Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by reversible phosphorylation: modulation of enzymatic activity by low density lipoprotein, sterols, and mevalonolactone.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase exists in interconvertible active and inactive forms in cultured fibroblasts from normal and familial hypercholesterolemic subjects. The inactive form can be activated by endogenous or added phosphoprotein phosphatase. Active or partially active HMG-CoA reductase in cell extracts was inactivated by a ATP-Mg-dependent reductase kinase. Incubation of phosphorylated (inactive) HMG-CoA reductase with purified phosphoprotein phosphatase was associated with dephosphorylation (reactivation) and complete restoration of HMG-CoA reductase activity. Low density lipoprotein, 25-hydroxycholesterol, 7-ketocholesterol, and mevalonolactone suppressed HMG-CoA reductase activity by a short-term mechanism involving reversible phosphorylation. 25-Hydroxycholesterol, which enters cells without the requirement of low density lipoprotein-receptor binding, inhibited the HMG-CoA reductase activity in familial hypercholesterolemic cells by reversible phosphorylation. Measurement of the short-term effects of inhibitors on the rate of cholesterol synthesis from radiolabeled acetate revealed that HMG-CoA reductase phosphorylation was responsible for rapid suppression of sterol synthesis. Reductase kinase activity of cultured fibroblasts was also affected by reversible phosphorylation. The active (phosphorylated) reductase kinase can be inactivated by dephosphorylation with phosphatase. Inactive reductase kinase can be reactivated by phosphorylation with ATP-Mg and a second protein kinase from rat liver, designated reductase kinase kinase. Reductase kinase kinase activity has been shown to be present in the extracts of cultured fibroblasts. The combined results represent the initial demonstration of a short-term regulation of HMG-CoA reductase activity and cholesterol synthesis in normal and receptor-negative cultured fibroblasts involving reversible phosphorylation of both HMG-CoA reductase and reductase kinase.
D055372:AMP-Activated Protein Kinases; D000085:Acetates / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D006938:Hyperlipoproteinemia Type II / Q000201:enzymology; D007653:Ketocholesterols / Q000494:pharmacology; D050356:Lipid Metabolism; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008798:Mevalonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D013261:Sterols / Q000494:pharmacology*; D014867:Water / Q000378:metabolism
D000085:Acetates; D006888:Hydroxycholesterols; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007653:Ketocholesterols; D008077:Lipoproteins, LDL; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D013261:Sterols; D014867:Water; C015367:mevalonolactone; C007997:25-hydroxycholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D010744:Phosphoric Monoester Hydrolases; C003001:7-ketocholesterol; D005459:Fluorides; D008798:Mevalonic Acid