domain can associate with the heterotrimer core (reaction 3) and adopt the active conformation (star shape) with the alignment of the regulatory spine (blue lines). Binding of low energy adenylate ligands to the active site and/or the subunit promotes formation of a phosphatase resistant conformation (reaction 5), indicated here as the phosphate (red P) burrowing into the KD. B, structure of the mammalian AMPK kinase domain (green cartoon representation) in the inactive and active states showing the position of the residues (blue spheres) that comprise the regulatory spine (residues Leu-68, Leu-79, Phe-158, and His-137 in mammalian AMPK). Upon association with the heterotrimer core (cyan surface representation), the kinase domain adopts an active conformation with the regulatory spine in alignment. The activation loop (shown in red) becomes structuredwhenit is bound in a cleft in the heterotrimer core. Protein Data Bank files used to generate this figure were 2Y94 and 2H6D. |