RESUMO
Callyspongiolide is a marine-derived macrolide that kills cells in a caspase-independent manner. NCI COMPARE analysis of human tumor cell line toxicity data for synthetic callyspongiolide indicated that its pattern of cytotoxicity correlated with that seen for concanamycin A, an inhibitor of the vacuolar-type H+-ATPase (V-ATPase). Using yeast as a model system, we report that treatment with synthetic callyspongiolide phenocopied a loss of V-ATPase activity including (1) inability to grow on a nonfermentable carbon source, (2) rescue of cell growth via supplementation with Fe2+, (3) pH-sensitive growth, and (4) a vacuolar acidification defect visualized using the fluorescent dye quinacrine. Crucially, in an in vitro assay, callyspongiolide was found to dose-dependently inhibit yeast V-ATPase (IC50 = 10 nM). Together, these data identify callyspongiolide as a new and highly potent V-ATPase inhibitor. Notably, callyspongiolide is the first V-ATPase inhibitor known to be expelled by Pdr5p.
Assuntos
Inibidores Enzimáticos/farmacologia , Macrolídeos/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Macrolídeos/química , Estrutura Molecular , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.
Assuntos
Adenoma/metabolismo , Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ácido Pirúvico/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Drosophila , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl-tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC.
Assuntos
Proteínas de Transporte/genética , Biossíntese de Proteínas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Controle de Qualidade , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
UNLABELLED: The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca(2+)/calmodulin-dependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1-AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 reexpression in these cells causes cell-cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca(2+) ionophore A23187 caused a G1 arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, Ca(2+)/calmodulin-independent CAMKK2 mutant also caused G1 arrest similar to that caused by expression of LKB1, while expression of a dominant-negative AMPK mutant, or a double knockout of both AMPK-α subunits, also prevented the cell-cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell-cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell-cycle arrest can be restored by reexpressing LKB1 or a constitutively active CAMKK2, or by pharmacologic agents that increase intracellular Ca(2+) and thus activate endogenous CAMKK2. IMPLICATIONS: Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented. Mol Cancer Res; 14(8); 683-95. ©2016 AACR.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Humanos , FosforilaçãoRESUMO
In pancreatic ß-cells, glucose induces the binding of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) to the insulin gene promoter to activate insulin gene transcription. At low glucose levels, glycogen synthase kinase 3ß (GSK3ß) is known to phosphorylate PDX-1 on C-terminal serine residues, which triggers PDX-1 proteasomal degradation. We previously showed that the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) regulates insulin gene transcription via PDX-1. However, the mechanisms underlying this regulation are unknown. In this study, we aimed to identify the role of PASK in the regulation of PDX-1 phosphorylation, protein expression, and stability in insulin-secreting cells and isolated rodent islets of Langerhans. We observed that glucose induces a decrease in overall PDX-1 serine phosphorylation and that overexpression of WT PASK mimics this effect. In vitro, PASK directly phosphorylates GSK3ß on its inactivating phosphorylation site Ser(9). Overexpression of a kinase-dead (KD), dominant negative version of PASK blocks glucose-induced Ser(9) phosphorylation of GSK3ß. Accordingly, GSK3ß Ser(9) phosphorylation is reduced in islets from pask-null mice. Overexpression of WT PASK or KD GSK3ß protects PDX-1 from degradation and results in increased PDX-1 protein abundance. Conversely, overexpression of KD PASK blocks glucose-induction of PDX-1 protein. We conclude that PASK phosphorylates and inactivates GSK3ß, thereby preventing PDX-1 serine phosphorylation and alleviating GSK3ß-mediated PDX-1 protein degradation in pancreatic ß-cells.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/metabolismo , Animais , Glucose/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Células Hep G2 , Proteínas de Homeodomínio/genética , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Mutação , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Edulcorantes/farmacologia , Transativadores/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
