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1.
J Cell Sci ; 134(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34553755

RESUMO

The ERK1/2 (also known as MAPK3 and MAPK1, respectively) signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins, such as Shoc2 to guide and fine-tune its signals. Mutations in SHOC2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here, we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We reveal that, in the Shoc2 module, USP7 functions as a molecular 'switch' that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH, thereby extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.


Assuntos
Síndrome dos Cabelos Anágenos Frouxos , Sistema de Sinalização das MAP Quinases , Síndrome de Noonan , Peptidase 7 Específica de Ubiquitina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Transdução de Sinais , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Peptidase 7 Específica de Ubiquitina/genética
2.
J Cell Sci ; 128(23): 4428-41, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26519477

RESUMO

The scaffold protein Shoc2 accelerates activity of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) pathway. Mutations in Shoc2 result in Noonan-like RASopathy, a developmental disorder with a wide spectrum of symptoms. The amplitude of the ERK1/2 signals transduced through the complex is fine-tuned by the HUWE1-mediated ubiquitylation of Shoc2 and its signaling partner RAF-1. Here, we provide a mechanistic basis of how ubiquitylation of Shoc2 and RAF-1 is controlled. We demonstrate that the newly identified binding partner of Shoc2, the (AAA+) ATPase PSMC5, triggers translocation of Shoc2 to endosomes. At the endosomes, PSMC5 displaces the E3 ligase HUWE1 from the scaffolding complex to attenuate ubiquitylation of Shoc2 and RAF-1. We show that a RASopathy mutation that changes the subcellular distribution of Shoc2 leads to alterations in Shoc2 ubiquitylation due to the loss of accessibility to PSMC5. In summary, our results demonstrate that PSMC5 is a new and important player involved in regulating ERK1/2 signal transmission through the remodeling of Shoc2 scaffold complex in a spatially-defined manner.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Mutação , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fatores de Transcrição/genética
3.
Hum Mutat ; 35(11): 1290-4, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25137548

RESUMO

Rasopathies are a group of genetic disorders caused by germline mutations in multiple genes of the Extracellular signal-Regulated Kinases 1 and 2 (ERK1/2) pathway. The only previously identified missense mutation in SHOC2, a scaffold protein of the ERK1/2 pathway, led to Noonan-like syndrome with loose anagen hair. Here, we report a novel mutation in SHOC2(c.519G>A; p.M173I) that leads to a Rasopathy with clinical features partially overlapping those occurring in Noonan and cardiofaciocutaneous syndromes. Studies to clarify the significance of this SHOC2 variant revealed that the mutant protein has impaired capacity to interact with protein phosphatase 1c (PP1c), leading to insufficient activation of RAF-1 kinase. This SHOC2 variant thus is unable to fully rescue ERK1/2 activity in cells depleted of endogenous SHOC2. We conclude that SHOC2 mutations can cause a spectrum of Rasopathy phenotypes in heterozygous individuals. Importantly, our work suggests that individuals with mild Rasopathy symptoms may be underdiagnosed.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Variação Genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Anormalidades Múltiplas/metabolismo , Pré-Escolar , Fácies , Feminino , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Mutação , Fenótipo , Ligação Proteica
4.
Carcinogenesis ; 34(5): 953-61, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23354304

RESUMO

Carcinoid tumors are rare neuroendocrine tumors (NETs) that are increasing in incidence. Mutation and altered expression of Wnt/ß-catenin signaling components have been described in many tumors but have not been well-studied in NETs. Here, we observed accumulation of ß-catenin in the cytoplasm and/or nucleus in 25% of clinical NET tissues. By mutational analysis, the mutations of ß-catenin (I35S) and APC (E1317Q, T1493T) were identified in NET cells and the tissues. Expression of representative Wnt inhibitors was absent or markedly decreased in BON, a human pancreatic carcinoid cell line; treatment with 5-aza-2'-deoxycytidine (5-aza-CdR) increased expression levels of the Wnt inhibitors. Methylation analyses demonstrated that CpG islands of SFRP-1 and Axin-2 were methylated, whereas the promoters of DKK-1, DKK-3 and WIF-1 were unmethylated in four NET cells. Aberrant methylation of SFRP-1 was particularly observed in most of clinical NET tissues. In addition, the repression of these unmethylated genes was associated with histone H3 lysine 9 dimethylation (H3K9me2) in BON cells. Together, 5-aza-CdR treatment inhibited cell proliferation and decreased the protein levels of H3K9me2 and G9a. Moreover, a novel G9a inhibitor, UNC0638, suppressed BON cell proliferation through inhibition of Wnt/ß-catenin pathway. Overexpression of the inhibitory genes, particularly SFRP-1 and WIF-1 in BON cells, resulted in suppression of anchorage-independent growth and inhibition of tumor growth in mice. Our findings suggest that aberrant Wnt/ß-catenin signaling, through either mutations or epigenetic silencing of Wnt antagonists, contributes to the pathogenesis and growth of NETs and have important clinical implications for the prognosis and treatment of NETs.


