ACTIVATION OF RAT-LIVER AMP-ACTIVATED PROTEIN-KINASE BY KINASE KINASEIN A PURIFIED, RECONSTITUTED SYSTEM EFFECTS OF AMP AND AMP ANALOGS

AMP-activated protein kinase, purified from rat liver as far as the di ethylaminoethyl - Sepharose step, is inactivated by treatment with pro tein phosphatase 2C, and reactivated by an endogenous 'kinase kinase'. Further purification of AMP-activated protein kinase on Blue Sepharos e removes the kinase kinase, but the system can be reconstituted by ad ding 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 t he most highly purified preparation of AMP-activated protein kinase, p hosphorylation of the 63-kDa polypeptide, previously identified as the catalytic subunit of AMP-activated protein kinase, can be demonstrate d. As previously shown in the partially purified system, phosphorylati on of the 63-kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concent ration. The structurally related AMP analogue, 8-aza-9-deazaadenosine- 5'-monophosphate, mimics the effect of AMP on both allosteric activati on and phosphorylation of the kinase, while adenosine (5')tetraphospho (5')adenosine antagonizes both effects. These results suggest that bot h the allosteric effect of AMP, and the promotion of phosphorylation a nd activation by the kinase kinase, are due to binding of AMP to a sin gle site on the kinase.