Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Filtros adicionais











Intervalo de ano
1.
Am J Hum Genet ; 102(3): 468-479, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29429572

RESUMO

Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity.

2.
J Immunol ; 195(10): 4922-32, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26432893

RESUMO

TRPMLs (or mucolipins) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the TRPML family includes three members: TRPML1-3. Although TRPML1 and TRPML3 have been well characterized, the cellular function of TRPML2 has remained elusive. To address TRPML2 function in a physiologically relevant cell type, we first analyzed TRPML2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of TRPML2 at the transcriptional level. Although TRPML2 expression was negligible in resting macrophages, TRPML2 mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, TRPML1 and TRPML3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous TRPML2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with TRPML1 and TRPML3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of TRPML2, we generated a TRPML2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in TRPML2-knockout mice. Furthermore, TRPML2-knockout mice displayed impaired recruitment of peripheral macrophages in response to i.p. injections of LPS or live bacteria, suggesting a potential defect in the immune response. Overall, our study reveals interesting differences in the regulation and distribution of the members of the TRPML family and identifies a novel role for TRPML2 in the innate immune response.


Assuntos
Quimiocina CCL2/imunologia , Imunidade Inata/fisiologia , Macrófagos Peritoneais/imunologia , Receptores Toll-Like/imunologia , Canais de Receptores Transientes de Potencial/imunologia , Animais , Quimiocina CCL2/genética , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Receptores Toll-Like/genética , Canais de Receptores Transientes de Potencial/genética
3.
Elife ; 42015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26247711

RESUMO

Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart.


Assuntos
Diferenciação Celular , Proliferação de Células , Centrossomo/metabolismo , Coração/embriologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Animais , Ratos , Salamandridae , Peixe-Zebra
4.
Biochim Biophys Acta ; 1843(11): 2556-62, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25014164

RESUMO

Apoptosis, or programmed cell death, is an essential physiological process for proper embryogenesis as well as for homeostasis during aging. In addition, apoptosis is one of the major mechanisms causing cell loss in pathophysiological conditions such as heart failure. Thus, inhibition of apoptosis is an important approach for preventive and therapeutic strategies. Here we show that the histone 3 lysine 4- and lysine 36-specific methyltransferase Smyd2 acts as an endogenous antagonistic player of p53-dependent cardiomyocyte apoptosis. Smyd2 protein levels were significantly decreased in cardiomyocytes upon cobalt chloride-induced apoptosis or myocardial infarction, while p53 expression was enhanced. siRNA-mediated knockdown of Smyd2 in cultured cardiomyocytes further enhanced cobalt chloride-induced cardiomyocyte apoptosis. In contrast, Smyd2 overexpression resulted in marked methylation of p53 and prevented its accumulation as well as apoptotic cell death in an Hsp90-independent manner. Moreover, overexpression, of Smyd2, but not Smyd2Y240F lacking a methyl transferase activity, significantly rescued CoCl2-induced apoptosis in H9c2 cardioblasts. Finally, Smyd2 cardiomyocyte-specific deletion in vivo promoted apoptotic cell death upon myocardial infarction, which correlated with enhanced expression of p53 and pro-apoptotic Bax. Collectively, our data indicate Smyd2 as a cardioprotective protein by methylating p53.

5.
EMBO J ; 33(2): 129-45, 2014 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-24421325

RESUMO

Establishing the correct orientation of the mitotic spindle is an essential step in epithelial cell division in order to ensure that epithelial tubules form correctly during organ development and regeneration. While recent findings have identified some of the molecular mechanisms that underlie spindle orientation, many aspects of this process remain poorly understood. Here, we have used the 3D-MDCK model system to demonstrate a key role for a newly identified protein complex formed by IQGAP1 and the epithelial growth factor receptor (EGFR) in controlling the orientation of the mitotic spindle. IQGAP1 is a scaffolding protein that regulates many cellular pathways, from cell-cell adhesion to microtubule organization, and its localization in the basolateral membrane ensures correct spindle orientation. Through its IQ motifs, IQGAP1 binds to EGFR, which is responsible for maintaining IQGAP1 in the basolateral membrane domain. Silencing IQGAP1, or disrupting the basolateral localization of either IQGAP1 or EGFR, results in a non-polarized distribution of NuMA, mitotic spindle misorientation and defects in single lumen formation.