Assuntos
Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ilhas de CpG , Citoplasma/genética , Citoplasma/metabolismo , Metilação de DNA , Análise Mutacional de DNA/métodos , Epigênese Genética , Epigenômica/métodos , Expressão Gênica/genética , Genes APC , Genes Supressores de Tumor , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Transcrição Gênica/genética
5.
Mol Pharm ; 10(1): 406-16, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23215050

RESUMO

OATP1B3 is a member of the OATP (organic anion transporting polypeptides) superfamily, responsible for mediating the transport of numerous endogenous and xenobiotic substances. Although initially reported to be exclusively expressed in the liver, several studies reported that OATP1B3 is frequently expressed in multiple types of cancers and may be associated with differing clinical outcomes. However, a detailed investigation on the expression and function of OATP1B3 protein in cancer has been lacking. In this study, we confirmed that colon and pancreatic cancer cells express variant forms of OATP1B3, different from OATP1B3 wild-type (WT) expressed in the normal liver. OATP1B3 variant 1 (V1), the most prevalent form among the variants, contains alternative exonic sequences (exon 2a) instead of exons 1 and 2 present in OATP1B3 WT. The translated product of OATP1B3 V1 is almost identical to OATP1B3 WT, with exception to the first 28 amino acids at the N-terminus. Exogenous expression of OATP1B3 V1 revealed that OATP1B3 V1 undergoes post-translational modifications and proteasomal degradation to a differing extent compared to OATP1B3 WT. OATP1B3 V1 showed only modest transport activity toward cholecystokin-8 (CCK-8, a prototype OATP1B3 substrate) in contrast to OATP1B3 WT showing a markedly efficient uptake of CCK-8. Consistent with these results, OATP1B3 V1 was localized mainly in the cytoplasm with a much lower extent of trafficking to the surface membrane compared to OATP1B3 WT. In summary, our results demonstrate that colon and pancreatic cancer cells express variant forms of OATP1B3 with only limited transport activity and different subcellular localization compared to OATP1B3 WT. These observed differences at the molecular and functional levels will be important considerations for further investigations of the biological and clinical significance of OATP1B3 expression in cancer.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Neoplasias Pancreáticas/genética , Transporte Biológico , Linhagem Celular Tumoral , Citoplasma/genética , Citoplasma/metabolismo , Éxons , Variação Genética , Células HCT116 , Humanos , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Neoplasias Pancreáticas/metabolismo , Processamento de Proteína Pós-Traducional , Sincalida/genética , Sincalida/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto
6.
Chembiochem ; 13(13): 1899-903, 2012 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-22807337

RESUMO

Probing the unknown: The immunoproteasome, an alternative form of the constitutive proteasome, has been implicated in a number of pathological states such as cancer and autoimmune diseases. In an effort to understand the role of the immunoproteasome in cells, the first immunoproteasome-specific near-infrared fluorescent probe has been developed.


Assuntos
Corantes Fluorescentes/análise , Complexo de Endopeptidases do Proteassoma/análise , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/imunologia , Proteômica/métodos
7.
Mol Pharm ; 9(8): 2197-205, 2012 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-22734651

