RESUMEN
PINK1, linked to familial Parkinson's disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for new therapeutics targeting mitochondrial dysfunction in Parkinson's disease.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Ginsenósidos/farmacología , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Enfermedad de Parkinson/enzimología , Extractos Vegetales/farmacología , Proteínas Quinasas/deficiencia , Transducción de Señal , Animales , Chaperonina 60/genética , Chaperonina 60/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.
Asunto(s)
Proliferación Celular , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transducción de Señal/fisiología , Catálisis , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Interferencia de ARN , Transfección , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
Although extracellular signal-regulated kinases (ERK1/2) have been shown to be required in Raf/MEK/ERK pathway signaling, its sufficiency for mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously described ERK2 mutants that exhibit enhanced autophosphorylation of TEY sites in the activation loop in terms of their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. We demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp substitutions that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation, whereas expression of ERK2-I84A and ERK2-R65S/D319N is not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D only mildly increased ERK kinase activity in cells, as assessed using the ERK substrates p90(RSK) and ETS domain-containing protein (ELK1). However, ERK2-L73P/S151D expression effectively induced down-regulation of androgen receptors, Retinoblastoma (Rb) protein and E2F1 transcription factor, and up-regulation of p16(INK4A) and p21(CIP1), accompanied by cell-cycle arrest and morphological differentiation in LNCaP cells and neurite-like processes in PC12 cells. These effects and the TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introduction of the active site-disabling Lys52Arg mutation, suggesting that its autoactivation drives this signaling. Moreover, introduction of mutations Asp316/319Ala or Asp319Asn, which impair the common docking site/D-domain-based physical interaction of ERK, did not significantly affect ERK2-L73P/S151D signaling, suggesting that ERK2 mediates growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Thus, our study presents convincing evidence of ERK sufficiency for Raf/MEK/ERK signaling.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Sistema de Señalización de MAP Quinasas , Animales , Sitios de Unión , Ciclo Celular , Línea Celular Tumoral , Senescencia Celular , Células HEK293 , Humanos , Masculino , Mutación , Células PC12 , Fosforilación , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Ratas , Receptores Androgénicos/metabolismo , Elementos de Respuesta , Quinasas raf/metabolismoRESUMEN
Proper subcellular localization of the extracellular signal-regulated kinases (ERKs) is important in regulating physiological functions such as proliferation and differentiation in the pheochromocytoma cell line (PC12 cells). Thus, a direct visualization method is necessary to observe ERK localization within the cell or in crude cellular extracts. In this paper, a determination method was established for the detection of ERK2 localization in PC12 cells using green fluorescent protein (GFP) and capillary electrophoresis with laser-induced fluorescence (LIF). GFP as a reporter or labeling tag for gene expression in biochemistry and cell biology was used for the detection of ERK2 localization in PC12 cells. PC12 cells were transfected with GFP-ERK2 plasmid construct that was inserted into a variant GFP gene (enhanced green fluorescent protein), and successfully expressed GFP-ERK2 fusion proteins. GFP-ERK2 fusion proteins were detected within 5 min by CE analysis using an uncoated fused-silica capillary with LIF. Optimum conditions for GFP-ERK2 fusion proteins detection were 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer containing 100 mM sodium dodecylsulfate, pH 11, running at 20 degrees C. This result offers new opportunity in screening for the determination of localization of intracellular components, protein-protein interactions and kinase activity within the cells.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Animales , Western Blotting , Proteínas Fluorescentes Verdes , Rayos Láser , Proteínas Luminiscentes/metabolismo , Células PC12 , Plásmidos , Ratas , TransfecciónRESUMEN
In this study, we developed a method to monitor the phosphorylation and translocation of the extracellular signal-regulated kinase (ERK2) proteins after PC12 cells have been stimulated by a mitogen. The method involves the use of green fluorescent protein (GFP), capillary electrophoresis and the measurement of laser-induced fluorescence (CE-LIF). We showed the prescence of the non-phosphorylated GFP-ERK2 and phosphorylated GFP-ERK2 in cell lysates by CE-LIF, and then compared the phosphorylations of GFP-ERK2 and GFP-183A. Phosphorylated GFP-ERK2 was detected at 6.7 min and the non-phosphorylated GFP-ERK2 at 5.3-5.5 min. The results were compared with confocal laser scanning microscope imaging and western blot results, and suggest that the developed method can be used to detect other enzymatic modifications.
