Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
| Members of the AMP-activated protein kinase (AMPK) family are activated by phosphorylation on a conserved threonine residue in the activation loop of the kinase domain. Mammalian AMPK adopts a phosphatase resistant conformation that is stabilized by binding low energy adenylate molecules. Similarly, binding of adenosine diphosphate to the Snf1 complex, the yeast AMPK, protects the kinase from dephosphorylation. Here we determine nucleotide specificity of the ligand-mediated protection from dephosphorylation and demonstrate the subunit and domain requirements for this reaction. Protection from dephosphorylation is highly specific for adenine nucleotides with adenosine diphosphate being the most effective ligand for mediating protection. The full-length α subunit (Snf1) was not competent for ADP-mediated protection confirming the requirement for the regulatory β and γ subunits. However, Snf1 heterotrimeric complexes which lacked either the glycogen binding domain of Gal83 or the linker region of α subunit were both competent for ADP-mediated protection. In contrast, adenylate-mediated protection of recombinant human AMPK was abolished when a portion of the linker region containing the α-hook domain was deleted. Therefore the exact means by which the different adenylate nucleotides are distinguished by the Snf1 enzyme may differ from its mammalian ortholog. |
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Human AMPK alpha-hook domain is required for adenylate-mediated
protection in vitro. A, structural model for human AMPK in the active conformation showing the alpha subunit (green), with its alpha-hook domain (red sticks) reaching into site 3 of the gamma subunit (cyan) bound to ADP (blue). B, Coomassie Blue-stained protein gel showing the purity of the recombinant AMPK (1:1:1) complex and the same complex with the alpha-hook deleted (AMPK d377–411). The mobility of molecular mass markers (M) is indicated in kilodaltons on the left. |