PAS kinase (PASK) is a glucose-regulated protein kinase involved in the control of pancreatic islet hormone release and insulin sensitivity. We aimed here to identify mutations in the PASK gene that may be associated with young-onset diabetes in humans. We screened 18 diabetic probands with unelucidated maturity-onset diabetes of the young (MODY). We identified two rare nonsynonymous mutations in the PASK gene (p.L1051V and p.G1117E), each of which was found in a single MODY family. Wild type or mutant PASKs were expressed in HEK 293 cells. Kinase activity of the affinity-purified proteins was assayed as autophosphorylation at amino acid Thr307 or against an Ugp1p-derived peptide. Whereas the PASK p.G1117E mutant displayed a â¼25% increase with respect to wild type PASK in the extent of autophosphorylation, and a â¼2-fold increase in kinase activity toward exogenous substrates, the activity of the p.L1051V mutant was unchanged. Amino acid Gly1117 is located in an α helical region opposing the active site of PASK and may elicit either: (a) a conformational change that increases catalytic efficiency or (b) a diminished inhibitory interaction with the PAS domain. Mouse islets were therefore infected with adenoviruses expressing wild type or mutant PASK and the regulation of insulin secretion was examined. PASK p.G1117E-infected islets displayed a 4-fold decrease in glucose-stimulated (16.7 versus 3 mM) insulin secretion, chiefly reflecting a 4.5-fold increase in insulin release at low glucose. In summary, we have characterized a rare mutation (p.G1117E) in the PASK gene from a young-onset diabetes family, which modulates glucose-stimulated insulin secretion.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Adulto , Animais , Linhagem Celular , Diabetes Mellitus/metabolismo , Genômica , Glucagon/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Secreção de Insulina , Masculino , Proteínas de Membrana/metabolismo , Modelos Genéticos , Mutagênese , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismoRESUMO
A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Dinitrofenóis/farmacologia , Metabolismo Energético , Ativação Enzimática , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Naftalimidas/farmacologia , Oligomicinas/farmacologia , Fosforilação , Subunidades Proteicas/metabolismoRESUMO
Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr172 is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKKbeta (Ca2+/calmodulin-dependent protein kinase kinase-beta). The sequence around Thr172 is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKbeta, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKalpha. Despite recent reports, we could not find any evidence that the alpha and/or beta subunits of AMPK formed a stable complex with CaMKKbeta. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect on basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP on phosphorylation of Thr172 being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/fisiologia , AMP Cíclico/farmacologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Células HeLa , Humanos , Imunoprecipitação , Ionóforos/farmacologia , Dados de Sequência Molecular , Fenformin/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
The tumor suppressor protein kinase LKB1 exerts its effects by phosphorylating and activating AMP-activated protein kinase (AMPK) and members of the AMPK-related kinase family, such as the brain-specific kinases BRSK1/BRSK2 (SAD-B/SAD-A). LKB1 contains a conserved serine residue near the C terminus (Ser-431 in mouse LKB1) that is phosphorylated by cyclic AMP-dependent protein kinase and p90-RSK. Although some studies suggest that LKB1 is constitutively active and is not rate-limiting for activation of AMPK, others have suggested that phosphorylation of Ser-431 is necessary to allow LKB1 to phosphorylate and activate AMPK and other downstream kinases. Prompted by our discovery of an LKB1 splice variant (LKB1S) that lacks Ser-431, we have reinvestigated this question. In HeLa cells (which lack endogenous LKB1), co-expression with STRADalpha and MO25alpha of wild type LKB1, the S431A or S431E mutants of LKB1, or LKB1(S) gave equal levels of activation of endogenous AMPK. Similarly, recombinant STRADalpha.MO25alpha complexes containing these LKB1 variants were equally effective at phosphorylating and activating AMPK, BRSK1, and BRSK2 in cell-free assays. Finally, all four LKB1 variants and a truncated LKB1 lacking the C-terminal region altogether were equally effective at causing cell cycle arrest when co-expressed with STRADalpha and MO25alpha in the G361 melanoma cell line. Our results do not support the idea that phosphorylation of Ser-431 increases the ability of LKB1 to phosphorylate downstream targets.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ciclo Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Processamento Alternativo/fisiologia , Substituição de Aminoácidos , Sistema Livre de Células , Ativação Enzimática/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação de Sentido Incorreto , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
LKB1 was discovered as a tumour suppressor mutated in Peutz-Jeghers syndrome, and is a gene involved in cell polarity as well as an upstream protein kinase for members of the AMP-activated protein kinase family. We report that mammals express two splice variants caused by alternate usage of 3'-exons. LKB1(L) is the previously described form, while LKB1(S) is a novel form in which the last 63 residues are replaced by a unique 39-residue sequence lacking known phosphorylation (Ser(431)) and farnesylation (Cys(433)) sites. Both isoforms are widely expressed in rodent and human tissues, although LKB1(S) is particularly abundant in haploid spermatids in the testis. Male mice in which expression of Lkb1(S) is knocked out are sterile, with the number of mature spermatozoa in the epididymis being dramatically reduced, and those spermatozoa that are produced have heads with an abnormal morphology and are non-motile. These results identify a previously undetected variant of LKB1, and suggest that it has a crucial role in spermiogenesis and male fertility.