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1.
Hum Mol Genet ; 25(24): 5460-5471, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27798111

RESUMO

Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC.


Assuntos
Proteína BRCA1/genética , Neoplasias Ovarianas/genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitinação/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA1/biossíntese , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Código das Histonas/genética , Histonas/genética , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Ubiquitina-Proteína Ligases/genética
2.
J Cell Sci ; 128(21): 3933-46, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26403202

RESUMO

ß-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate ß-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing ß-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of ß-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1-ß-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear ß-catenin-lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of ß-catenin at specific serines, which when mutated (S191A and S605A) reduced ß-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of ß-catenin stimulates Wnt-dependent gene transactivation by enhancing ß-catenin-LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/ß-catenin signaling.


Assuntos
Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Western Blotting , Linhagem Celular , Células HCT116 , Humanos , Imunoprecipitação , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Células NIH 3T3 , Reação em Cadeia da Polimerase em Tempo Real , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genética , Proteínas rac1 de Ligação ao GTP/genética
3.
Exp Cell Res ; 348(2): 132-145, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27658570

RESUMO

ß-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of ß-catenin stimulate nuclear entry, the N- and C-terminal "tail" sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of ß-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocation of ß-catenin through the nuclear pore complex. Extensive alanine mutagenesis of the tail sequences revealed that nuclear translocation of ß-catenin was dependent on specific uniformly distributed patches of hydrophobic residues, whereas the mutagenesis of acidic amino acids had no effect. Moreover, the mutation of hydrophobic patches within the N-tail and C-tail of full length ß-catenin reduced nuclear transport rate and diminished its ability to activate transcription. We propose that the tail sequences can contribute to ß-catenin transport and suggest a possible similar role for hydrophobic unstructured regions in other proteins.


Assuntos
Núcleo Celular/metabolismo , Interações Hidrofóbicas e Hidrofílicas , beta Catenina/química , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Sobrevivência Celular , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Humanos , Camundongos , Mutagênese/genética , Células NIH 3T3 , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Ativação Transcricional/genética
4.
Exp Cell Res ; 341(2): 196-206, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26844628

RESUMO

Beta-catenin plays a key role in transducing Wnt signals from the plasma membrane to the nucleus. Here we characterize an unusual subcellular distribution of beta-catenin in MCF-7 breast cancer cells, wherein beta-catenin localizes to the cytoplasm and membrane but atypically did not relocate to the nucleus after Wnt treatment. The inability of Wnt or the Wnt agonist LiCl to induce nuclear localization of beta-catenin was not due to defective nuclear transport, as the transport machinery was intact and ectopic GFP-beta-catenin displayed rapid nuclear entry in living cells. The mislocalization is explained by a shift in the retention of beta-catenin from nucleus to cytoplasm. The reduced nuclear retention is caused by unusually low expression of lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors. The reconstitution of LEF-1 or TCF4 expression rescued nuclear localization of beta-catenin in Wnt treated cells. In the cytoplasm, beta-catenin accumulated in recycling endosomes, golgi and beta-COP-positive coatomer complexes. The peripheral association with endosomes diminished after Wnt treatment, potentially releasing ß-catenin into the cytoplasm for nuclear entry. We propose that in MCF-7 and perhaps other breast cancer cells, beta-catenin may contribute to cytoplasmic functions such as ER-golgi transport, in addition to its transactivation role in the nucleus.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , beta Catenina/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Células MCF-7 , Ativação Transcricional/fisiologia , Proteínas Wnt/metabolismo
5.
J Biol Chem ; 290(11): 7304-13, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25645916

RESUMO

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.


Assuntos
Apoptose , Proteína BRCA1/metabolismo , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismo
6.
Semin Cancer Biol ; 27: 20-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24820952

RESUMO

The nuclear localization of specific proteins is critical for cellular processes such as cell division, and in recent years perturbation of the nuclear transport cycle of key proteins has been linked to cancer. In particular, specific gene mutations can alter nuclear transport of tumor suppressing and oncogenic proteins, leading to cell transformation or cancer progression. This review will focus on one such factor, ß-catenin, a key mediator of the canonical wnt signaling pathway. In response to a wnt stimulus or specific gene mutations, ß-catenin is stabilized and translocates to the nucleus where it binds TCF/LEF-1 transcription factors to transactivate genes that drive tumor formation. Moreover, the nuclear import and accumulation of ß-catenin correlates with clinical tumor grade. Recent evidence suggests that the primary nuclear transport route of ß-catenin is independent of the classical Ran/importin import machinery, and that ß-catenin directly contacts the nuclear pore complex to self-regulate its own entry into the nucleus. Here we propose that the ß-catenin nuclear import pathway may provide an opportunity for identification of specific drug targets and inhibition of ß-catenin nuclear function, much like the current screening of drugs that block binding of ß-catenin to LEF-1/TCFs. Here we will discuss the diverse mechanisms regulating nuclear localization of ß-catenin and their potential as targets for anticancer agent development.


