Regulation of liver hydroxymethylglutaryl-CoA reductase by a bicyclic phosphorylation system.

Protein phosphatase C was purified 1500-fold from rat liver by a six-step procedure including a fractionation step with 80% ethanol at room temperature and two successive chromatographic separations on DEAESephadex. This preparation restored hydrox~ethyl glutaryl-CoA (HMG-CoA) reductase activity in liver microsomes pretreated with MgATP and also inactivated HMG-CoA reductase kinase. The relative rates of activation of HMG-CoA reductase, inactivation of reductase kinase, and dephosphorylation of phosphorylase a were constant throughout each step in the purification. Each of the reactions catalyzed by the purified phosphatase were inhibited in parallel by sodium fluoride (Kj = 3 to 4 mM), Inactivated reductase kinase was fully reactivated by MgATP and an enzyme, termed HMG-CoA reductase kinase kinase, which was separated from HMG-CoA reductase kinase by chromatography on DEAE-cellulose. These new studies support the thesis (Ingebritsen, T. S., Lee, H.-S., Parker, R. A., and Gibson, D. M. (1978) Biochem. Biophys. Res. Commun. 81, 1268-1277) that liver HMG-CoA reductase is controlled by a bicyclic system in which both HMG-CoA reductase and HMGCoA reductase kinase are regulated by reversible phosphorylation. More than 80% of the HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase activities in rat liver extracts were found in the cytosol in contrast to HMGCoA reductase which is firmly bound to liver microsomes. The activities of HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase were not influenced by CAMP or the specific heat-stable inhibitor of CAMP-dependent protein kinase. Similarly, high concentrations of CAMP-dependent protein kinase (in the presence of CAMP) were unable to catalyze either the inactivation of HMG-CoA reductase or the reactivation of HMG-CoA reductase kinase. These results are discussed in the light of the recent observation (Ingebritsen, T. S., Geelen, M. J. H., Parker, R. A., Evenson, K. J., and Gibson, D. M. (1979) J. Biol. Chern. 254, 99869989) that HMG-CoA reductase was inactivated, and HMG-CoA reductase kinase activity was activated in isolated hepatocytes in response to glucagon and CAMP.