Assuntos
Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Epitélio/crescimento & desenvolvimento , Receptores ErbB/metabolismo , Morfogênese/genética , Fuso Acromático/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Células Cultivadas , Cães , Epitélio/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Domínios e Motivos de Interação entre Proteínas , Distribuição Tecidual , Proteínas Ativadoras de ras GTPase/química
6.
J Cell Physiol ; 228(9): 1907-21, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23460338

RESUMO

We report here that C6orf89, which encodes a protein that interacts with bombesin receptor subtype-3 and accelerates cell cycle progression and wound repair in human bronchial epithelial cells (Liu et al., 2011, PLoS ONE 6: e23072), encodes one soluble and two type II membrane proteins that function as histone deacetylases (HDAC) enhancers. Soluble 34/64sp is selectively targeted to the nucleolus and is retained in nucleolar organiser regions (NORs) in mitotic cells. Nucleolar 34/64sp is integrated into the ribosomal gene transcription machinery, colocalises and coimmunoprecipitates with the Pol I transcription factor UBF, and undergoes a dramatic relocalisation to the nucleolus upon the arrest of rDNA transcription, protein synthesis and PI3K/mTORC2 signalling. Membrane 42/116mp localises to the Golgi and the midbody, and its controlled ectopic expression provokes the disruption of the Golgi cisternae and hinders the separation of daughter cells and the completion of mitosis. The latter effect is also produced by the microinjection of an affinity-purified amfion antibody. The identification of C60rf89 as a gene that encodes three distinct proteins with the capacity to enhance the activity of histone deacetylases (HDACs) in the nucleolus, the Golgi and the midbody provides new information regarding the components of the acetylome and their capacity to interact with different functional groups in the cell.


Assuntos
Núcleo Celular/metabolismo , Complexo de Golgi/metabolismo , Região Organizadora do Nucléolo/metabolismo , Proteínas/genética , Ciclo Celular/genética , Núcleo Celular/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Complexo de Golgi/genética , Células HeLa , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Mitose/genética , Região Organizadora do Nucléolo/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas/metabolismo , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismo
7.
Nat Cell Biol ; 14(8): 838-49, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22820376

RESUMO

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.


Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Sinaptotagminas/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Imunofluorescência , Humanos , Análise em Microsséries , Microscopia Confocal , Reação em Cadeia da Polimerase
8.
J Cell Sci ; 124(Pt 3): 459-68, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21224396

RESUMO

Loss-of-function mutations in mucolipin 1 (MCOLN1) result in mucolipidosis type IV (MLIV), a lysosomal storage disorder characterized by severe mental and psychomotor retardation. MCOLN1 is a lysosomal ion channel that belongs to the transient receptor potential (TRP) superfamily. To better understand the cellular function of MCOLN1, a split-ubiquitin yeast two-hybrid screen was performed with the purpose of revealing new MCOLN1 interaction partners. The screen identified two members of the lysosome-associated protein transmembrane (LAPTM) family as novel interaction partners of MCOLN1. The binding between MCOLN1 and LAPTM members (LAPTMs) was confirmed by co-immunoprecipitation and yeast two-hybrid assays. In addition, MCOLN1 and LAPTMs extensively colocalize at late endosomes and lysosomes. Overexpression of LAPTM4b caused enlargement of lysosomes and defective lysosomal degradation, indicating that LAPTMs are important for proper lysosomal function. Interestingly, lysosomal swelling induced by LAPTM4b was rescued by expression of MCOLN1, suggesting a functional connection between the two proteins. Finally, depletion of endogenous LAPTMs by siRNA induced accumulation of concentric multi-lamellar structures and electron-dense inclusions that closely resemble the structures found in MLIV cells. Overall, our data provide new insight into the molecular mechanisms of MCOLN1 function and suggest a potential role for LAPTMs in MLIV pathogenesis.


Assuntos
Proteínas de Membrana/fisiologia , Mucolipidoses/fisiopatologia , Proteínas Oncogênicas/fisiologia , Canais de Cátion TRPM/fisiologia , Endossomos/metabolismo , Células Epiteliais , Células HeLa , Humanos , Imunoprecipitação , Lisossomos/metabolismo , Lisossomos/patologia , Lisossomos/ultraestrutura , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Ligação Proteica , Canais de Cátion TRPM/genética , Canais de Receptores Transientes de Potencial , Técnicas do Sistema de Duplo-Híbrido
9.
J Cell Biol ; 189(4): 725-38, 2010 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-20479469

RESUMO

Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin-Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects.