Assuntos
Neoplasias/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos
7.
8.
Traffic ; 13(6): 800-14, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22420687

RESUMO

The breast cancer associated gene 1 (BRCA1)-A protein complex assembles at DNA damage-induced nuclear foci to facilitate repair of double-stranded breaks. Here, we describe the first systematic comparison of the dynamics, copy number and organization of its core components at foci. We show that the protein pools at individual foci generally comprise a small immobile fraction (∼20%) and larger mobile fraction (∼80%), which together occupy the same focal space but exist at different densities. In the mobile fraction, Abraxas (CCDC98) and the heterodimer BARD1-BRCA1 share similar rates of dynamic exchange (complete turnover in ∼500 seconds). In contrast, RAP80, which is required for initial foci assembly, was more dynamic with 25-fold faster turnover at mature foci. In addition, Abraxas, BARD1, BRCA1 and Merit40 (NBA1) were stably retained in the immobile fraction of foci under conditions causing loss of BRCC36 and RAP80, suggesting a shift to RAP80-independent localization after foci formation. These results, combined with our finding that RAP80 (∼1200 copies per focus) is twofold more abundant than Abraxas/BARD1/BRCA1 at foci, suggest new models defining the dynamic organization of BRCA1-A complex at mature foci, wherein the unusually fast turnover of RAP80 may contribute to its regulation of BRCA1-dependent DNA repair.


Assuntos
Proteína BRCA1/metabolismo , Núcleo Celular/metabolismo , Dano ao DNA , Genes BRCA1 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA , Enzimas Desubiquitinantes , Chaperonas de Histonas , Humanos , Proteínas de Membrana/metabolismo , Modelos Teóricos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Radiação Ionizante , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
Biochim Biophys Acta ; 1833(10): 2334-47, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23770048

RESUMO

Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Rac1 with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization.


Assuntos
Actinas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Actinas/antagonistas & inibidores , Actinas/genética , Transporte Ativo do Núcleo Celular , Western Blotting , Reparo do DNA/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Humanos , Hidroxiureia/farmacologia , Imunoprecipitação , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Transdução de Sinais , Timidina/farmacologia
10.
Adv Exp Med Biol ; 773: 353-72, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24563356

RESUMO

Several components of the Wnt signaling pathway have in recent years been linked to the nuclear pore complex. ß-catenin, the primary transducer of Wnt signals from the plasma membrane to the nucleus, has been shown to transiently associate with different FG-repeat containing nucleoporins (Nups) and to translocate bidirectionally through pores of the nuclear envelope in a manner independent of classical transport receptors and the Ran GTPase. Two key regulators of ß-catenin, IQGAP1 and APC, have also been reported to bind specific Nups or to locate at the nuclear pore complex. The interaction between these Wnt signaling proteins and different Nups may have functional implications beyond nuclear transport in cellular processes that include mitotic regulation, centrosome positioning and cell migration, nuclear envelope assembly/disassembly, and the DNA replication checkpoint. The broad implications of interactions between Wnt signaling proteins and Nups will be discussed in the context of cancer.


Assuntos
Neoplasias/metabolismo , Poro Nuclear/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Genes APC , Humanos , Neoplasias/patologia , Transporte Proteico , beta Catenina/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo
11.
Traffic ; 12(8): 983-99, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21496192

RESUMO

Nuclear localization of ß-catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase-3ß (GSK-3ß), causing nuclear accumulation of ß-catenin and transactivation of genes that transform cells. ß-catenin is a shuttling protein; however, the mechanism by which GSK-3ß regulates ß-catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the ß-catenin-green fluorescent protein dynamics in NIH 3T3 cells before and after GSK-3ß inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in ß-catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing ß-catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks ß-catenin phosphorylation by GSK-3ß, did not alter nuclear retention or transport, implying that GSK-3ß acts through an independent regulator. We compared five nuclear binding partners and identified LEF-1 as the key mediator of Wnt3a and LiCl-induced nuclear retention of ß-catenin. Thus, Wnt stimulation triggered a LEF-1 positive feedback loop to enhance the nuclear chromatin-retained pool of ß-catenin by 100-300%. These findings shed new light on regulation of ß-catenin nuclear dynamics.


