Characterization and regulation of reductase kinase, a protein kinase that modulates the enzymic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Abstract

was associated with significant loss of radioactivity and enzymic activity in the protein band ascribed to reductase kinase. These results provide evidence that the activity of reductase kinase, like HMG-CoA reductase, is modulated by a reversible phosphorylation-dephosphorylation reaction sequence. Recently, we (1) demonstrated that the enzymic activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA reductase; mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.341 was modulated by phosphorylation-dephosphorylation. Phosphorylation of HMG-CoA reductase was catalyzed by the microsomal enzyme reductase kinase and ATP plus Mg2+ (1). Ingebritsen et al. (2) suggested that the enzyme that inactivates HMG-CoA reductase also exists in active and inactive forms.Reductase kinase has now been further characterized and evidence of a reductase kinase kinase has been obtained. Evidence is provided for the regulation of the activity of reductase kinase by an enzyme-mediated phosphorylation-dephosphorylation reaction sequence. Thus, two protein kinases are involved in the regulation of the catalytic activity of HMG-CoA reductase. with a gradient of 0.3 M NaCl in buffer A (150 ml) and buffer A (150 ml). Reductase kinase activity eluted between 1.4 and 3.8 mmho. These fractions (49 mg of protein) were pooled, dialyzed against buffer A, and chromatographed on a second DE-52 column [1.2 X 9 cm, gradient of buffer A containing 0.27 M NaCl (120 ml) and buffer A (120 ml)]. The partially purified reductase kinase used in the present studies eluted between 1.7 and 3.8 mmho; it was dialyzed against 10 mM KH2PO4, pH 7.4/0.1 mM EDTA/5 mM dithiothreitol and stored frozen (0.2 mg/ml). Selected preparations of partially purified reductase kinase were purified to homogeneity by utilizing phosphocellulose chromatography, gel filtration (Sepharose 6B), adsorption chromatography on alumina C&y, and thin-layer isoelectric focusing (pH 4-6.5; pI =5.6 + 0.2). MATERIALS ANDIsolation of Inactivated Reductase Kinase. Rat liver microsomes (15 mg/ml in 50 mM imidazole, pH 7.4/250 mM NaCl/1 mM EDTA/5 mM dithiothreitol) containing endogeneous phosphoprotein phosphatase were incubated at 370 for 2 hr and then extracted three times to solubilize inactive reductase kinase. Alternatively, solubilized (6.2 mg/ml) or purified reductase kinase (0.1 mg/ml) was incubated for 30 min at 370C with partially purified phosphoprotein phosphatase (4.5 mg). The incubation was terminated by the addition of 50 mM NaF.Isolation of Reductase Kinase Kinase. Rat liver cytosol (100,000 X g supernatant) was fractionated with ammonium sulfate; the protein (5.2 g) precipitated at 60% saturation. It was dialyzed against buffer A, applied to a column of phosphocellulose (2.5 X 15 cm), and eluted with a gradient of 1 M NaCl in buffer A plus 50 ,uM phenylmethylsulfonyl fluoride (400 ml) and buffer A with 50 ,AM phenylmethylsulfonyl fluoride (400 ml). Fractions were assayed for protein kinase activity and the Abbreviations: HMG-CoA reductase, 3-...

THE AMP-ACTIVATED/SNF1 PROTEIN KINASE SUBFAMILY: Metabolic Sensors of the Eukaryotic Cell?

“…could be resolved from AMPK on DEAE-Sepharose (66). Fractions containing AMPK and the putative upstream kinase were also separated by Beg et al (21). These studies indicated that the system was a protein kinase cascade in which an upstream kinase (now termed AMPKK) phosphorylated and activated the downstream kinase (AMPK).…”

Somatic cell genetic analysis of regulation of expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

“…The procedure used was essentially the bromodeoxyuridine-visible light method of Kao and Puck (8). CHO-KI cells grown in F12 medium/8% FC were mutagenized by 0.4 ,l ethyl methane sulfonate/ml of medium in 100-mm Lux petri dishes containing 2.5 X 106 cells for 16 hr and then incubated and passed when necessary for a period of [5][6][7] days to permit expression of a mutant phenotype. The cells were pooled, and 25,000 cells were inoculated into each of forty 60-mm petri dishes in F12 medium/8% FC and incubated for 4 hr to permit their attachment.…”

“…These observations, combined with others on the regulatory effects of low-density lipoprotein, have led to the hypothesis (2) that the regulation of HMG-CoA reductase by sterols involves transcriptional or translational control of enzyme biosynthesis. Other studies (5) have provided evidence for a phosphorylation-dependent inactivation process for HMG-CoA reductase and have suggested that enzyme inactivation may also play a role in regulation of HMG-CoA reductase (6).…”

Nitrite Activates 5′AMP-Activated Protein Kinase-Endothelial Nitric Oxide Synthase Pathway in Human Glomerular Endothelial Cells

“…AMPK is allosterically activated in response to an elevation in AMP-to-ATP ratio, and phosphorylation of threonine 172 residue of the α subunit by upstream kinases such as Liver Kinase B1 (LKB1) or calcium/calmodulin-dependent kinase kinase β increases the activity. [20][21][22][23][24][25][26][27] Recent studies have established a concept of AMPK as a key enzyme regulating metabolism. A variety of metabolic stresses and hormonal stimulations such as physical exercise, leptin and adiponectin have been reported to activate AMPK, which leads to lots of metabolic responses metabolome-widely including glucose uptake, fatty acid oxidation, and insulin sensitization etc.…”