RESUMO

Carfilzomib is a novel class of peptidyl epoxyketone proteasome inhibitor and has demonstrated promising activity in multiple clinical trials to treat patients with multiple myeloma and other types of cancers. Here, we investigated molecular mechanisms underlying acquired resistance to carfilzomib and a potential strategy to restore cellular sensitivity to carfilzomib. H23 and DLD-1 cells (human lung and colon adenocarcinoma cell lines) with acquired resistance to carfilzomib displayed marked cross-resistance to YU-101, a closely related proteasome inhibitor, and paclitaxel, a known substrate of Pgp. However, carfilzomib-resistant cells remained sensitive to bortezomib, a clinically used dipeptide with boronic acid pharmacophore. In accordance with these observations, carfilzomib-resistant H23 and DLD-1 cells showed marked upregulation of P-glycoprotein (Pgp) as compared to their parental controls, and coincubation with verapamil, a Pgp inhibitor, led to an almost complete restoration of cellular sensitivity to carfilzomib. These results indicate that Pgp upregulation plays a major role in the development of carfilzomib resistance in these cell lines. In developing a potential strategy to overcome carfilzomib resistance, we as a proof of concept prepared a small library of peptide analogues derived from the peptide backbone of carfilzomib and screened these molecules for their activity to restore carfilzomib sensitivity when cotreated with carfilzomib. We found that compounds as small as dipeptides are sufficient in restoring carfilzomib sensitivity. Taken together, we found that Pgp upregulation plays a major role in the development of resistance to carfilzomib in lung and colon adenocarcinoma cell lines and that small peptide analogues lacking the pharmacophore can be used as agents to reverse acquired carfilzomib resistance. Our findings may provide important information in developing a potential strategy to overcome drug resistance.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Peptídeos/química , Inibidores de Proteassoma/farmacologia , Pirazinas/farmacologia , Verapamil/farmacologia
8.
J Biol Chem ; 285(5): 2986-95, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-19948725

RESUMO

We screened a protoberberine backbone derivative library for compounds with anti-proliferative effects on p53-defective cancer cells. A compound identified from this small molecule library, cadein1 (cancer-selective death inducer 1), an isoquinolinium derivative, effectively leads to a G(2)/M delay and caspase-dependent apoptosis in various carcinoma cells with non- functional p53. The ability of cadein1 to induce apoptosis in p53-defective colon cancer cells was tightly linked to the presence of a functional DNA mismatch repair (MMR) system, which is an important determinant in chemosensitivity. Cadein1 was very effective in MMR(+)/p53(-) cells, whereas it was not effective in p53(+) cells regardless of the MMR status. Consistently, when the function of MMR was blocked with short hairpin RNA in SW620 (MMR(+)/p53(-)) cells, cadein1 was no longer effective in inducing apoptosis. Besides, the inhibition of p53 increased the pro-apoptotic effect of cadein1 in HEK293 (MMR(+)/p53(+)) cells, whereas it did not affect the response to cadein1 in RKO (MMR(-)/p53(+)) cells. The apoptotic effects of cadein1 depended on the activation of p38 but not on the activation of Chk2 or other stress-activated kinases in p53-defective cells. Taken together, our results show that cadein1 may have a potential to be an anti-cancer chemotherapeutic agent that is preferentially effective on p53-mutant colon cancer cells with functional MMR.


Assuntos
Apoptose , Genes p53 , Isoquinolinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos/farmacologia , Pareamento Incorreto de Bases , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Reparo do DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Químicos
9.
Biochem Biophys Res Commun ; 397(4): 637-43, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20471956

RESUMO

Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon gamma-irradiation in ATM(+) cells but not ATM(-) cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteína p300 Associada a E1A/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína p300 Associada a E1A/genética , Células HeLa , Humanos , Fosforilação , Estabilidade Proteica , Serina/genética , Serina/metabolismo
10.
Mol Biol Cell ; 30(14): 1655-1663, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091164

RESUMO

Valosin-containing protein (VCP), also named p97, is an essential hexameric AAA+ ATPase with diverse functions in the ubiquitin system. Here we demonstrate that VCP is critical in controlling signals transmitted via the essential Shoc2-ERK1/2 signaling axis. The ATPase activity of VCP modulates the stoichiometry of HUWE1 in the Shoc2 complex as well as HUWE1-mediated allosteric ubiquitination of the Shoc2 scaffold and the RAF-1 kinase. Abrogated ATPase activity leads to augmented ubiquitination of Shoc2/RAF-1 and altered phosphorylation of RAF-1. We found that in fibroblasts from patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) that harbor germline mutations in VCP, the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced. This study provides a mechanistic basis for the critical role of VCP in the regulation of the ERK1/2 pathway and reveals a previously unrecognized function of the ERK1/2 pathway in the pathogenesis of IBMPFD.


Assuntos
Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Osteíte Deformante/metabolismo , Osteíte Deformante/patologia , Proteína com Valosina/metabolismo , Endossomos/metabolismo , Humanos , Modelos Biológicos , Ubiquitinação
11.
Cancer Res ; 65(24): 11400-10, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16357148

RESUMO

Histone deacetylase inhibitors (HDI) have been reported to inhibit the growth and survival of cancer cells while leaving normal cells untouched. However, the mechanisms underlying this selective cell death are poorly understood. Gene expression analysis revealed that HDI treatment induced up-regulation of p21(WAF1/Cip1) and down-regulation of ErbB2 in cancer cells but not normal cells. Overexpression of p21(WAF1/Cip1) and/or silencing of ErbB2 enhanced cancer cell growth inhibition, suggesting that HDI-induced up-regulation/down-regulation of these genes play critical roles in HDI-induced growth inhibition of cancer cells. Most importantly, we found that the gene silencing factor methyl CpG-binding domain protein 3 (MBD3) was not only released from cancer-selective promoter of the HDI up-regulated p21(WAF1/Cip1) gene but also recruited to that of the HDI-down-regulated ErbB2 gene. Furthermore, silencing of MBD3 by small interfering RNA abrogated the HDI-induced gene regulation and growth inhibition in lung cancer but not in normal cells. Together, our results support the critical potential of MBD3 in HDI-induced cancer-selective cell death via cancer differential gene expression.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Inibidores de Histona Desacetilases , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/farmacologia , Ativação Transcricional , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia
12.
Commun Integr Biol ; 9(4): e1188241, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27574535

RESUMO

The extracellular signal-regulated kinase (ERK1/2) cascade regulates a myriad of functions in multicellular organisms. Scaffold proteins provide critical spatial and temporal control over the specificity of signaling. Shoc2 is a scaffold that accelerates activity of the ERK1/2 pathway. Loss of Shoc2 expression in mice results in embryonic lethality, thus highlighting the essential role of Shoc2 in embryogenesis. In agreement, patients carrying mutated Shoc2 suffer from a wide spectrum of developmental deficiencies. Efforts to understand the mechanisms by which Shoc2 controls ERK1/2 activity revealed the intricate machinery that governs the ability of Shoc2 to transduce signals of the ERK1/2 pathway. Understanding the mechanisms by which Shoc2 contributes to a high degree of specificity of ERK1/2 signaling as well as deciphering the biological functions of Shoc2 in development and human disorders are major unresolved questions.

13.
Data Brief ; 7: 770-8, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27077079

RESUMO

The Suppressor of Clear, Caenorhabditis elegans Homolog (SHOC2) is a scaffold protein that positively modulates activity of the RAS/ERK1/2 MAP kinase signaling cascade. We set out to understand the ERK1/2 pathway transcriptional response transduced through the SHOC2 scaffolding module. This data article describes raw gene expression within triplicates of kidney fibroblast-like Cos1 cell line expressing non-targeting shRNA (Cos-NT) and triplicates of Cos1 cells depleted of SHOC2 using shRNA (Cos-LV1) upon activation of ERK1/2 pathway by the Epidermal Growth Factor Receptor (EGFR). The data referred here is available in NCBI׳s Gene Expression Omnibus (GEO), accession GEO: GSE67063 as well as NCBI׳s Sequence Read Archive (SRA), accession SRA: SRP056324. A complete analysis of the results can be found in "Shoc2-tranduced ERK1/2 motility signals - Novel insights from functional genomics"(Jeoung et al., 2016) [1].

14.
Cell Signal ; 28(5): 448-459, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26876614

RESUMO

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway plays a central role in defining various cellular fates. Scaffold proteins modulating ERK1/2 activity control growth factor signals transduced by the pathway. Here, we analyzed signals transduced by Shoc2, a critical positive modulator of ERK1/2 activity. We found that loss of Shoc2 results in impaired cell motility and delays cell attachment. As ERKs control cellular fates by stimulating transcriptional response, we hypothesized that the mechanisms underlying changes in cell adhesion could be revealed by assessing the changes in transcription of Shoc2-depleted cells. Using quantitative RNA-seq analysis, we identified 853 differentially expressed transcripts. Characterization of the differentially expressed genes showed that Shoc2 regulates the pathway at several levels, including expression of genes controlling cell motility, adhesion, crosstalk with the transforming growth factor beta (TGFß) pathway, and expression of transcription factors. To understand the mechanisms underlying delayed attachment of cells depleted of Shoc2, changes in expression of the protein of extracellular matrix (lectin galactoside-binding soluble 3-binding protein; LGALS3BP) were functionally analyzed. We demonstrated that delayed adhesion of the Shoc2-depleted cells is a result of attenuated expression and secretion of LGALS3BP. Together our results suggest that Shoc2 regulates cell motility by modulating ERK1/2 signals to cell adhesion.