Assuntos
Núcleo Celular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , beta Catenina/metabolismo , Animais , Núcleo Celular/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Cloreto de Lítio/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Mutação , Células NIH 3T3 , Fosforilação , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
12.
J Biol Chem ; 287(10): 7701-16, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22262852

RESUMO

BRCA1 is a DNA damage response protein and functions in the nucleus to stimulate DNA repair and at the centrosome to inhibit centrosome overduplication in response to DNA damage. The loss or mutation of BRCA1 causes centrosome amplification and abnormal mitotic spindle assembly in breast cancer cells. The BRCA1-BARD1 heterodimer binds and ubiquitinates γ-tubulin to inhibit centrosome amplification and promote microtubule nucleation; however regulation of BRCA1 targeting and function at the centrosome is poorly understood. Here we show that both N and C termini of BRCA1 are required for its centrosomal localization and that BRCA1 moves to the centrosome independently of BARD1 and γ-tubulin. Mutations in the C-terminal phosphoprotein-binding BRCT domain of BRCA1 prevented localization to centrosomes. Photobleaching experiments identified dynamic (60%) and immobilized (40%) pools of ectopic BRCA1 at the centrosome, and these are regulated by the nuclear export receptor CRM1 (chromosome region maintenance 1) and BARD1. CRM1 mediates nuclear export of BRCA1, and mutation of the export sequence blocked BRCA1 regulation of centrosome amplification in irradiated cells. CRM1 binds to undimerized BRCA1 and is displaced by BARD1. Photobleaching assays implicate CRM1 in driving undimerized BRCA1 to the centrosome and revealed that when BRCA1 subsequently binds to BARD1, it is less well retained at centrosomes, suggesting a mechanism to accelerate BRCA1 release after formation of the active heterodimer. Moreover, Aurora A binding and phosphorylation of BRCA1 enhanced its centrosomal retention and regulation of centrosome amplification. Thus, CRM1, BARD1 and Aurora A promote the targeting and function of BRCA1 at centrosomes.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Carioferinas/metabolismo , Sinais de Exportação Nuclear , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Aurora Quinases , Proteína BRCA1/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Dano ao DNA/genética , Feminino , Humanos , Carioferinas/genética , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Exportina 1
13.
J Biol Chem ; 287(2): 819-31, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22110128

RESUMO

ß-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. ß-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10-12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3-8 of ß-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of ß-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of ß-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10-12, and ß-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of ß-catenin to a greater extent than that of importin-ß. The Arm R10-12 sequence facilitated transport even when ß-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated ß-catenin transport through direct interaction with the NPC.


Assuntos
Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos , Animais , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Chaperonas Moleculares/genética , Células NIH 3T3 , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação/fisiologia , Ligação Proteica , Via de Sinalização Wnt/fisiologia , beta Catenina/genética
14.
IUBMB Life ; 64(3): 209-14, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22162224

RESUMO

Genetic mutations of adenomatous polyposis coli (APC) predispose to high risk of human colon cancer. APC is a large tumor suppressor protein and truncating mutations disrupt its normal roles in regulating cell migration, DNA replication/repair, mitosis, apoptosis, and turnover of oncogenic ß-catenin. APC is targeted to multiple subcellular sites, and here we discuss recent evidence implicating novel protein interactions and functions of APC in the nucleus and at centrosomes and mitochondria. The ability of APC to shuttle between these and other cell locations is hypothesized to be integral to its cellular function.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Mitocôndrias/metabolismo , Animais , Humanos
15.
J Biol Chem ; 285(24): 18144-54, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20378546

RESUMO

Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B'/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56alpha, -beta, and -epsilon isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56alpha subcellular localization. We detected B56alpha in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56alpha. Conversely, CRM1 overexpression shifted B56alpha to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451-469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56alpha. Active NESs were identified at similar positions in the cytoplasmic B56-beta and epsilon isoforms, but not in the nuclear-localized B56-delta or gamma isoforms. The transient expression of B56alpha induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56alpha co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56alpha-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56alpha can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.


Assuntos
Núcleo Celular/metabolismo , Centrossomo/metabolismo , Proteína Fosfatase 2/metabolismo , Células 3T3 , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Camundongos , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína
16.
Am J Pathol ; 175(2): 580-91, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19590041

RESUMO

Epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis, including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however, whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown, especially in the renal system. In this study, we show that transforming growth factor (TGF)-beta1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-catenin, the transcriptional induction of Slug, and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.