Assuntos
Células Epiteliais/ultraestrutura , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fuso Acromático/ultraestrutura , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Ciclo Celular , Células Cultivadas , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Morfogênese/fisiologia , Interferência de RNA , Transdução de Sinais , Fuso Acromático/metabolismo , Tubulina (Proteína)/análise
10.
J Biol Chem ; 284(52): 36357-66, 2009 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19864416

RESUMO

Loss of function mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmological abnormalities. MCOLN1 is an ion channel that regulates membrane transport along the endolysosomal pathway. It has been suggested that MCOLN1 participates in several Ca(2+)-dependent processes, including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Here, we searched for proteins that interact with MCOLN1 in a Ca(2+)-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca(2+), and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37 and 49. We further show that MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by overexpression of an ATPase-defective dominant-negative form of Vps4B (Vps4B(E235Q)). In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that overexpression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosome markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. Overall, our data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca(2+) sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Substituição de Aminoácidos , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Fusão de Membrana/genética , Mucolipidoses/genética , Mucolipidoses/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica/genética , Canais de Cátion TRPM/genética , Canais de Receptores Transientes de Potencial
11.
J Cell Physiol ; 218(2): 416-26, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18932217

RESUMO

We have previously reported the physical interaction between Daxx, the adaptor protein that mediates activation of the Jun amino-terminal kinase (JNK), and GLUT4, the insulin-dependent glucose transporter, interaction that involves their C-domains. Co-immunoprecipitation and two-hybrid-based protein-protein interaction studies show now that Daxx and GLUT4 interact with JNK1 through D-sites in their NH(2)-(aa 1-501) and large endofacial loop, respectively. Serum deprivation strongly enhances the association of JNK1 with Daxx and dissociates the kinase from GLUT4. SP600125, a potent JNK1 inhibitor, reduces the JNK1 activity associated with GLUT4 and the phosphorylation of two minor GLUT4 species in serum-starved 3T3-L1 adipocytes. In addition, Daxx interacts with kinesin KIF5B through the 6xTPR domain of the kinesin light chain, a domain engaged in the grab hold of protein cargo by kinesin motors that codistribute with JNK. Depletion of Daxx in 3T3-L1 adipocytes provokes the partial translocation of the GLUT4 retained in the GLUT4 storage compartment to endosomes.


Assuntos
Proteínas de Transporte/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesina/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Animais , Antracenos/farmacologia , Proteínas de Transporte/química , Transportador de Glucose Tipo 4/química , Humanos , Imunoprecipitação , Proteínas Substratos do Receptor de Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/química , Proteínas Nucleares/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Soro
12.
Autophagy ; 4(6): 832-4, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18635948

RESUMO

Mucolipidosis IV (MLIV) is a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. In contrast with most lysosomal storage disorders, which are attributed to the absence of specific lysosomal hydrolases, accumulation of material in MLIV results from defects in membrane transport along the late endocytic pathway. Mutations in MCOLN1 are the cause of MLIV; however, how the lack of MCOLN1 function ultimately leads to neurodegeneration remains largely unknown. We found that MCOLN1 is required for efficient fusion of both late endosomes and autophagosomes with lysosomes. Impaired autophagosome degradation results in accumulation of autophagosomes in MLIV fibroblasts. In addition, we found increased levels and aggregation of p62, suggesting that abnormal accumulation of ubiquitinated protein inclusions may contribute to the neurodegenerative phenotype observed in MLIV patients. These findings corroborate recent evidence indicating that defects in autophagy may be a common feature of many neurodegenerative disorders.


Assuntos
Autofagia/fisiologia , Lisossomos/metabolismo , Mucolipidoses , Endossomos/metabolismo , Humanos , Mucolipidoses/genética , Mucolipidoses/metabolismo , Mucolipidoses/fisiopatologia , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Canais de Receptores Transientes de Potencial
13.
Hum Mol Genet ; 17(17): 2723-37, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18550655

RESUMO

Mutations in Mucolipin 1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a lysosomal storage disease characterized by several neurological and ophthalmological abnormalities. It has been proposed that MCOLN1 might regulate transport of membrane components in the late endosomal-lysosomal pathway; however, the mechanisms by which defects of MCOLN1 function result in mental and psychomotor retardation remain largely unknown. In this study, we show constitutive activation of autophagy in fibroblasts obtained from MLIV patients. Accumulation of autophagosomes in MLIV cells was due to the increased de novo autophagosome formation and to delayed fusion of autophagosomes with late endosomes/lysosomes. Impairment of the autophagic pathway led to increased levels and aggregation of p62, suggesting that abnormal accumulation of ubiquitin proteins may contribute to the neurodegeneration observed in MLIV patients. In addition, we found that delivery of platelet-derived growth factor receptor to lysosomes is delayed in MCOLN1-deficient cells, suggesting that MCOLN1 is necessary for efficient fusion of both autophagosomes and late endosomes with lysosomes. Our data are in agreement with recent evidence showing that autophagic defects may be a common characteristic of many neurodegenerative disorders.