Assuntos
Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Células COS , Movimento Celular/genética , Chlorocebus aethiops , Genômica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Transcriptoma
15.
Oncogene ; 23(9): 1724-36, 2004 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-14676837

RESUMO

Many cases of breast cancer show loss of estrogen receptor (ER) alpha expression, which leads to unresponsiveness to antihormonal treatment even though there is no loss of the structurally and biochemically similar ER beta. ER activity is positively and negatively regulated by transcriptional regulators such as histone deacetylase (HDAC), which is known to be a negative ER regulator. Here, we evaluated using ER beta as an alternative target for tamoxifen therapy by treating ER alpha-negative, beta-positive breast cancer cells with the HDAC inhibitor trichostatin A (TSA), and testing whether tamoxifen responsiveness increased following upregulation of ER beta. TSA enhanced the overall ER transcriptional activity in these cells, as visualized by estrogen response element-regulated reporter and the expression of progesterone receptor, a known ER target, without ER alpha restoration. Additionally, TSA induced the expression and nuclear translocation of ER beta but not alpha, suggesting that these actions leading to increase of ER transcriptional activity are mediated through ER beta rather than alpha. Furthermore, following treatment with TSA, the formerly unresponsive MDA-MB-231 and Hs578T breast cancer cells became responsive to tamoxifen. However, reduction of ER beta expression by short interfering RNA abrogated this TSA-induced sensitization effect in these cells. Together, these results show that the HDAC inhibitor TSA sensitized ER alpha-negative, antihormone-unresponsive breast cancer cells to tamoxifen treatment possibly by upregulating ER beta activity.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Receptores de Estrogênio/deficiência , Tamoxifeno/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Antagonistas de Estrogênios/farmacologia , Antagonistas de Estrogênios/uso terapêutico , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Células HeLa , Histona Desacetilases/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Elementos de Resposta/genética , Tamoxifeno/uso terapêutico
16.
J Cancer Res Clin Oncol ; 130(4): 225-34, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14745549

RESUMO

PURPOSE: Ataxia-telangiectasia (A-T) is a progressive, degenerative, complex autosomal recessive disease characterized by cerebellar degeneration, immunodeficiency, premature aging, radiosensitivity, and a predisposition to cancer. Mutations in the ataxia-telangiectasia mutated (atm) gene, which phosphorylates downstream effector proteins, are linked to A-T. One of the proteins phosphorylated by the ATM protein is Nijmegen Breakage Syndrome protein (NBS, p95/nibrin), which was recently shown to be encoded by a gene mutated in the Nijmegen breakage syndrome (nbs), an autosomal recessive disease with a phenotype virtually similar to that of A-T. The similarities in the clinical and cellular features of NBS and A-T have led us to hypothesize that the two corresponding gene products may function in similar ways in the cellular signaling pathway. Thus, we sought to identify genes whose expression is mediated by the atm and nbs gene products. MATERIAL AND METHODS: To identify genes, we performed an analysis of oligonucleotide microarrays using the appropriate cell lines, isogenic A-T (ATM-) and control cells (ATM+), and isogenic NBS (NBS-) and control cells (NBS+). RESULTS: We examined genes regulated by ATM and NBS, respectively. To determine the effect of ATM and NBS on gene expression in detail, we classified these genes into different functional categories, including those involved in apoptosis, cell cycle/DNA replication, growth/differentiation, signal transduction, cell-cell adhesion, and metabolism. In addition, we compared the genes regulated by the ATM and NBS to determine the relationship of their signaling pathways and to better understand their functional relationship. CONCLUSIONS: We found that, while ATM and NBS regulate several genes in common, both of these proteins also have distinct patterns of gene regulation, findings consistent with the functional overlap and distinctiveness of these two conditions. Due to the role of ATM and NBS in tumor suppression and the response to chemotherapy and radiotherapy, these findings may assist in the development of a more rational approach to cancer treatment, as well as a better understanding of tumorigenesis.


