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González E Montañez C Ray PN Howard PL García-Sierra F Mornet D Cisneros B 《FEBS letters》2000,482(3):209-214
The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function. 相似文献
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We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization. 相似文献
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Aleman V Osorio B Chavez O Rendon A Mornet D Martinez D 《Histochemistry and cell biology》2001,115(3):243-254
It has been suggested that the absence or altered structure of Dp71, a C-terminal dystrophin gene encoded protein, is responsible for mental alterations observed in about 30% of Duchenne muscular dystrophy patients. Most of these patients have premature translational termination or point mutations at the C-terminal domain of this gene. In brain, Dp71 is the major protein product of the dystrophin gene. To determine the function of Dp71 isoforms in this organ, it is important to document their presence and intracellular localization in brain cells. Extracts from cultured hippocampal neurons and forebrain astrocytes and 5F3 and Dys 2 monoclonal antibodies were thus used for western blots. In these conditions, two Dp71 isoforms spliced or not at exon 78 were detected in both cells (Dp71f and Dp71d, respectively). By immunocytochemistry, we mapped Dp71f and Dp71d in the Golgi complex (GC) and in neuronal nuclei. Only Dp71d was found in cytoplasmic neurofilaments. In astrocytes, these isoforms were detected in the GC. These cell localization data suggest that these Dp71 isoforms may have different functions in the same cell or organelle, as well as in the two different cells analyzed. 相似文献
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The function of dystrophin Dp71 in neuronal cells remains to be established. Previously, we revealed the involvement of this protein in both nerve growth factor (NGF)-induced neuronal differentiation and cell adhesion by isolation and characterization of PC12 neuronal cells with depleted levels of Dp71. In this work, a novel phenotype of Dp71-knockdown cells was characterized, which is their delayed growth rate. Cell cycle analyses revealed an altered behavior of Dp71-depleted cells, which consists of a delay in G0/G1 transition and an increase in apoptosis during nocodazole-induced mitotic arrest. Dp71 associates with lamin B1 and β-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and β-dystroglycan in PC12 cells at mitosis and cytokinesis by means of immunofluorescence and confocal microscopy analysis. All of these three proteins exhibited a similar immunostaining pattern, localized at mitotic spindle, cleavage furrow, and midbody. It is noteworthy that a drastic decreased staining in mitotic spindle, cleavage furrow, and midbody was observed for both lamin B1 and β-dystroglycan in Dp71-depleted cells. Furthermore, we demonstrated the interaction of Dp71 with lamin B1 in PC12 cells by immunoprecipitation and pull-down assays, and importantly, we revealed that knockdown of Dp71 expression caused a marked reduction in lamin B1 levels and altered localization of the nuclear envelope protein emerin. Our data indicate that Dp71 is a component of the mitotic spindle and cytokinesis multi-protein apparatuses that might modulate the cell division cycle by affecting lamin B1 and β-dystroglycan levels. 相似文献
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Hwang YH Tani M Nakagawa T Okino N Ito M 《Biochemical and biophysical research communications》2005,331(1):37-42
We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence. 相似文献
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xCT, the core subunit of the system x(c)(-) high affinity cystine transporter, belongs to a superfamily of glycoprotein-associated amino acid transporters. Although xCT was shown to promote cystine transport in Xenopus oocytes, little work has been done with mammalian cells (Sato, H., Tamba, M., Ishii, T., and Bannai, S. J. Biol. Chem. 274, 11455-11458, 1999). Therefore, we have constructed mammalian expression vectors for murine xCT and its accessory subunit 4F2hc and transfected them into HEK293 cells. We report that this transporter binds cystine with high affinity (81 microM) and displays a pharmacological profile expected for system x(c)(-). Surprisingly, xCT transport activity in HEK293 cells is not dependent on the co-expression of the exogenous 4F2hc. Expression of GFP-tagged xCT indicated a highly clustered plasma membrane and intracellular distribution suggesting the presence of subcellular domains associated with combating oxidative stress. Our results indicate that HEK293 cells transfected with the xCT subunit would be a useful vehicle for future structure-function and pharmacology experiments involving system x(c)(-). 相似文献
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《FEBS letters》1998,441(2):337-341
The Dp71 dystrophin isoform has recently been shown to localize to actin filament bundles in early myogenesis. We have identified an actin binding motif within Dp71 that is not found in other dystrophin isoforms. Actin overlay assays and transfection of COS-7 cells with fusion proteins of wild type and mutated Flag epitope-tagged Dp71 demonstrate that this motif is necessary and sufficient to direct localization of Dp71 to actin stress fibers. Furthermore, this localization is independent of alternative splicing which alters the C-terminus of the protein. The identification of an actin binding site suggests Dp71 may function to anchor membrane receptors to the cytoskeleton. 相似文献
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Rodríguez-Muñoz R Villarreal-Silva M González-Ramírez R García-Sierra F Mondragón M Mondragón R Cerna J Cisneros B 《Biochemical and biophysical research communications》2008,375(3):303-307
The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in the center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation. 相似文献
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Romo-Yáñez J Ceja V Ilarraza-Lomelí R Coral-Vázquez R Velázquez F Mornet D Rendón A Montañez C 《Journal of cellular biochemistry》2007,102(1):82-97
