Allosteric Activation of Rat Liver Microsomal [Hydroxymethylglutaryl-CoA Reductase (NADPH)]Kinase by Nucleoside Phosphates

386

234

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine.2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of-63 kDa, is labelled using [L4C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [y3'P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity.3. In the absence of AMP the purified kinase has apparent K , values for ATP and acetyl-CoA carboxylase of 86 pM and 1.9 pM respectively. AMP increases the V,,, 3 -5-fold without a significant change in the K, for either protein or ATP substrates.4. The response to AMP depends on thc ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 yM. Studics with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations.5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.Cholesterol and fatty acids in mammals are derived either from the diet, or from de nuvu synthesis via branching pathways which diverge from a common pool of cytoplasmic acetyl-CoA. There arc scveral situations in which the two biosynthetic pathways are regulated in parallel. They exhibit a synchronous diurnal rhythm in rat liver [l, 21, a finding which may perhaps be explained by diurnal variation in the insuliniglucagon ratio, since insulin stimulates and glucagon inhibits both pathways in isolated rat hepatocytes and in vivo [3-51. The peak diurnal rates of both paihways are also reduced by 60-70% when rats are fed a diet high in polyunsaturated fatty acids [6], a treatment which also depresses plasma cholesierol and triglyccridc levels 171.The important sites of regulation within the pathways of fatty acid and cholesterol synthesis are believed to be those catalyzed by acetyl-CoA carboxylase and HMG-CoA reductase, respectively, and both enzymes are known to be regu- lated by reversible phosphorylaiion. Acetyl-CoA carboxylase is phosphorylated at multiplc sites by a variety of protein kinases, some of which inactivate the enzyme 181. These protein kinases include a ...

Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Purification and characterization of the AMP‐activated protein kinase

Carling¹,

Clarke²,

Zammit³

et al. 1989

386

234

“…Inactivation exhibits linear plots when the logarithm of activity remaining is plotted against time, with half times of 3.5 min for peak A and 17 min for peak B. This is consistent with a single site of reaction for both peaks, in contrast to results with mammalian AMP-activated protein kinase, where the plots are biphasic due to reaction at both the AMP (allosteric) and ATP (catalytic) sites [18,21,221. In the presence of Mg2+, ATP (2.5 mM) markedly reduces the rate of inactivation for both peaks.…”

Section: Inactivation Of Kinases a And B By Fs0phcoado And Protein Pmentioning

confidence: 59%

Biochemical characterization of two forms of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase kinase from cauliflower (Brassica oleracia)

Ball

Dale

Weekes

et al. 1994

We recently reported the existence of a protein kinase cascade in higher plants, of which the central component is a 3-hydroxy-3-methylglutaryl(HMG-)-CoA reductase kinase functionally related to mammalian AMP-activated protein kinase [MacKintosh, R. W., Davies, S. p., Clarke P. R., Weekes, J., Gillespie, S. G., Gibb Using a series of variants of the synthetic peptide substrate, the substrate specificities of the two forms are similar but not identical. Form B does not appear to be a proteolytic fragment of form A, and we therefore propose that it represents a closely related member of the same protein kinase sub-family.It is now clear that reversible protein phosphorylation is the major mechanism for the regulation of protein function in animal cells, its functions being particularly to mediate the response to extracellular signals such as hormones and cytokines, and to control events which occur discontinuously in the cell cycle such as DNA replication and mitosis. A third function which is just emerging may be to mediate the response of cells to environmental stresses such as heat shock or hypoxia [I]. At least in the case of cell-cycle control, the protein kinases involved appear to be highly conserved between plants and animals [3, 41. Plant cells also respond to a variety of external stimuli such as hormones, light and environmental stress. The systems which mediate these responses inside the plant cell remain largely undefined, although by analogy with animal systems it appears that protein kinases and phosphatases will play an important role. phosphoenolpyruvate carboxylase [8,9]. A number of higher plant protein kinases [lo-131 and protein phosphatases [14] have also been defined by cDNA cloning.Recently we obtained the first direct biochemical evidence for a protein kinase cascade in higher plants [15]. The protein kinase which is the central component of this cascade appears by functional criteria to be a homologue of the AMPactivated protein kinase, an activity which in mammalian cells is a component of the response to cellular stress [l] such as heat shock and hypoxia (unpublished results). Both the animal and plant kinases inactivate mammalian acetylCoA carboxylase and 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase, apparently by phosphorylation at the same sites. The system also appears to be highly conserved in that, like the mammalian kinase, the plant kinase is inactivated by mammalian protein phosphatases and reactivated by mammalian kinase kinase. The one difference found to date is that the plant protein kinase is not activated by AMP. Since the plant lunase inactivates HMG-CoA reductase from potato tubers, and the phosphorylation site defined on mammalian HMG-CoA reductase is conserved in a number of plant HMG-CoA reductases, we have provisionally named the plant kinase HMG-CoA reductase kinase [15].In this study we report the further purification and characterization of the HMG-CoA reductase kinase protein from cauliflower inflorescences. During the course of this work we identified a seco...

“…As its name suggests, the kinase is allosterically activated by low concentrations of 5'-AMP [lo-121, and studies with the reactive nucleotide analogue, 5'-fluorosulphonylbenzoyladenosine [13], suggest that the 63-kDa kinase subunit contains distinct allosteric (AMP) and catalytic (ATP) sites [12]. High concentrations of ATP can also bind at the allosteric site and hence antagonize activation by AMP [12, 131.…”

mentioning

confidence: 99%

Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP‐activated protein kinase

Moore

Weekes

Hardie

1991

208

163

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5 -10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation.2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (z 1 : 1 S). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1 : 15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity.3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase.4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaicacid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays.5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP.6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes. Abbreviations. Fmoc, fluorenylmethoxycarbonyl; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; TosLysCH2C1, tosyllysinechloromethane; TosPheCH2C1, tosylphenylalaninechloromethane.Enzymes. AMP-activated protein kinase (EC 2.7.1.109); acetylCoA carboxylase (EC 6.4.1.2); HMG-CoA reductase (EC 1.1.1.34); hormone-sensitive lipase (EC 3.1.1.3; 3.1.1.13); adenylate kinase (EC 2.7.4.3); cyclic-AMP-dependent protein kina...