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1.
EMBO J ; 33(16): 1784-801, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-24975362

RESUMO

Mitogen-activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular-signal-regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three-tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5-ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3-ERK5-dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5-MAPK module and human muscle cell differentiation.


Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , MAP Quinase Quinase 5/metabolismo , MAP Quinase Quinase Quinase 3/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Ubiquitina/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA , Ativação Enzimática , Humanos , Proteínas Inibidoras de Apoptose/genética , MAP Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinase 3/genética , MAP Quinase Quinase Quinases/genética , Fatores de Transcrição MEF2/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
2.
J Biol Chem ; 287(34): 28445-55, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22711539

RESUMO

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proteínas Inibidoras de Apoptose/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Estabilidade Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Melanoma/genética , Melanoma/patologia , Proteínas de Neoplasias/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-raf/genética
3.
Nanoscale ; 11(26): 12517-12529, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31188378

RESUMO

Peptide nucleic acids (PNAs) have gained considerable attention due to their remarkable potential in gene editing and targeting-based strategies. However, cellular delivery of PNAs remains a challenge in developing their broader therapeutic applications. Here, we investigated a novel complex made of lipid bicelles and PNA-based carriers for the efficient delivery of PNAs. For proof of concept, PNAs targeting microRNA (miR) 210 and 155 were tested. Comprehensive evaluation of positive as well as negative charge-containing bicelles with PNA : lipid ratios of 1 : 100, 1 : 1000, and 1 : 2500 was performed. The negatively charged bicelles with a PNA : lipid molar ratio of 1 : 2500 yielded a discoidal shape with a uniform diameter of ∼30 nm and a bilayer thickness of 5 nm, while the positively charged bicellar system contained irregular vesicles after the incorporation of PNA. Small-angle X-ray scattering (SAXS) analysis was performed to provide insight into how the hydrophobic PNAs interact with bicelles. Further, flow cytometry followed by confocal microscopy analyses substantiate the superior transfection efficiency of bicelles containing dye-conjugated antimiR PNAs. Functional analysis also confirmed miR inhibition by PNA oligomers delivered by bicelles. The nanodiscoidal complex opens a new pathway to deliver PNAs, which, on their own, are a great challenge to be endocytosed into cells.


Assuntos
Lipídeos , Ácidos Nucleicos Peptídicos , Transfecção , Células HeLa , Humanos , Lipídeos/química , Lipídeos/farmacologia , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , Espalhamento a Baixo Ângulo , Difração de Raios X
4.
Methods Mol Biol ; 1120: 43-54, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24470018

RESUMO

Ubiquitination of proteins has emerged as a vital posttranslational modification at the crux of numerous signalling pathways, regulating them in various ways. Most members of the small GTPase family including Ras and Rho proteins are regulated by GEFs, GAPs, and RhoGDIs that modulate their cycling between the active and inactive states. Ubiquitination has added another layer to the regulation of small GTPases. Recently, we have uncovered that inhibitors of apoptosis (IAPs) function as direct E3 ubiquitin ligases for Rho GTPase Rac1 and target it for proteasomal degradation. Here, we describe in vitro and in vivo ubiquitination assays for detecting the conjugation of ubiquitin to Rac1 by XIAP and cIAP1.


Assuntos
Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células HEK293 , Células HeLa , Histidina/química , Humanos , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/química , Compostos Organometálicos/química , Ligação Proteica
5.
Sci Signal ; 7(337): ra73, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25097033

RESUMO

The RAF family of kinases mediates RAS signaling, and RAF inhibitors can be effective for treating tumors with BRAF(V600E) mutant protein. However, RAF inhibitors paradoxically accelerate metastasis in RAS-mutant tumors and become ineffective in BRAF(V600E) tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling. We found that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and cell migration in a cell type-dependent manner. Knocking down ARAF prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and decreased the number of protrusions from tumor cell spheroids in three-dimensional culture that were induced by BRAF(V600E)-specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors induced the homodimerization of ARAF and the heterodimerization of BRAF with CRAF and the scaffolding protein KSR1. In a purified protein solution, recombinant proteins of the three RAF isoforms competed for binding to MEK1. In cells in culture, overexpressing mutants of ARAF that could not homodimerize impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent activation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade and promoting tumor cell invasion and suggest a new therapeutic target for RAS- and RAF-mediated cancers.


Assuntos
Movimento Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Moleculares , Proteínas Proto-Oncogênicas A-raf/metabolismo , Análise de Variância , Ligação Competitiva , Western Blotting , Dimerização , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Indenos/farmacologia , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Invasividade Neoplásica , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas A-raf/química , Proteínas Proto-Oncogênicas A-raf/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Pirazóis/farmacologia , RNA Interferente Pequeno/genética , Sorafenibe , Imagem com Lapso de Tempo , Células Tumorais Cultivadas
6.
Small GTPases ; 3(2): 131-6, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22790203

RESUMO

Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination-dependent inactivation of Rac1 signaling.


Assuntos
Movimento Celular , Proteínas Inibidoras de Apoptose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Forma Celular , Humanos , Metástase Neoplásica/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
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