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Proc Natl Acad Sci U S A. Oct 15, 1993; 90(20): 9261–9265.
PMCID: PMC47547

Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion.

Abstract

An AMP-activated protein kinase has been reported to phosphorylate rodent 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA reductase; (S)-mevalonate:-NAD+ oxidoreductase (CoA-acylating), EC 1.1.1.88] at Ser-871, thereby lowering its catalytic activity [Clarke, P. R. & Hardie, D. G. (1990) EMBO J. 9, 2439-2446]. To explore the physiologic role of this reaction, we prepared a cDNA encoding a mutant form of hamster HMG-CoA reductase with alanine substituted for serine at residue 871. When overexpressed in transfected cells, the wild-type enzyme, but not the Ser-871 to Ala mutant, was labeled with [32P]phosphate, confirming Ser-871 as the site of phosphorylation. The wild-type enzyme, but not the mutant enzyme, showed reduced activity when the cells were harvested with the phosphatase inhibitor KF, confirming phosphorylation as a mechanism for inactivation within the cell. Despite the lack of phosphorylation, the posttranscriptional feedback regulation of the mutant enzyme was normal, as indicated by reduced activity when cells were incubated with mevalonate, 25-hydroxycholesterol, or low density lipoprotein. Moreover, the mutant enzyme showed a normal acceleration of degradation when the transfected cells were incubated with sterols. Cells expressing the wild-type enzyme showed a decreased incorporation of [14C]pyruvate into sterols when ATP was depleted by incubation with 2-deoxy-D-glucose. No such reduction was seen in cells expressing the Ser-871 to Ala mutant enzyme. We conclude that the AMP-activated protein kinase does not play a role in end-product feedback regulation of HMG-CoA reductase, but rather it comes into play when cellular ATP levels are depleted, thereby lowering the rate of cholesterol synthesis and preserving the energy stores of the cell.

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  • Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990 Feb 1;343(6257):425–430. [PubMed]
  • Clarke PR, Hardie DG. Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver. EMBO J. 1990 Aug;9(8):2439–2446. [PMC free article] [PubMed]
  • Hardie DG. Regulation of fatty acid and cholesterol metabolism by the AMP-activated protein kinase. Biochim Biophys Acta. 1992 Feb 12;1123(3):231–238. [PubMed]
  • Hardie DG, MacKintosh RW. AMP-activated protein kinase--an archetypal protein kinase cascade? Bioessays. 1992 Oct;14(10):699–704. [PubMed]
  • Parker RA, Miller SJ, Gibson DM. Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme. J Biol Chem. 1989 Mar 25;264(9):4877–4887. [PubMed]
  • Marrero PF, Haro D, Hegardt FG. Phosphorylation of HMG-CoA reductase induced by mevalonate accelerates its rate of degradation in isolated rat hepatocytes. FEBS Lett. 1986 Mar 3;197(1-2):183–186. [PubMed]
  • Zammit VA, Caldwell AM. Direct demonstration that increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase does not increase its rate of degradation in isolated rat hepatocytes. Biochem J. 1992 Jun 15;284(Pt 3):901–904. [PMC free article] [PubMed]
  • Gillespie JG, Hardie DG. Phosphorylation and inactivation of HMG-CoA reductase at the AMP-activated protein kinase site in response to fructose treatment of isolated rat hepatocytes. FEBS Lett. 1992 Jul 13;306(1):59–62. [PubMed]
  • Ingebritsen TS, Geelen MJ, Parker RA, Evenson KJ, Gibson DM. Modulation of hydroxymethylglutaryl-CoA reductase activity, reductase kinase activity, and cholesterol synthesis in rat hepatocytes in response to insulin and glucagon. J Biol Chem. 1979 Oct 25;254(20):9986–9989. [PubMed]
  • Easom RA, Zammit VA. Diurnal changes in the fraction of 3-hydroxy-3-methylglutaryl-CoA reductase in the active form in rat liver microsomal fractions. Biochem J. 1984 Jun 15;220(3):739–745. [PMC free article] [PubMed]
  • Beg ZH, Brewer HB., Jr Regulation of liver 3-hydroxy-3-methylglutaryl-CoA reductase. Curr Top Cell Regul. 1981;20:139–184. [PubMed]
  • Brown MS, Goldstein JL, Dietschy JM. Active and inactive forms of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver of the rat. Comparison with the rate of cholesterol synthesis in different physiological states. J Biol Chem. 1979 Jun 25;254(12):5144–5149. [PubMed]
  • Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. [PubMed]
  • Luskey KL, Stevens B. Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation. J Biol Chem. 1985 Aug 25;260(18):10271–10277. [PubMed]
  • Liscum L, Finer-Moore J, Stroud RM, Luskey KL, Brown MS, Goldstein JL. Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum. J Biol Chem. 1985 Jan 10;260(1):522–530. [PubMed]
  • Nakanishi M, Goldstein JL, Brown MS. Multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme. J Biol Chem. 1988 Jun 25;263(18):8929–8937. [PubMed]
  • Chin DJ, Gil G, Russell DW, Liscum L, Luskey KL, Basu SK, Okayama H, Berg P, Goldstein JL, Brown MS. Nucleotide sequence of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, a glycoprotein of endoplasmic reticulum. Nature. 1984 Apr 12;308(5960):613–617. [PubMed]
  • Andersson S, Davis DL, Dahlbäck H, Jörnvall H, Russell DW. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed]
  • Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. [PubMed]
  • Mosley ST, Brown MS, Anderson RG, Goldstein JL. Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3 -methylglutaryl coenzyme A reductase. J Biol Chem. 1983 Nov 25;258(22):13875–13881. [PubMed]
  • Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed]
  • Brown MS, Faust JR, Goldstein JL, Kaneko I, Endo A. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase. J Biol Chem. 1978 Feb 25;253(4):1121–1128. [PubMed]
  • Nordstrom JL, Rodwell VW, Mitschelen JJ. Interconversion of active and inactive forms of rat liver hydroxymethylglutaryl-CoA reductase. J Biol Chem. 1977 Dec 25;252(24):8924–8934. [PubMed]
  • Brown MS, Dana SE, Dietschy JM, Siperstein MD. 3-Hydroxy-3-methylglutaryl coenzyme A reductase. Solubilization and purification of a cold-sensitive microsomal enzyme. J Biol Chem. 1973 Jul 10;248(13):4731–4738. [PubMed]

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