Subunit and domain requirements for adenylate-mediated protection of Snf1 kinase activation loop from dephosphorylation.
ABSTRACT Members of the AMP-activated protein kinase (AMPK) family are activated by phosphorylation at 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 ADP to the Snf1 complex, yeast AMPK, protects the kinase from dephosphorylation. Here, we determined the nucleotide specificity of the ligand-mediated protection from dephosphorylation and demonstrate the subunit and domain requirements for this reaction. Protection from dephosphorylation was highly specific for adenine nucleotides, with ADP 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 that lacked either the glycogen-binding domain of Gal83 or the linker region of the α subunit were 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 compared with its mammalian ortholog.
Article: Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases.[show abstract] [hide abstract]
ABSTRACT: The SNF1/AMP-activated protein kinases are αβγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the β and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking β- and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.Proceedings of the National Academy of Sciences 05/2012; 109(22):8652-7. · 9.68 Impact Factor