RESUMO
The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.
Assuntos
Proteína Quinase C , Piruvato Desidrogenase Quinase de Transferência de Acetil , Humanos , Regulação Alostérica , Proteína Quinase C/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Domínio Catalítico , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/química , Ligação ProteicaRESUMO
PURPOSE: Phosphoinositide-dependent kinase 1 (PDK1) is highly expressed in many solid tumors. And several studies have demonstrated that PDK1 has been an emerging and promising target for anti-cancer therapies. However, the role of PDK1 has not been studied so far in malignant pheochromocytoma (PCC). METHODS: In this study, immunohistochemical staining was performed to investigate the protein level of PDK1 in 63 PCC tissue samples, of which 49 were benign and 14 were malignant. In addition, we evaluated the effect of inhibition of PDK1 with siRNA on cell growth, apoptosis and invasive capacity in PC12 cells and identified the underlying mechanisms. RESULTS: We found that PDK1 was overexpressed in malignant PCC tissues, and knockdown of PDK1 with siRNA significantly inhibited cell proliferation, increased apoptosis induction, and attenuated cell migration and invasive capacity in PC12 cells. We also showed that knockdown of PDK1 significantly reduced the phosphorylation of Akt at threonine 308 (p-Akt T308) but did not alter the serine phosphorylation of Akt on the S473 site (p-Akt S473). Furthermore, we found that the p-Akt expression was noticeably decreased after knockdown of PDK1, but the t-Akt expression did not show a significant decrease. CONCLUSION: We have demonstrated for the first time that PDK1 is overexpressed in human malignant PCC and plays an important role in the malignant biological behaviors of PC12 cell. Specifically, we have revealed that knockdown of PDK1 could attenuate activation of the Akt signaling. These data suggest that PDK1 could be a new promising potential therapeutic target in human cancer treatment for malignant PCC.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/análise , Neoplasias das Glândulas Suprarrenais/enzimologia , Proteínas de Neoplasias/análise , Feocromocitoma/enzimologia , Glândulas Suprarrenais/química , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno , TransfecçãoRESUMO
Purpose:To investigate the role of PI3k/Akt signal pathway in the protective effects of propofol on intestinal and lung injury induced by intestinal ischemia/reperfusion(I/R).Methods:Male Sprague-Dawley rats were subjected to 45 min of ischemia by occluding the superior mesenteric artery and to 2h of reperfusion to establish the model of I/R. Twenty four rats were randomly divided into four groups: Sham, intestinal I/R (II/R), propofol (P), wortmannin (W). In groups P, W, propofol was injected intravenously and continuously at the onset of reperfusion via infusion pump. PI3K inhibitor (wortmannin) was administered intravenously in group W 25 min before ischemia. Intestinal tissues and lung tissues were obtained for determination of histologic injury, wet/dry weight ratio, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and myeloperoxidase (MPO) activities. Meanwhile, the expressions of caspase-3 and phosphorylated Akt (p-Akt) in intestines and lungs were detected by western blot.Results:Propofol treatment alleviated intestinal and lung morphological changes which were observed in II/R group，Moreover, wet/dry weight ratio, the MDA level, MPO activity and expression of caspase-3 were significantly decreased whereas the SOD activity and p-Akt expression were significantly increased. Notably, the protections were significantly reversed by pretreatment of wortmannin.Conclusion:PI3K/Akt pathway activation play a critical role in the protective effects of propofol on intestinal and lung injury induced by ischemia/reperfusion.(AU)
Assuntos
Animais , Ratos , Propofol/análise , Propofol/uso terapêutico , Isquemia , Traumatismo por Reperfusão , Lesão Pulmonar , Intestinos/lesões , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Apoptose , Estresse OxidativoRESUMO
Estradiol (E2) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERß. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E2. In conclusion, E2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy.
Assuntos
Cardiomegalia/prevenção & controle , Endotelina-1/toxicidade , Estradiol/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Animais Recém-Nascidos , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiotoxicidade , Células Cultivadas , Citoproteção , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Visceral leishmaniasis (VL) or Kala-Azar (KA) is one of the most deadly forms of disease among all neglected tropical diseases. There are no satisfactory drugs or vaccine candidates available for this dreaded disease. Our previous studies showed promising therapeutic and prophylactic efficacy of the live, radio-attenuated parasites through intramuscular (I.M.) and intraperitoneal (I.P.) route in BALB/c mice model. METHODS: The T-cell proliferation level, the mRNA expression level of inducible nitric oxide synthase (iNOS) and tumor growth factor-beta (TGF-ß) genes and finally the phosphorylation levels of phosphoinositide dependent kinase 1 (PDK1), phosphoinositide 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) molecules were checked in BALB/c mice model immunized with radio-attenuated Leishmania donovani parasites through I.M. route. RESULTS: Higher T-cell proliferation, increased iNOS level, and suppressed TGF-ß level were found in treated infected animal groups (100 and 150Gy) in relation to untreated infected animals. Likewise, phosphorylation levels of PDK1, PI3K and p38MAPK of these two groups were increased when compared to untreated infected controls. CONCLUSION: The clearance of the parasites from treated infected groups of animals may be mediated by the restoration of T-cell due to therapy with radio-attenuated L. donovani parasites. The killing of parasites was mediated by increase in nitric oxide release through PDK1, PI3K and p38MAPK signaling pathways. A lower TGF-ß expression has augmented the restored Th1 ambience in the 100 and 150Gy treated animal groups proving further the efficacy of the candidate vaccine.
