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

Research output: Contribution to journalArticle

115 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)9261-9265
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number20
StatePublished - Oct 15 1993

Fingerprint

Hydroxymethylglutaryl CoA Reductases
AMP-Activated Protein Kinases
Sterols
Cricetinae
Serine
Adenosine Triphosphate
Phosphorylation
Enzymes
Mevalonic Acid
NAD-Dependent Hydroxymethylglutaryl-CoA Reductases
Oxidoreductases
Deoxyglucose
Coenzyme A
Pyruvic Acid
LDL Lipoproteins
Phosphoric Monoester Hydrolases
Alanine
Rodentia
Complementary DNA
Phosphates

Keywords

  • Cholesterol
  • End-product feedback regulation
  • Energy metabolism
  • Protein degradation

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

@article{62bb37158f6a4167b1b574d3d79370e6,
title = "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.",
keywords = "Cholesterol, End-product feedback regulation, Energy metabolism, Protein degradation",
author = "Ryuichiro Sato and Goldstein, {Joseph L.} and Brown, {Michael S.}",
year = "1993",
month = "10",
day = "15",
language = "English (US)",
volume = "90",
pages = "9261--9265",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "20",

}

TY - JOUR

T1 - 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

AU - Sato, Ryuichiro

AU - Goldstein, Joseph L.

AU - Brown, Michael S.

PY - 1993/10/15

Y1 - 1993/10/15

N2 - 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.

AB - 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.

KW - Cholesterol

KW - End-product feedback regulation

KW - Energy metabolism

KW - Protein degradation

UR - http://www.scopus.com/inward/record.url?scp=0027428833&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027428833&partnerID=8YFLogxK

M3 - Article

VL - 90

SP - 9261

EP - 9265

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 20

ER -