Asunto(s)
Electroforesis Capilar/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas/metabolismo , Animales , Western Blotting , Microscopía Electrónica , Fosforilación , Transporte de Proteínas , RatasRESUMEN
The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcio/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Activación Enzimática , Células HeLa , Humanos , MAP Quinasa Quinasa Quinasa 2/genética , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 7 Activada por Mitógenos/genética , Unión Proteica , Transporte de ProteínasRESUMEN
OBJECTIVE: The aim of this study was to elucidate the role of ERK1/2 on cisplatin resistance in human ovarian cancer cells. METHODS: The relationship between nuclear levels of ERK2 and cisplatin-induced apoptosis in human ovarian carcinoma cell line, OVCAR-3, and in cells of the cisplatin-resistant subclone, OVCAR-3/CDDP, was examined using immunoblotting and immunocytochemistry. RESULTS: Cisplatin treatment resulted in the activation of ERK2, both in OVCAR-3 and OVCAR-3/CDDP cells. However, considerable levels of activated ERK2 existed in the nuclei of OVCAR-3/CDDP cells during serum starvation and in the early period (1-3 h) after cisplatin treatment. Conversely, phospho-ERK2 was marginally detected in the nuclei of OVCAR-3 cells prior to cisplatin treatment. These phenomena were confirmed by immunofluorescence staining of the phosphorylated ERK2 in the nuclei of both cells. High basal phospho-ERK2 in the nuclei of OVCAR-3/CDDP cells contributed to cisplatin resistance, and was supported by several observations; (1) treatment of U0126, an inhibitor of MEK/ERK signaling pathway, partially sensitized OVCAR-3/CDDP cells to cisplatin; (2) pretreatment of OVCAR-3 cells with phorbol 12-myristate 13-acetate (PMA), an activator of ERK, induced nuclear translocation of activated ERK2, which led to the suppression of cisplatin-induced apoptosis. CONCLUSIONS: These results collectively indicate that prelocalization of activated ERK2 in the nuclei contribute to cisplatin resistance in OVCAR-3/CDDP cells.
Asunto(s)
Cisplatino/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Butadienos/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Núcleo Celular/enzimología , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Nitrilos/farmacología , Neoplasias Ováricas/patología , Fosforilación , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The extracellular signal-regulated kinase (ERK) cascade is a central pathway that transmits signals from many extracellular agents to regulate cellular processes such as proliferation, differentiation and cell cycle progression. The signaling via the ERK cascade is mediated by sequential phosphorylation and activation of protein kinases in the different tiers of the cascade. Although the main core phosphorylation chain of the cascade includes Raf kinases, MEK1/2, ERK1/2 (ERKs) and RSKs, other alternatively spliced forms and distinct components exist in the different tiers, and participate in ERK signaling under specific conditions. These components enhance the complexity of the ERK cascade and thereby, enable the wide variety of functions that are regulated by it. Another factor that is important for the dissemination of ERKs' signals is the multiplicity of the cascade's substrates, which include transcription factors, protein kinases and phosphatases, cytoskeletal elements, regulators of apoptosis, and a variety of other signaling-related molecules. About 160 substrates have already been discovered for ERKs, and the list of these substrates, as well as the function and mechanism of activation of representative substrates, are described in the current review. Many of these substrates are localized in the nucleus, and seem to participate in the regulation of transcription upon stimulation. However, other substrates are found in the cytosol as well as in other cellular organelles, and those are responsible for processes such as translation, mitosis and apoptosis. Understanding of these processes may provide a full picture of the distinct, and even opposing cellular processes that are regulated by the ERK cascade.
Asunto(s)
Ciclo Celular , Diferenciación Celular , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Transducción de Señal , Animales , Activación Enzimática , Humanos , Fosforilación , Unión Proteica , Factores de Transcripción , Quinasas raf/metabolismoRESUMEN
SB203580 is a p38 MAPK inhibitor that has been implicated in the activation of c-Raf. This study shows that the addition of SB203580 to PC12 cells causes the sustained activation of B-Raf but not of ERK. The addition of SB203580 prolonged the transient activation of both B-Raf and ERK by EGF alone. No significant change was detected in MAPKAPK-2 activity at low concentrations of SB203580, which induced neurite outgrowth in the EGF-stimulated PC12 cells. Therefore, these results indicate that SB203580 influences not only c-Raf as previously reported, but can also induce the activation of B-Raf, which in conjunction with EGF causes the sustained activation of ERK and differentiation in PC12 cells.