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
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
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DOI：
10.1073/pnas.90.20.9261
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发表时间：
1993-10-15
影响因子：
11.1
通讯作者：
BROWN, MS
中科院分区：
文献类型：
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作者：
SATO, R;GOLDSTEIN, JL;BROWN, MS
据报道，一种amp激活的蛋白激酶可以磷酸化啮齿动物的3-羟基-3-甲基戊二酰辅酶A还原酶[HMG-CoA还原酶；[5] -甲羟戊酸：NAD+氧化还原酶（coa -酰化），EC 1.1.1.88]在Ser-871的催化活性降低[Clarke， P. R. and Hardie， D. G.（1990）环境化学学报，9,24 -24]。为了探索这一反应的生理作用，我们制备了一种编码仓鼠HMG-CoA还原酶突变体的cDNA，该突变体在871残基上用丙氨酸取代了丝氨酸。当在转染细胞中过表达时，野生型酶，而不是Ser-871到Ala突变体，被标记为[P-32]磷酸，证实Ser-871是磷酸化位点。当用磷酸酶抑制剂KF收获细胞时，野生型酶，而不是突变型酶，显示出活性降低，证实磷酸化是细胞内失活的一种机制。尽管缺乏磷酸化，但突变酶的转录后反馈调节是正常的，当细胞与甲羟酸盐、25-羟基胆固醇或低密度脂蛋白孵育时，活性降低。此外，当转染的细胞与甾醇孵育时，突变酶表现出正常的加速降解。表达野生型酶的细胞显示，当ATP与2-脱氧-d -葡萄糖孵育耗尽时，[C-14]丙酮酸与甾醇的结合减少。在表达Ser-871 to Ala突变酶的细胞中没有观察到这种减少。我们得出的结论是，amp激活的蛋白激酶在HMG-CoA还原酶的最终产物反馈调节中不起作用，而是在细胞ATP水平耗尽时发挥作用，从而降低胆固醇合成速率并保持细胞的能量储存。
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 [P-32]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 [C-14]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.