Assuntos
Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Animais , Western Blotting , Proliferação de Células , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Injeções Intramusculares , Injeções Intraperitoneais , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/prevenção & controle , Masculino , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/genética , Carga Parasitária , Fosforilação , RNA Mensageiro , Células Th1/imunologia , Fator de Crescimento Transformador beta/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
This study was undertaken in order to provide further insight into the role of leucine supplementation in the skeletal muscle regeneration process, focusing on myofiber size and strength recovery. Young (2-month-old) rats were subjected or not to leucine supplementation (1.35 g/kg per day) started 3 days prior to cryolesion. Then, soleus muscles were cryolesioned and continued receiving leucine supplementation until 1, 3 and 10 days later. Soleus muscles from leucine-supplemented animals displayed an increase in myofiber size and a reduction in collagen type III expression on post-cryolesion day 10. Leucine was also effective in reducing FOXO3a activation and ubiquitinated protein accumulation in muscles at post-cryolesion days 3 and 10. In addition, leucine supplementation minimized the cryolesion-induced decrease in tetanic strength and increase in fatigue in regenerating muscles at post-cryolesion day 10. These beneficial effects of leucine were not accompanied by activation of any elements of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signalling pathway in the regenerating muscles. Our results show that leucine improves myofiber size gain and strength recovery in regenerating soleus muscles through attenuation of protein ubiquitination. In addition, leucine might have therapeutic effects for muscle recovery following injury and in some muscle diseases.
Assuntos
Suplementos Nutricionais , Leucina/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Distrofias Musculares/dietoterapia , Regeneração/efeitos dos fármacos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Administração Oral , Animais , Temperatura Baixa , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Membro Posterior , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The role of phosphoinositide-dependent protein kinase-1 (PDK-1) activity on α(1B)-adrenoceptor phosphorylation and function was explored using pharmacological inhibitors and expression of a dominant-negative mutant of this enzyme. Noradrenaline-, phorbol myristate acetate-, lysophosphatidic acid- and epidermal growth factor-mediated α(1B)-adrenoceptor phosphorylation were markedly reduced by the two inhibitors used: UCN-01 [(7-hydroxystaurosporine; (3R*,8S*, 9R*, 10R*,12R*)-2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)-8,12-epoxy-1H, 8H-2,7b,12a-triazadibenzo[a,g]-cyclonona[cde]triden-1-one)] and OSU-03012 [(2-amino-N-[4-[5-(2-phenanthrenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide)]. A similar effect was observed in cells expressing a PDK-1 dominant-negative mutant. Phosphorylated PDK-1 (S241) and protein kinase C α (T497) were associated with cell membranes in the basal state which increased in response to the hormonal stimuli mentioned previously. UCN-01 essentially abolished phospho-PDK-1 membrane-association and markedly attenuated that of protein kinase C α. Consistent with the findings, UCN-01 reduced lysophosphatidic acid- and epidermal growth factor-induced α(1B-)adrenoceptor desensitization. Our data suggest that PDK-1 plays a permissive role in α(1B)-adrenoceptor desensitization and phosphorylation and participates in the formation of signaling complexes, which delicately modulate receptor function and regulation.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Cricetinae , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Mutação , Norepinefrina/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ratos , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3ß, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases.
Assuntos
Proliferação de Células , Ciclina D1/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Trombina/farmacologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Trombina/metabolismo , Trombina/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Protein restriction at early stages of life reduces ß-cell volume, number of insulin-containing granules, insulin content and release by pancreatic islets in response to glucose and other secretagogues, abnormalities similar to those seen in type 2 diabetes. Amino acids are capable to directly modulate insulin secretion and/or contribute to the maintenance of ß-cell function, resulting in an improvement of insulin release. Animal models of protein malnutrition have provided important insights into the adaptive mechanisms involved in insulin secretion in malnutrition. In this review, we discuss studies focusing on the modulation of insulin secretion by amino acids, specially leucine and taurine, in rodent models of protein malnutrition. Leucine supplementation increases insulin secretion by pancreatic islets in malnourished mice. This effect is at least in part due to increase in the expression of proteins involved in the secretion process, and the activation of the PI3K/PKB/mTOR pathway seems also to contribute. Mice supplemented with taurine have increased insulin content and secretion as well as increased expression of genes essential for ß-cell functionality. The knowledge of the mechanisms through which amino acids act on pancreatic ß-cells to stimulate insulin secretion is of interest for clinical medicine. It can reveal new targets for the development of drugs toward the treatment of endocrine diseases, in special type 2 diabetes.