RESUMO
Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor ß1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of ß1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a ß1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT ß1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of ß1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, ß1Pix knockdown, or overexpression of WT ß1Pix or the ß1Pix-Δ602-611 mutant. Moreover, we found that ß1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of ß1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Células Epiteliais/citologia , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Camundongos , FosforilaçãoRESUMO
The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by beta-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKalpha1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKalpha1 by LKB1 in response to lipolytic signals. Activation of AMPKalpha1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKalpha1 in vitro. Functional analysis of an AMPKalpha1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA-activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Lipólise , Proteínas Quinases Ativadas por AMP/genética , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Ácidos Graxos/metabolismo , Glicerol/metabolismo , Isoproterenol/farmacologia , Camundongos , Fosforilação , Mutação Puntual , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain alphaG-helix. Mutation of the corresponding AMPK alpha-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the alphaG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (alpha1(-/-), alpha2(-/-)) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the alphaG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Calpha was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic alphaG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic alphaG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Multimerização Proteica , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , TreoninaRESUMO
AMPK is a metabolic stress-sensing kinase with important functions for red blood cell (RBC) survival. By using a proteomic approach, we identified putative AMPK targets in hemoglobin-depleted lysates of RBC, including metabolic enzymes, cytoskeletal proteins and enzymes involved in the oxidative stress response. These data tie in with the phenotypic observations of AMPKalpha1-deficient RBC and provide reference for future studies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Eritrócitos/enzimologia , Proteínas Quinases Ativadas por AMP/química , Animais , Cromatografia de Afinidade , Ativação Enzimática , Hemoglobinas/química , Hemoglobinas/isolamento & purificação , Humanos , Camundongos , Níquel/química , Especificidade por SubstratoRESUMO
Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.
Assuntos
Proteínas Quinases Ativadas por AMP/análise , Cromatografia Líquida/métodos , Fosfopeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/isolamento & purificação , Autorradiografia , Escherichia coli/genética , Radioisótopos de Fósforo , Fosforilação , Mutação PuntualRESUMO
AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets have been identified. Recent work suggests that AMPK has specialized functions in the brain, such as involvement in appetite control. Nevertheless, brain-specific substrates of AMPK are unknown. Here, we performed a proteomic in vitro screen to identify new putative AMPK targets in brain. Prefractionation of murine brain lysates by liquid chromatography, utilizing four different, serially connected columns with different chemistries was found to be superior to a single column method. A pilot screen involving incubation of small volumes of individual fractions with radiolabeled ATP in the presence or absence of active AMPK, followed by one-dimensional SDS-PAGE and autoradiography, revealed the presence of potential AMPK substrates in a number of different fractions. On the basis of these results, several kinase assays were repeated with selected fractions on a preparative scale. Following separation of the radiolabeled proteins by two-dimensional electrophoresis and comparison of samples with or without added AMPK by differential autoradiography, 53 AMPK-specific phospho-spots were detected and excised. Thereof, 26 unique proteins were identified by mass spectrometry and were considered as new potential downstream targets of AMPK. Kinase assays with 14 highly purified candidate substrate proteins confirmed that at least 12 were direct targets of AMPK in vitro. Although the physiological consequences of these phosphorylation events remain to be established, hypotheses concerning the most intriguing potential targets of AMPK that have been identified by this search are discussed herein. Our data suggests that signaling by AMPK in brain is likely to be involved in the regulation of pathways that have not yet been linked to this kinase.