Assuntos
Caderinas/metabolismo , Desdiferenciação Celular , Epitélio/patologia , Túbulos Renais/patologia , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Mesoderma/patologia , Transporte Ativo do Núcleo Celular , Animais , Caderinas/genética , Linhagem Celular , Núcleo Celular/metabolismo , Fibrose , Ratos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima , beta Catenina/metabolismo
17.
Cancer Res ; 67(13): 6230-7, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17616680

RESUMO

Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/química , Proteínas Nucleares/genética , Motivos de Aminoácidos , Animais , Transformação Celular Neoplásica , Citoplasma/metabolismo , Análise Mutacional de DNA , Fibroblastos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Nucleofosmina , Plasmídeos/metabolismo , Estrutura Terciária de Proteína
18.
Biochem Biophys Rep ; 19: 100662, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31338436

RESUMO

Beta-catenin is well-known as a key effector of Wnt signalling and aberrant expression is associated with several human cancers. Stabilisation of and atypical subcellular localisation of beta-catenin, regulated in part through specific protein-protein interactions has been linked to cancer development, however the mechanisms behind these pathologies is yet to be fully elucidated. Affinity purification and mass spectrometry were used to identify potential ß-catenin interacting proteins in SW480 colon cancer cells. Recombinant ß-catenin constructs were used to co-isolate interacting proteins from stable isotope labelled cells followed by detection using mass spectrometry. Several known and new putative interactors were observed. In particular, we identified interaction with a set of coatomer complex I subunits implicated in retrograde transport at the Golgi, and confirmed endogenous interaction of ß-catenin with coatomer subunit COPB using immunoprecipitation assays and immunofluorescence microscopy. These observations suggest a hitherto unrecognised role for ß-catenin in the secretory pathway and warrant further functional studies to unravel its activity at this cellular location.

19.
Cancer Lett ; 263(2): 189-96, 2008 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-18243530

RESUMO

BRCA1 regulates gene transcription as part of its tumor suppressor function. Prior studies on BRCA1 transactivation did not account for the impact of its binding partner, BARD1. Here we tested the effect of BARD1 on BRCA1 transactivation of the p21 and Gadd45 promoters. We show that BARD1 promoted nuclear accumulation of BRCA1, but repressed BRCA1-mediated transactivation by up to 75% in transfected cells normalized for nuclear BRCA1 levels. The BRCA1 (C61G) RING mutant transactivation function was not regulated by BARD1. We propose that BARD1 reduces BRCA1 transcriptional activity, and that this at least partly involves BRCA1/BARD1 E3 ubiquitin ligase activity, which is disrupted by the C61G mutation.


Assuntos
Proteína BRCA1/farmacologia , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas Nucleares/genética , Ativação Transcricional/efeitos dos fármacos , Proteínas Supressoras de Tumor/farmacologia , Ubiquitina-Proteína Ligases/farmacologia , Proteína BRCA1/antagonistas & inibidores , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Regiões Promotoras Genéticas , Transfecção , Ubiquitina-Proteína Ligases/metabolismo
20.
Cell Signal ; 19(9): 1879-92, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17531442

RESUMO

BRCA1 is a tumor suppressor involved in the maintenance of genome integrity. BRCA1 co-localizes with DNA repair proteins at nuclear foci in response to DNA double-strand breaks caused by ionizing radiation (IR). The response of BRCA1 to agents that elicit DNA single-strand breaks (SSB) is poorly defined. In this study, we compared chemicals that induce SSB repair and observed the most striking nuclear redistribution of BRCA1 following treatment with the alkylating agent methyl methanethiosulfonate (MMTS). In MCF-7 breast cancer cells, MMTS induced movement of endogenous BRCA1 into distinctive nuclear foci that co-stained with the SSB repair protein XRCC1, but not the DSB repair protein gamma-H2AX. XRCC1 did not accumulate in foci after ionizing radiation. Moreover, we showed by deletion mapping that different sequences target BRCA1 to nuclear foci induced by MMTS or by ionizing radiation. We identified two core MMTS-responsive sequences in BRCA1: the N-terminal BARD1-binding domain (aa1-304) and the C-terminal sequence aa1078-1312. These sequences individually are ineffective, but together they facilitated BRCA1 localization at MMTS-induced foci. Site-directed mutagenesis of two SQ/TQ motif serines (S1143A and S1280A) in the BRCA1 fusion protein reduced, but did not abolish, targeting to MMTS-inducible foci. This is the first report to describe co-localization of BRCA1 with XRCC1 at SSB repair foci. Our results indicate that BRCA1 requires BARD1 for targeting to different types of DNA lesion, and that distinct C-terminal sequences mediate selective recruitment to sites of double- or single-strand DNA damage.


Assuntos
Alquilantes/farmacologia , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dano ao DNA , Metanossulfonato de Metila/análogos & derivados , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos da radiação , Quebras de DNA de Cadeia Simples/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Metanossulfonato de Metila/farmacologia , Dados de Sequência Molecular , Mutação/genética , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Radiação Ionizante , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
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