RESUMEN
The AMP-activated protein kinase (AMPK) is a metabolic-stress-sensing protein kinase that regulates metabolism in response to energy demand and supply by directly phosphorylating rate-limiting enzymes in metabolic pathways as well as controlling gene expression.
Asunto(s)
Complejos Multienzimáticos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Quinasas Activadas por AMP , Animales , Diabetes Mellitus/genética , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucólisis , Humanos , Lípidos , Modelos Biológicos , Modelos Moleculares , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación , Obesidad/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endotelio Vascular/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteína Quinasa C/metabolismo , Animales , Bovinos , Células Cultivadas , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Transducción de SeñalRESUMEN
The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Hígado/metabolismo , Factores de Ribosilacion-ADP , Animales , Brefeldino A , Ciclopentanos/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacología , Transporte Iónico , Hígado/citología , Hígado/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Ratas , Tapsigargina/farmacología , Vasopresinas/farmacologíaRESUMEN
The 5'-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either alpha1 or alpha2 catalytic subunits together with beta and gamma noncatalytic subunits in a trimeric complex. The alpha1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK alpha1 with a Km of 3.8 microM and a Vmax of 4.8 micromol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 microM and a Vmax of 8.1 micromol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha1 isoform has a Kcat approximately 250-fold higher than the AMPK alpha2 isoform isolated from rat liver. The AMPK alpha1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Phi(X, beta)XXS/TXXXPhi (Phi, hydrophobic; beta, basic). In good AMPK alpha1 peptide substrates, a hydrophobic residue at the P-5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.
Asunto(s)
Isoenzimas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación , Proteínas Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Isoenzimas/metabolismo , Cinética , Complejos Multienzimáticos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Ratas , Especificidad por Sustrato , Porcinos , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca2+ inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein alpha subunits, and carboxyl-terminal alpha-subunit synthetic peptides. An anti-Gi1-2 alpha antibody and a Gi2 alpha peptide (Gi2 alpha) Ile345-Phe355), but not a Gi3 alpha peptide (Gi3 alpha Ile344-Phe354), inhibited vasopressin- and thapsigargin-stimulated Ca2+ inflow, had no effect on vasopressin-stimulated release of Ca2+ from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca2+. An anti-Gq alpha antibody also inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. Immunofluorescence measurements showed that Gi2 alpha is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, Gq/11 alpha was found principally at the cell periphery. It is concluded that the trimeric G-protein, Gi2, is required for store-activated Ca2+ inflow in hepatocytes and acts between the release of Ca2+ from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca2+ channel protein(s) in the plasma membrane.
Asunto(s)
Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP/metabolismo , Hígado/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Anticuerpos/farmacología , Transporte Biológico/efectos de los fármacos , Agonistas de los Canales de Calcio/farmacología , Compartimento Celular , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Proteínas de Unión al GTP/inmunología , Hígado/citología , Datos de Secuencia Molecular , Toxina del Pertussis , Proteínas Proto-Oncogénicas/inmunología , Terpenos/farmacología , Tapsigargina , Vasopresinas/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. It may also regulate cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. We have purified the AMP-activated protein kinase 14,000-fold from porcine liver. The 63-kDa catalytic subunit co-purifies with two proteins of 40 and 38 kDa that may function as subunits. Partial amino acid sequence of the 63-kDa subunit revealed a striking homology with the catalytic domain of the yeast protein kinase transcriptional regulator Snf1 and its plant homologs. The Snf1 (72 kDa) and Snf4 (36 kDa) complex was also purified and found to phosphorylate the AMP-activated protein kinase peptide substrate, HMRSAMSGLHLVKRR-amide, but was not activated by AMP. Both Snf1/4 and the AMP-activated protein kinase phosphorylate and inactivate yeast acetyl-CoA carboxylase in vitro. These results indicate that during evolution the catalytic domain sequences of the Snf1 protein kinase subfamily have been exploited in the control of mammalian lipid metabolism and raise the possibilities that the AMP-activated protein kinase may have other substrates involved in regulating gene expression pathways, as well as Snf1 homologs participating in the control of lipid metabolism in many eukaryotic organisms.
Asunto(s)
Hígado/enzimología , Complejos Multienzimáticos/química , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Saccharomyces cerevisiae/enzimología , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Ratas , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The reported cDNA structure of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc. Natl. Acad. Sci USA, 87:2284-2288, 1990). The calculated M(r) is 107,534 whereas the estimate by SDS-PAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors in the cDNA sequence for non-muscle MLCK and that the NH2-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH2-terminally blocked. A CNBr peptide derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating that Met-1 is present. Using a limited thermolysin digest we isolated an NH2-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH2-terminal Met being blocked by acetylation. The results demonstrate that the NH2-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating Met is not removed, but modified by alpha-NH2 acetylation of the translation product.