Assuntos
Mucolipidoses/fisiopatologia , Autofagia , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Lisossomos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Receptores Transientes de Potencial
14.
Biochem J ; 410(2): 417-25, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17988215

RESUMO

Mucolipins constitute a family of cation channels with homology with the transient receptor potential family. Mutations in MCOLN1 (mucolipin 1) have been linked to mucolipidosis type IV, a recessive lysosomal storage disease characterized by severe neurological and ophthalmologic abnormalities. At present, little is known about the mechanisms that regulate MCOLN1 activity. In the present paper, we addressed whether MCOLN1 activity is regulated by phosphorylation. We identified two PKA (protein kinase A) consensus motifs in the C-terminal tail of MCOLN1, containing Ser(557) and Ser(559). Ser(557) was the principal phosphorylation site, as mutation of this residue to alanine caused a greater than 75% reduction in the total levels of phosphorylated MCOLN1 C-terminal tail. Activation of PKA with forskolin promoted MCOLN1 phosphorylation, both in vitro and in vivo. In contrast, addition of the PKA inhibitor H89 abolished MCOLN1 phosphorylation. We also found that PKA-mediated phosphorylation regulates MCOLN1 channel activity. Forskolin treatment decreased MCOLN1 channel activity, whereas treatment with H89 increased MCOLN1 channel activity. The stimulatory effect of H89 on MCOLN1 function was not observed when Ser(557) and Ser(559) were mutated to alanine residues, indicating that these two residues are essential for PKA-mediated negative regulation of MCOLN1. This paper presents the first example of regulation of a member of the mucolipin family by phosphorylation.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Cátion TRPM/metabolismo , Linhagem Celular , Eletrofisiologia , Glutationa Transferase/metabolismo , Humanos , Rim/embriologia , Rim/fisiologia , Cinética , Lisossomos , Mucolipidoses/genética , Mutação , Fosforilação , Plasmídeos , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPM/genética , Canais de Receptores Transientes de Potencial
15.
Traffic ; 7(6): 686-98, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16683917

RESUMO

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.


Assuntos
Receptores ErbB/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anisomicina/farmacologia , Sequência de Bases , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptores ErbB/genética , Genes erbB-1 , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
16.
Traffic ; 7(3): 337-53, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16497227

RESUMO

Mutations in the mucolipin-1 gene have been linked to mucolipidosis type IV, a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. Mucolipin-1 is a membrane protein containing six putative transmembrane domains with both its N- and C-termini localized facing the cytosol. To gain information on the sorting motifs that mediate the trafficking of this protein to lysosomes, we have generated chimeras in which the N- and C- terminal tail portions of mucolipin-1 were fused to a reporter gene. In this article, we report the identification of two separate di-leucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes. One di-leucine motif is positioned at the N-terminal cytosolic tail and mediates direct transport to lysosomes, whereas the other di-leucine motif is found at the C-terminal tail and functions as an adaptor protein 2-dependent internalization motif. We have also found that the C-terminal tail of mucolipin-1 is palmitoylated and that this modification might regulate the efficiency of endocytosis. Finally, the mutagenesis of both di-leucine motifs abrogated lysosomal accumulation and resulted in cell-surface redistribution of mucolipin-1. Taken together, these results reveal novel information regarding the motifs that regulate mucolipin-1 trafficking and suggest a role for palmitoylation in protein sorting.


Assuntos
Leucina/química , Lisossomos/metabolismo , Canais de Cátion TRPM/metabolismo , Alanina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Cisteína/química , Citosol/metabolismo , Endocitose , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Palmítico/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética , Canais de Receptores Transientes de Potencial
17.
J Biol Chem ; 277(22): 19783-91, 2002 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-11842083

RESUMO

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464-509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-beta (TbetaRII) receptors (1,2 ). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu(489)-Leu(490)) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ligases/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Enzimas de Conjugação de Ubiquitina , Células 3T3 , Sequência de Aminoácidos , Animais , Western Blotting , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Clonagem Molecular , Detergentes/farmacologia , Fibroblastos/metabolismo , Transportador de Glucose Tipo 4 , Leucina/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/metabolismo , Polietilenoglicóis/farmacologia , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA