RESUMO
Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in â¼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM(SA/SA)) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM(SA/SA)-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM(SA/SA) mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.
Assuntos
Aneuploidia , Citocinese , Envelhecimento da Pele/patologia , Gordura Subcutânea/patologia , Tetraploidia , Vimentina/fisiologia , Animais , Western Blotting , Ciclo Celular , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/fisiologia , Fosforilação , Gordura Subcutânea/metabolismo , Proteína Supressora de Tumor p53/metabolismo , CicatrizaçãoRESUMO
Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM(SA/SA)) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM(WT/SA)) were indistinguishable from WT (VIM(WT/WT)) mice. In VIM(SA/SA) mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM(SA/SA), reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM(SA/SA), the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.
Assuntos
Aneuploidia , Catarata/etiologia , Catarata/metabolismo , Senescência Celular , Endoftalmite/etiologia , Endoftalmite/metabolismo , Células Epiteliais/patologia , Mitose , Vimentina/metabolismo , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Catarata/genética , Catarata/patologia , Núcleo Celular/patologia , Endoftalmite/genética , Endoftalmite/patologia , Células Epiteliais/metabolismo , Técnicas de Introdução de Genes , Cristalino/patologia , Camundongos , Dados de Sequência Molecular , Fosforilação , Vimentina/química , Vimentina/genéticaRESUMO
DNA-damaging strategies, such as radiotherapy and the majority of chemotherapeutic therapies, are the most frequently used non-surgical anti-cancer therapies for human cancers. These therapies activate DNA damage/replication checkpoints, which induce cell-cycle arrest to provide the time needed to repair DNA damage. Due to genetic defect(s) in the ATM (ataxia-telangiectasia mutated)-Chk2-p53 pathway, an ATR (ATM- and Rad3-related)-Chk1-Cdc25 route is the sole checkpoint pathway in a majority of cancer cells. Chk1 inhibitors are expected to selectively induce the mitotic cell death (mitotic catastrophe) of cancer cells. However, recent new findings have pointed out that Chk1 is essential for the maintenance of genome integrity even during unperturbed cell-cycle progression, which is controlled by a variety of protein kinases. These observations have raised concerns about a possible risk of Chk1 inhibitors on the clinics. In this review, we summarize recent advances in Chk1 regulation by phosphorylation, and discuss Chk1 as a molecular target for cancer therapeutics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fosfatases cdc25/metabolismo , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
LAP (leucine-rich repeats (LRR) and PSD-95/Dlg/ZO-1 (PDZ)) family proteins, including Scribble, LET-413, ERBIN, Densin-180 and Lano, are involved in the regulation of cell polarity. The LRR domains of LAP proteins were reported to mediate their basolateral membrane localization and to be essential for their function. To further dissect the mechanism of the plasma membrane localization of ERBIN, we introduced various mutants of ERBIN into cultured cells and observed the intracellular localization. When an LRR domain mutant lacking amino acid residues 1-32 at the amino (N) terminal region was over-expressed in cells, the mutant did not localize at the plasma membrane, but localized in the cytoplasm. We found that cysteines 14 and 16 at the N-terminal region of ERBIN are in vivo palmitoylated. Over-expressed mutants in which cysteine 14 and/or cysteine 16 were changed to serines did not localize at the plasma membrane, indicating that the palmitoylation of ERBIN is necessary for its plasma membrane localization. The over-expressed 1-196 amino acids fragment of ERBIN, which lacked the latter half of LRR, was palmitoylated but did not localize at the plasma membrane. These results suggest that both palmitoylation and LRR are required for the plasma membrane localization of ERBIN.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Lipoilação/fisiologia , Ácido Palmítico/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cães , Células HeLa , Humanos , Leucina/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , RatosRESUMO
Protein aggregate formation in muscle is thought to be pathogenic and associated with clinical weakness. Over-expression of either wild type or a mutant form of myeloid leukemia factor 1 (MLF1) in transgenic mouse skeletal muscle and in cultured cells resulted in aggregate formation. Aggregates were detected in MLF1 transgenic mice at 6 weeks of age, and increased in size with age. However, histological examination of skeletal muscles of MLF1 transgenic mice revealed no pathological changes other than the aggregates, and RotaRod testing did not detect functional deficits. MLF1 has recently been identified as a protein that could neutralize the toxicity of intracellular protein aggregates in a Drosophila model of Huntington's disease (HD). We also demonstrate that MLF1 interacts with MRJ, a heat shock protein, which can independently neutralize the toxicity of intracellular protein aggregates in the Drosophila HD model. Our data suggest that over-expression of MLF1 has no significant impact on skeletal muscle function in mice; that progressive formation of protein aggregates in muscle are not necessarily pathogenic; and that MLF1 and MRJ may function together to ameliorate the toxic effects of polyglutamine or mutant proteins in myodegenerative diseases such as inclusion body myositis and oculopharyngeal muscular dystrophy, as well as neurodegenerative disease.
Assuntos
Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Citoproteção/genética , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Corpos de Inclusão/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Chaperonas Moleculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Debilidade Muscular/genética , Debilidade Muscular/metabolismo , Debilidade Muscular/fisiopatologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/antagonistas & inibidores , Peptídeos/metabolismoRESUMO
Primary cilia, microtubule-based sensory structures, orchestrate various critical signals during development and tissue homeostasis. In view of the rising interest into the reciprocal link between ciliogenesis and cell cycle, we discuss here several recent advances to understand the molecular link between the individual step of ciliogenesis and cell cycle control. At the onset of ciliogenesis (the transition from centrosome to basal body), distal appendage proteins have been established as components indispensable for the docking of vesicles at the mother centriole. In the initial step of axonemal extension, CP110, Ofd1, and trichoplein, key negative regulators of ciliogenesis, are found to be removed by a kinase-dependent mechanism, autophagy, and ubiquitin-proteasome system, respectively. Of note, their disposal functions as a restriction point to decide that the axonemal nucleation and extension begin. In the elongation step, Nde1, a negative regulator of ciliary length, is revealed to be ubiquitylated and degraded by CDK5-SCF(Fbw7) in a cell cycle-dependent manner. With regard to ciliary length control, it has been uncovered in flagellar shortening of Chlamydomonas that cilia itself transmit a ciliary length signal to cytoplasm. At the ciliary resorption step upon cell cycle re-entry, cilia are found to be disassembled not only by Aurora A-HDAC6 pathway but also by Nek2-Kif24 and Plk1-Kif2A pathways through their microtubule-depolymerizing activity. On the other hand, it is becoming evident that the presence of primary cilia itself functions as a structural checkpoint for cell cycle re-entry. These data suggest that ciliogenesis and cell cycle intimately link each other, and further elucidation of these mechanisms will contribute to understanding the pathology of cilia-related disease including cancer and discovering targets of therapeutic interventions.
RESUMO
Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Proteínas de Drosophila , Genes Supressores de Tumor , Mitose , Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático , Sequência de Aminoácidos , Western Blotting , Cromatografia em Gel , Células HeLa , Humanos , Microscopia de Fluorescência , Fosforilação , Testes de Precipitina , Proteínas Serina-Treonina Quinases/imunologia , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1-Thr210 phosphorylation. Plk1-Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.
Assuntos
Proteínas 14-3-3/metabolismo , Anáfase , Proteínas de Ciclo Celular/metabolismo , Metáfase , Fosfatidilinositol 3-Quinases/metabolismo , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Biocatálise , Caenorhabditis elegans , Drosophila melanogaster , Ativação Enzimática , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Modelos Biológicos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinase 1 Polo-LikeRESUMO
The primary cilium is an antenna-like organelle that modulates differentiation, sensory functions, and signal transduction. After cilia are disassembled at the G0/G1 transition, formation of cilia is strictly inhibited in proliferating cells. However, the mechanisms of this inhibition are unknown. In this paper, we show that trichoplein disappeared from the basal body in quiescent cells, whereas it localized to mother and daughter centrioles in proliferating cells. Exogenous expression of trichoplein inhibited primary cilia assembly in serum-starved cells, whereas ribonucleic acid interference-mediated depletion induced primary cilia assembly upon cultivation with serum. Trichoplein controlled Aurora A (AurA) activation at the centrioles predominantly in G1 phase. In vitro analyses confirmed that trichoplein bound and activated AurA directly. Using trichoplein mutants, we demonstrate that the suppression of primary cilia assembly by trichoplein required its ability not only to localize to centrioles but also to bind and activate AurA. Trichoplein or AurA knockdown also induced G0/G1 arrest, but this phenotype was reversed when cilia formation was prevented by simultaneous knockdown of IFT-20. These data suggest that the trichoplein-AurA pathway is required for G1 progression through a key role in the continuous suppression of primary cilia assembly.
Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Centríolos/metabolismo , Cílios/fisiologia , Fase G1/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinases , Western Blotting , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Diferenciação Celular , Células Cultivadas , Imunofluorescência , Humanos , Imunoprecipitação , Microtúbulos/metabolismo , Morfogênese , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transdução de SinaisAssuntos
Anticorpos Fosfo-Específicos , Engenharia de Proteínas , Animais , Aurora Quinases , Técnicas Citológicas , Desenho de Fármacos , Proteína Glial Fibrilar Ácida , Humanos , Filamentos Intermediários , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Engenharia de Proteínas/métodos , Proteínas Quinases/análise , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Coloração e Rotulagem , Quinases Associadas a rhoRESUMO
The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell-cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/fisiologia , Polaridade Celular/fisiologia , Células Epiteliais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Estruturas Animais/metabolismo , Animais , Canalículos Biliares/metabolismo , Sítios de Ligação , Caderinas/metabolismo , Proteínas de Transporte/genética , Moléculas de Adesão Celular/metabolismo , Agregação Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Desmogleína 2/metabolismo , Células Epiteliais/citologia , Humanos , Junções Intercelulares/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Ligação Proteica , Interferência de RNA , Proteína da Zônula de Oclusão-1RESUMO
We present a 60-year-old woman with chest pain preceded by emotional stress. Trans-thoracic echocardiography demonstrated localized left ventricular hypokinesis around the apical area. Multi-detector row computed tomography (MDCT) revealed no significant stenosis in the coronary arteries, which comfirmed Takotsubo-like cardiomyopathy. We show here the usefulness of MDCT for the noninvasive differentiation of Takotsubo cardiomyopathy from acute coronary syndrome.
Assuntos
Cardiomiopatia de Takotsubo/diagnóstico por imagem , Eletrocardiografia , Feminino , Humanos , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X/métodosRESUMO
BACKGROUND: The ability to evaluate coronary stenosis using multi-detector computed tomography (MDCT) has been well discussed. In contrast, several studies demonstrated that the plaque burden measured by intravascular ultrasound (IVUS) has a relationship to the risk of cardiovascular events. the accuracy of MDCT was studied to determine plaque and vessel size compared with IVUS. METHODS AND RESULTS: Fifty-six proximal lesions (American College of Cardiology/American Heart Association classification: segment 1, 5, 6) from 33 patients were assessed using MDCT and IVUS. The plaque and vessel area were measured from the cross-sectional image using both MDCT and IVUS. Eight coronary artery lesions with motion artifacts and heavily calcified plaques were excluded from the analysis. The vessel and lumen size evaluated using MDCT were closely correlated with those evaluated by IVUS (R(2)=0.614, 0.750 respectively). Furthermore, there was a strong correlation between percentage plaque area assessed by MDCT and IVUS (R(2)=0.824). CONCLUSION: MDCT can noninvasively quantify coronary atherosclerotic plaque with good correlation compared with IVUS in patients with atherosclerosis.
Assuntos
Doença da Artéria Coronariana/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Idoso , Vasos Coronários/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Ultrassonografia de IntervençãoRESUMO
Intermediate filaments (IF) form the structural framework of the cytoskeleton. Although histopathological detection of IF proteins is utilized for examining cancer specimens as reliable markers, the molecular mechanisms by which IF are involved in the biology of cancer cells are still unclear. We found that site-specific phosphorylation of IF proteins induces the disassembly of filament structures. To further dissect the in vivo spatiotemporal dynamics of IF phosphorylation, we developed site- and phosphorylation state-specific antibodies. Using these antibodies, we detected kinase activities that specifically phosphorylate type III IF, including vimentin, glial fibrillary acidic protein and desmin, during mitosis. Cdk1 phosphorylates vimentin-Ser55 from prometaphase to metaphase, leading to the recruitment of Polo-like kinase 1 (Plk1) to vimentin. Upon binding to Phospho-Ser55 of vimentin, Plk1 is activated, and then phosphorylates vimentin-Ser82. During cytokinesis, Rho-kinase and Aurora-B specifically phosphorylate IF at the cleavage furrow. IF phosphorylation by Cdk1, Plk1, Rho-kinase and Aurora-B plays an important role in the local IF breakdown, and is essential for the efficient segregation of IF networks into daughter cells. As another part of our research on IF, we have set out to find the binding partners with simple epithelial keratin 8/18. We identified tumor necrosis factor receptor type 1-associated death domain protein (TRADD) as a keratin 18-binding protein. Together with data from other laboratories, it is proposed that simple epithelial keratins may play a role in modulating the response to some apoptotic signals. Elucidation of the precise molecular functions of IF is expected to improve our understanding of tumor development, invasion and metastasis.
Assuntos
Anticorpos , Transformação Celular Neoplásica , Filamentos Intermediários/química , Filamentos Intermediários/fisiologia , Animais , Humanos , Filamentos Intermediários/imunologia , Queratinas/fisiologia , Mitose/fisiologia , FosforilaçãoRESUMO
In astrocytes, the PGF(2alpha) or ionomycin treatment induces the phosphorylation at Ser38 and Ser82 of vimentin, a type III intermediate filament, by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We found here that vimentin phospho-Ser82 was dephosphorylated much slower than phospho-Ser38. Vimentin phospho-Ser38 was dephosphorylated quickly by purified PP1 catalytic subunit (PP1c) in vitro, whereas phospho-Ser82 was insensitive to PP1c. Because PP1c directly bound to vimentin through a VxF motif (Val83-Asp84-Phe85), the PP1c active site appeared to be unable to approach phospho-Ser82, leading to the prolongation of the phosphorylation at Ser-82. In astrocytes, PP1calpha was in vivo associated with vimentin filaments. The repetitive treatment by ionomycin at a short interval resulted in the sustained elevation of Ser82 phosphorylation, leading to the marked disassembly of vimentin filaments. Taken together, these results suggest that vimentin is a novel member of binding partner of PP1c in astrocytes, and vimentin-Ser82 may act as a memory phosphorylation site.
Assuntos
Astrócitos/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Serina/metabolismo , Vimentina/metabolismo , Motivos de Aminoácidos , Animais , Astrócitos/citologia , Sítios de Ligação , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Imuno-Histoquímica , Ionomicina/metabolismo , Ionomicina/farmacologia , Modelos Biológicos , Fosforilação , Proteína Fosfatase 1 , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, (125) I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.
Assuntos
Adesinas Bacterianas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Yersinia pseudotuberculosis/genética , Adesinas Bacterianas/genética , Western Blotting , Citoplasma/química , Vesículas Citoplasmáticas/química , Imunoprecipitação , Isótopos de Iodo , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/genéticaRESUMO
Cytokinesis is regulated by several protein kinases, such as Aurora-B and Rho-kinase/ROCK. We have indicated that these two kinases are the cleavage furrow (CF) kinases that accumulate at the cleavage furrow and phosphorylate several intermediate filament (IF) proteins into two daughter cells. It has been reported that Aurora-B phosphorylates MgcRacGAP to functionally convert to a RhoGAP during cytokinesis. Therefore, we investigated here the relationship between Aurora-B and Rho-kinase/ROCK in cytokinesis, by using small interfering RNA (siRNA) technique. Aurora-B depletion did not alter the cleavage furrow-specific localization of Rho-kinase/ROCK and vice versa. Treatment of Aurora-B or Rho-kinase/ROCK siRNA increased multinucleate cells, and the effect of double depletion was additive. Aurora-B depletion induced the reduction of cleavage furrow-specific phosphorylation of vimentin at Ser72 but not vimentin at Ser71, myosin light chain (MLC) at Ser19, and myosin binding subunit of myosin phosphatase (MBS) at Ser852. In contrast, Rho-kinase/ROCK depletion led to the reduction of cleavage furrow-specific phosphorylation of MLC at Ser19, MBS at Ser852, and vimentin at Ser71 but not vimentin at Ser72. Cleavage furrow-specific ezrin/radixin/moesin (ERM) phosphorylation was not altered in the Aurora-B- and/or Rho-kinase/ROCK-depleted cells. In addition, C3 or toxin B treatment did not abolish ERM phosphorylation at the cleavage furrow in cells attaining cytokinesis. These results suggest that Aurora-B and Rho-kinase/ROCK regulate the progression of cytokinesis without communicating to each other, and there may exist a novel protein kinase which phosphorylates ERM at the cleavage furrow.
Assuntos
Proteínas Sanguíneas/metabolismo , Citocinese/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Aurora Quinase B , Aurora Quinases , Toxinas Bacterianas/farmacologia , Complemento C3/farmacologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Vimentina/genética , Vimentina/metabolismo , Quinases Associadas a rhoRESUMO
PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.
Assuntos
Movimento Celular/fisiologia , Endocitose/fisiologia , Integrinas/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C-épsilon/metabolismo , Vimentina/metabolismo , Animais , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Humanos , Integrinas/genética , Camundongos , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Vimentina/genéticaRESUMO
Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.
Assuntos
Proteínas de Transporte/fisiologia , Filamentos Intermediários/metabolismo , Queratinas/química , Queratinas/metabolismo , Sequência de Aminoácidos , Animais , Células CACO-2 , Proteínas de Transporte/química , Linhagem Celular Tumoral , Clonagem Molecular , Proteínas do Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Desmoplaquinas , Desmossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Filamentos Intermediários/química , Queratina-18 , Queratina-8 , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Microtúbulos/química , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Peptídeos/química , Plectina , Reação em Cadeia da Polimerase , Ligação Proteica , Precursores de Proteínas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Transfecção , Tubulina (Proteína)/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. delta-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of delta-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with delta-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with delta-catenin/NPRAP at synapses in delta-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the delta-catenin/NPRAP-N-cadherin complex.