An AMP-activated protein kinase has been reported to phosphorylate rodent 3-hydroxy-3-methylglutarylcoenzyme A reductase [HMG-CoA reductase; (S)-mevalonate:-NAD+ oxidoreductase (CoA-acylating), EC 1.1. In addition to this classic end-product feedback regulation, which affects the level of enzyme protein, the activity of HMG-CoA reductase can be altered reversibly by a cycle of phosphorylation and dephosphorylation (2). Phosphorylation occurs on a serine (residue 871 in the hamster sequence) thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.is near the C terminus of the protein (3). The reaction is catalyzed by a unique kinase that uses ATP as a phosphate donor and requires AMP as an activator (4,5). Phosphorylation decreases the catalytic activity of the enzyme by ==80%. Although some groups have speculated that phosphorylation accelerates the degradation of HMG-CoA reductase protein (6, 7), other groups have found no evidence for such an effect (8).The AMP-activated kinase also phosphorylates and inactivates acetyl-CoA carboxylase, thereby potentially inhibiting the synthesis of fatty acids as well as cholesterol (4, 5). Hardie (4, 5) has suggested that the AMP-activated kinase functions to conserve energy by inhibiting anabolic pathways when cellular ATP levels are depleted and AMP levels increase. This conclusion is supported by the finding of increased phosphorylation of HMG-CoA reductase in hepatocytes that have been incubated with fructose, which raises AMP levels (9). This hypothesis is also consistent with the presence of a similar AMP-activated kinase in organisms as primitive as insects and plants, which suggests a fundamental cell-autonomous role such as energy conservation (4, 5).Other investigators (2, 10-12) have provided evidence to support the idea that phosphorylation of HMG-CoA reductase in liver may mediate responses to insulin or may counteract the pattern of diurnal rhythm of HMG-CoA reductase in this organ. To date, however, there are no convincing data to correlate a change in the phosphorylation state of HMGCoA reductase with an overall change in the rate of cholesterol biosynthesis in liver or any other tissue (13).To explore the regulatory role of phosphorylation, in the current experiments we have prepared a cDNA encoding a mutant form of hamster HMG-CoA reductase with Ala substituted for Ser-871 and transfected it into several cell types. As expected, this enzyme is no longer phosphorylated significantly in cultured cells. Nevertheless