title: 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.
authors: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors: Dakshayini G
authors: DG
authors: Chandrashekarappa
authors: Rhonda R
authors: RR
authors: McCartney
authors: Martin C
authors: MC
authors: Schmidt
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D011494:Protein Kinases; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D000091162:AMP-Activated Protein Kinase Kinases