Activation of Rat Liver AMP-activated Protein Kinase by Kinase Kinase in a Purified, Reconstituted System. Effects of AMP and AMP Analogues

Eur J Biochem. 1994 Feb 1;219(3):751-7. doi: 10.1111/j.1432-1033.1994.tb18554.x.

Abstract

AMP-activated protein kinase, purified from rat liver as far as the diethylaminoethyl-Sepharose step, is inactivated by treatment with protein phosphatase 2C, and reactivated by an endogenous 'kinase kinase'. Further purification of AMP-activated protein kinase on Blue Sepharose removes the kinase kinase, but the system can be reconstituted by adding back the flow-through from the Blue-Sepharose column. The kinase kinase can be further purified by subjecting the flow-through from the Blue-Sepharose column to chromatography on a Mono-Q column. A single peak of kinase kinase activity is obtained. Using this fraction, and the most highly purified preparation of AMP-activated protein kinase, phosphorylation of the 63-kDa polypeptide, previously identified as the catalytic subunit of AMP-activated protein kinase, can be demonstrated. As previously shown in the partially purified system, phosphorylation of the 63-kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concentration. The structurally related AMP analogue, 8-aza-9-deazaadenosine-5'-monophosphate, mimics the effect of AMP on both allosteric activation and phosphorylation of the kinase, while adenosine (5')tetraphospho(5')adenosine antagonizes both effects. These results suggest that both the allosteric effect of AMP, and the promotion of phosphorylation and activation by the kinase kinase, are due to binding of AMP to a single site on the kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases
  • Adenosine Monophosphate / analogs & derivatives
  • Adenosine Monophosphate / metabolism
  • Adenosine Monophosphate / pharmacology*
  • Animals
  • Binding Sites
  • Dinucleoside Phosphates / pharmacology
  • Enzyme Activation / drug effects
  • Formycins / pharmacology
  • Liver / enzymology*
  • Molecular Weight
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism*
  • Phosphorylation
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases*
  • Rats
  • Ribonucleotides / pharmacology

Substances

  • Dinucleoside Phosphates
  • Formycins
  • Multienzyme Complexes
  • Ribonucleotides
  • formycin 5'-phosphate
  • Adenosine Monophosphate
  • diadenosine tetraphosphate
  • formycin
  • Protein Kinases
  • AMP-activated protein kinase kinase
  • Protein-Serine-Threonine Kinases
  • AMP-Activated Protein Kinases