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1.
EMBO J ; 31(4): 817-28, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22258620

RESUMO

Development of nervous tissue is a coordinated process of neural progenitor cell (NPC) proliferation and neuronal differentiation. Intracellular signalling events that regulate the balance between NPC proliferation and neuronal differentiation, therefore, determine the size and composition of nervous tissues. Here, we demonstrate that negative regulation of phosphoinosite 3-kinase (PI3K)-Akt signalling by phosphatase tensin homologue (Pten) is essential for maintaining NPC population in mouse retina. We found that mouse retinal progenitor cells (RPCs) lacking the Pten gene complete neurogenesis earlier than their normal developmental schedule, resulting in their premature depletion in the mature retina. We further discover that Notch intracellular domain (NICD) fails to form transcription activator complex in Pten-deficient RPCs, and thereby unable to support RPC maintenance. Taken together, our results suggest that Pten plays a pivotal role in retinal neurogenesis by supporting Notch-driven RPC maintenance against neurogenic PI3K-Akt signalling.


Assuntos
Neurogênese/fisiologia , Neurônios/citologia , PTEN Fosfo-Hidrolase/fisiologia , Receptores Notch/metabolismo , Retina/citologia , Transdução de Sinais , Animais , Linhagem Celular , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica
2.
J Biol Chem ; 287(31): 25954-63, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22692211

RESUMO

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Psoríase/metabolismo , Actinas/metabolismo , Estudos de Casos e Controles , Hipóxia Celular , Movimento Celular , Proliferação de Células , Regulação para Baixo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP40/genética , Células HeLa , Proteínas de Choque Térmico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Chaperonas Moleculares , Fosforilação , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Psoríase/patologia , Transdução de Sinais , Pele/metabolismo , Pele/patologia
3.
J Cell Biochem ; 112(9): 2566-73, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21590709

RESUMO

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.


Assuntos
Alcalose/fisiopatologia , Autofagia , Proteínas/metabolismo , Estresse Fisiológico , Alcalose/metabolismo , Tamanho Celular , Regulação para Baixo , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Mutação de Sentido Incorreto , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagossomos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR
4.
Biochem Biophys Res Commun ; 404(2): 672-7, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21147067

RESUMO

Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.


Assuntos
Autofagia , Corpos de Inclusão/metabolismo , Lisossomos , Inibidores de Proteassoma , Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Corpos de Inclusão/genética , Doenças Neurodegenerativas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
5.
Biochem Biophys Res Commun ; 404(2): 728-34, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21163252

RESUMO

Liver cancer development follows a multistep process that includes epigenetic changes beginning at the initiation stage, changes that have been studied for their potential diagnostic value. Here, we examined long-term, cancer-associated epigenetic changes during carcinogenesis using a mouse model of liver cancer. WW45-haploinsufficient (WW45(+/-)) mice developed liver cancer after 12 months due to dysregulation of the Hippo pathway and consequent Yap overexpression. There was no pathological sign of neoplastic regions in the livers of 10-month-old WW45(+/-) mice but whole-gene expression patterns statistically proved the resemblance between 10-month-old livers and hepatomas from WW45(+/-) mice. We found epigenetic features in the livers of 10-month-old WW45(+/-) mice which were already distinctive from the wild-type counterparts prior to tumorigenesises. H19 ICR showed loss-of-imprinting in two steps and allelic histone marker signature during tumorigenesis showed similarity with ES cells. Progressive cancer pathognomonic global hypomethylation was a characteristic post-10-month feature and was well reflected in retrotransposons. Heterochromatic histone modifications also decreased in retrotransposons after 10 months in the liver of WW45(+/-) mice. This study showed potential epigenetic features for cancer prognostic use and supported the epigenetic progenitor model of cancer.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Impressão Genômica , Haploinsuficiência , Neoplasias Hepáticas/genética , Retroelementos , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Histonas/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Mutantes
6.
Mol Cell Biol ; 27(15): 5565-74, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17526737

RESUMO

Integrin-linked kinase (ILK) is a scaffold and protein kinase that acts as a pivotal effector in integrin signaling for various cellular functions. In this study, we found that ILK remarkably reduced the protein stability of Notch1 through Fbw7. The kinase activity of ILK was essential for the inhibition of Notch1 signaling. Notably, the protein level and transcriptional activity of the endogenous Notch1 intracellular domain (Notch1-IC) were higher in ILK-null cells than in ILK wild-type cells, and the level of endogenous Notch1-IC was increased by the blocking of the proteasome, suggesting that ILK enhances the proteasomal degradation of Notch1-IC. ILK directly bound and phosphorylated Notch1-IC, thereby facilitating proteasomal protein degradation through Fbw7. Furthermore, we found down-regulation of Notch1-IC and up-regulation of ILK in basal cell carcinoma and melanoma patients but not in squamous cell carcinoma patients. These results suggest that ILK down-regulated the protein stability of Notch1-IC through the ubiquitin-proteasome pathway by means of Fbw7.


Assuntos
Regulação para Baixo/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Notch1/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Animais , Carcinoma Basocelular/enzimologia , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Núcleo Celular/enzimologia , Humanos , Melanoma/enzimologia , Camundongos , Células NIH 3T3 , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Receptor Notch1/genética , Serina/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Termodinâmica , Transcrição Gênica , Regulação para Cima/genética
7.
Cell Signal ; 20(7): 1368-74, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18450422

RESUMO

We have investigated the effect of Akt on estrogen receptor (ER) alpha protein level and its transcriptional activity. Transient transfection studies revealed that constitutively active Akt1 up-regulated ERalpha at the post-transcriptional level. Studies using Akt inhibitor and dominant-negative Akt1 showed that Akt1 kinase activity is required for the up-regulation of ERalpha. Cycloheximide decay assays and studies with proteasome inhibitor indicated that Akt1-mediated up-regulation of ERalpha was maintained by inhibiting proteasome-mediated degradation of ERalpha. When Akt consensus phosphorylation site mutant, ERalphaS167A was tested for Akt1-mediated up-regulation, increase of ERalphaS167A by Akt1 was significantly impaired as compared to wild type ERalpha. In addition, dominant-negative glycogen synthase kinase (GSK) 3beta and LiCl could also partially up-regulate ERalpha protein level, suggesting that concerted action of Akt1-mediated phosphorylation on S167 and kinase activity of Akt-downstream GSK3beta could affect ERalpha protein level. Paradoxically, co-expression of Akt1 could down-regulate transcriptional activity of ERalpha. The inhibitory effect of Akt1 on ERalpha transcriptional activity was not attributable to changes in subcellular distribution of ERalpha. Transfection studies using increasing amount of Akt1 and ERalpha indicated that the transcriptional activity of ERalpha was negatively regulated by ERalpha protein quantities at higher ERalpha concentrations. Chromatin immunoprecipitation assays revealed that at Akt1 concentration high enough to induce up-regulation of ERalpha, association of ERalpha to promoter region of ERalpha target pS2 gene was impaired. Taken together, these data suggest that Akt1 could increase ERalpha protein level with simultaneous reduction in its transcriptional activity, possibly by modulating association of ERalpha to the target gene promoters.


Assuntos
Receptor alfa de Estrogênio/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Linhagem Celular , Ativação Enzimática , Receptor alfa de Estrogênio/química , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfosserina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Frações Subcelulares/metabolismo , Termodinâmica , Regulação para Cima
8.
J Mol Biol ; 368(2): 388-97, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17362989

RESUMO

We propose a biochemical mechanism by which Daxx modulates NF-kappaB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-kappaB in HeLa cells. Overexpression of Daxx repressed the expression of NF-kappaB-regulated genes such as I kappa B alpha and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-kappaB stimulated by TNFalpha. Here, we suggest that Daxx-mediated repression of NF-kappaB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-kappaB transcriptional activity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Ativação Transcricional/genética , Acetilação , Núcleo Celular/metabolismo , Proteínas Correpressoras , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares , Fosforilação , Ligação Proteica , Transporte Proteico , Frações Subcelulares/metabolismo , Fator de Transcrição RelA/metabolismo
9.
Cell Signal ; 19(11): 2379-87, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17728103

RESUMO

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.


Assuntos
Proteínas 14-3-3/metabolismo , Actinas/química , Actinas/metabolismo , Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP27 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Modelos Biológicos , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
10.
Nat Commun ; 9(1): 2502, 2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29950673

RESUMO

The numbers and types of cells constituting vertebrate neural tissues are determined by cellular mechanisms that couple neurogenesis to the proliferation of neural progenitor cells. Here we identified a role of mammalian target of rapamycin complex 1 (mTORC1) in the development of neural tissue, showing that it accelerates progenitor cell cycle progression and neurogenesis in mTORC1-hyperactive tuberous sclerosis complex 1 (Tsc1)-deficient mouse retina. We also show that concomitant loss of immunoproteasome subunit Psmb9, which is induced by Stat1 (signal transducer and activator of transcription factor 1), decelerates cell cycle progression of Tsc1-deficient mouse retinal progenitor cells and normalizes retinal developmental schedule. Collectively, our results establish a developmental role for mTORC1, showing that it promotes neural development through activation of protein turnover via a mechanism involving the immunoproteasome.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neurogênese/fisiologia , Retina/crescimento & desenvolvimento , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Cisteína Endopeptidases/metabolismo , Embrião de Mamíferos , Feminino , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Retina/citologia , Retina/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/fisiologia , Proteína 1 do Complexo Esclerose Tuberosa/genética
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