Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme.
Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [gamma-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the "linker" segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.
Parker
R A
RA
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
Miller
S J
SJ
Gibson
D M
DM
D000818:Animals; D001665:Binding Sites; D002154:Calpain; D002384:Catalysis; D002940:Circadian Rhythm; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D008297:Male; D008798:Mevalonic Acid; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D011500:Protein Synthesis Inhibitors; D006903:Hydroxymethylglutaryl CoA Reductases; D002154:Calpain; D008798:Mevalonic Acid