Assuntos
Proteínas de Ciclo Celular/genética , Quebra Cromossômica , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Mutadas de Ataxia Telangiectasia , Quebra Cromossômica/genética , Análise por Conglomerados , Proteínas de Ligação a DNA , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Supressoras de Tumor
17.
Mol Cell Biol ; 34(19): 3579-93, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25022756

RESUMO

Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Células COS , Chlorocebus aethiops , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Proteínas Supressoras de Tumor
18.
PLoS One ; 8(6): e66067, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23805200

RESUMO

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Sequência Conservada , Fator de Crescimento Epidérmico/farmacologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , Domínios Proteicos/fisiologia , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos
19.
PLoS One ; 8(9): e73732, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040045

RESUMO

The proteasome is a key regulator of cellular protein homeostasis and is a clinically validated anticancer target. The immunoproteasome, a subtype of proteasome expressed mainly in hematopoietic cells, was initially recognized for its role in antigen presentation during the immune response. Recently, the immunoproteasome has been implicated in several disease conditions including cancer and autoimmune disorders, but many of the factors contributing to these pathological processes remain unknown. In particular, the codon 60 polymorphism of the PSMB9 gene encoding the ß1i immunoproteasome catalytic subunit has been investigated in the context of a variety of diseases. Despite this, previous studies have so far reported inconsistent findings regarding the impact of this polymorphism on proteasome activity. Thus, we set out to investigate the impact of the PSMB9 codon 60 polymorphism on the expression and activity of the ß1i immunoproteasome subunit in a panel of human cancer cell lines. The ß1i-selective fluorogenic substrate Acetyl-Pro-Ala-Leu-7-amino-4-methylcoumarin was used to specifically measure ß1i catalytic activity. Our results indicate that the codon 60 Arg/His polymorphism does not significantly alter the expression and activity of ß1i among the cell lines tested. Additionally, we also examined the expression of ß1i in clinical samples from colon and pancreatic cancer patients. Our immunohistochemical analyses showed that ≈ 70% of clinical colon cancer samples and ≈ 53% of pancreatic cancer samples have detectable ß1i expression. Taken together, our results indicate that the ß1i subunit of the immunoproteasome is frequently expressed in colon and pancreatic cancers but that the codon 60 genetic variants of ß1i display similar catalytic activities and are unlikely to contribute to the significant inter-cell-line and inter-individual variabilities in the immunoproteasome activity.


Assuntos
Códon/genética , Cisteína Endopeptidases/genética , Neoplasias/genética , Polimorfismo Genético , Substituição de Aminoácidos , Biocatálise , Domínio Catalítico/genética , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Cisteína Endopeptidases/metabolismo , Corantes Fluorescentes/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genótipo , Células HCT116 , Humanos , Hidrólise , Immunoblotting , Imuno-Histoquímica , Células MCF-7 , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Análise Serial de Tecidos
20.
Mol Biosyst ; 8(9): 2295-302, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22722901

RESUMO

The discovery of NF-κB signaling pathways has greatly enhanced our understanding of inflammatory and immune responses. In the canonical NF-κB pathway, the proteasomal degradation of IκBα, an inhibitory protein of NF-κB, is widely accepted to be a key regulatory step. However, contradictory findings have been reported as to whether the immunoproteasome plays an obligatory role in the degradation of IκBα and activation of the canonical NF-κB pathway. Such results were obtained mainly using traditional gene deletion strategies. Here, we have revisited the involvement of the immunoproteasome in the canonical NF-κB pathway using small molecule inhibitors of the immunoproteasome, namely UK-101 and LKS01 targeting ß1i and ß5i, respectively. H23 and Panc-1 cancer cells were pretreated with UK-101, LKS01 or epoxomicin (a prototypic inhibitor targeting both the constitutive proteasome and immunoproteasome). We then examined whether these pretreatments lead to any defect in activating the canonical NF-κB pathway following TNFα exposure by monitoring the phosphorylation and degradation of IκBα, nuclear translocation of NF-κB proteins and DNA binding and transcriptional activity of NF-κB. Our results consistently indicated that there is no defect in activating the canonical NF-κB pathway following selective inhibition of the immunoproteasome catalytic subunits ß1i, ß5i or both using UK-101 and LKS01, in contrast to epoxomicin. In summary, our current results using chemical genetic approaches strongly support that the catalytic activity of the immunoproteasome subunits ß1i and ß5i is not required for canonical NF-κB activation in lung and pancreatic adenocarcinoma cell line models.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Western Blotting , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Imunofluorescência , Humanos , Compostos de Organossilício/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos
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