PC12 cells express different Dp71 isoforms originated from alternative splicing; one of them, Dp71ab lacks exons 71 and 78. To gain insight into the function of Dp71 isoforms we identified dystrophin associated proteins (DAPs) that associate in vivo with Dp71ab during nerve growth factor (NGF) induced differentiation of PC12 cells. DAPs expression was analyzed by RT-PCR, Western blot and indirect immunofluorescence, showing the presence of each mRNA and protein corresponding to alpha-, beta-, gamma-, delta-, and epsilon-sarcoglycans as well as zeta-sarcoglycan mRNA. Western blot analysis also revealed the expression of beta-dystroglycan, alpha1-syntrophin, alpha1-, and beta-dystrobrevins. We have established that Dp71ab forms a complex with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and alpha-, beta- and gamma-sarcoglycans in undifferentiated PC12 cells. In differentiated PC12 cells, the complex composition changes since Dp71ab associates only with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and delta-sarcoglycan. Interestingly, neuronal nitric oxide synthase associates with the Dp71ab/DAPs complex during NGF treatment, raising the possibility that Dp71ab may be involved in signal transduction events during neuronal differentiation. 相似文献
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González-Ramírez R Morales-Lázaro SL Tapia-Ramírez V Mornet D Cisneros B 《Journal of cellular biochemistry》2008,105(3):735-745
Dystrophin and dystrophin-associated proteins (DAPs) form a complex around the sarcolemma, which gives stability to the sarcolemma and leads signal transduction. Recently, the nuclear presence of dystrophin Dp71 and DAPs has been revealed in different non-muscle cell types, opening the possibility that these proteins could also be present in the nucleus of muscle cells. In this study, we analyzed by Immunofluorescence assays and Immunoblotting analysis of cell fractions the subcellular localization of Dp71 and DAPs in the C(2)C(12) muscle cell line. We demonstrated the presence of Dp71, alpha-sarcoglycan, alpha-dystrobrevin, beta-dystroglycan and alpha-syntrophin not only in plasma membrane but also in the nucleus of muscle cells. In addition, we found by Immunoprecipitation assays that these proteins form a nuclear complex. Interestingly, myogenesis modulates the presence and/or relative abundance of DAPs in the plasma membrane and nucleus as well as the composition of the nuclear complex. Finally, we demonstrated the presence of Dp71, alpha-sarcoglycan, beta-dystroglycan, alpha-dystrobrevin and alpha-syntrophin in the C(2)C(12) nuclear envelope fraction. Interestingly, alpha-sarcoglycan and beta-dystroglycan proteins showed enrichment in the nuclear envelope, compared with the nuclear fraction, suggesting that they could function as inner nuclear membrane proteins underlying the secondary association of Dp71 and the remaining DAPs to the nuclear envelope. Nuclear envelope localization of Dp71 and DAPs might be involved in the nuclear envelope-associated functions, such as nuclear structure and modulation of nuclear processes. 相似文献
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Sara Luz Morales‐Lázaro Ricardo González‐Ramírez Pablo Gómez Victor Tapia‐Ramírez Mario Bermúdez De León Bulmaro Cisneros 《Journal of neurochemistry》2010,112(2):474-485
In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E‐115 cell line. We demonstrated that Dp71 expression is up‐regulated in response to cAMP‐mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5′‐flanking 159‐bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA‐less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2α bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over‐expression of Sp1 and AP2α, as well as knock‐down experiments on Sp1 and AP2α gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2α, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2α binding, which ultimately releases the promoter from repression. 相似文献
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RELT is a recently identified Tumor Necrosis Factor Receptor that possess two homologues in humans named RELL1 and RELL2. We investigated whether RELT and its homologues could induce cellular death when transiently transfected into HEK 293 epithelial cells. Transfection of RELT family members into HEK 293 epithelial cells induced cell death characterized by rounding and lifting of cells accompanied by DNA fragmentation, characteristics that are consistent with the activation of an apoptotic pathway. Overexpression of RELT in COS-7 cells resulted in cell rounding and lifting without DNA fragmentation, suggesting that the effects of RELT signaling may vary among different cell types. In summary, we report that overexpression of RELT or its homologues RELL1 and RELL2 in HEK 293 epithelial cells results in cell death with morphological characteristics consistent with the activation of an apoptotic pathway. 相似文献
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Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins. After rhodopsin bleaching in cells loaded with 11-cis-retinal, ERC signals recover in minutes and recurrently over a period of hours by simple dark adaptation, with no added chromophore. The purpose of this study is to investigate the source of ERC signal recovery in these cells. Giant HEK293S cells expressing normal wild-type (WT)-human rod opsin (HEK293S) were regenerated by solubilized 11-cis-retinal, all-trans-retinal, or Vitamin A in darkness. ERCs were elicited by flash photolysis and measured by whole-cell recording. Visible flashes initially elicit bimodal (R(1), R(2)) ERC signals in WT-HEK293S cells loaded with 11-cis-retinal for 40 min or overnight. In contrast, cells regenerated for 40 min with all-trans-retinal or Vitamin A had negative ERCs (R(1)-like) or none at all. After these were placed in the dark overnight, ERCs with outward R(2) signals were recorded the following day. This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells. These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye. 相似文献
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Tang KF Wang Y Wang P Chen M Chen Y Hu HD Hu P Wang B Yang W Ren H 《Biochimica et biophysica acta》2007,1770(5):820-825
It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer depletion rather than the consequence of growth arrest induced by Dicer knockdown. Considering the reports that there is consistent placental outgrowth in PHLDA2 knockout mice and that PHLDA2 overexpression in mice causes growth inhibition, we speculate that PHLDA2 may be a candidate for contributing to the reduced growth rate of Dicer-deficient cells and the very early embryonic lethality in Dicer knockout mice. 相似文献
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Wu X Currall B Yamashita T Parker LL Hallworth R Zuo J 《Developmental neurobiology》2007,67(4):483-497
The remarkable hearing sensitivity and frequency selectivity in mammals is attributed to cochlear amplifier in the outer hair cells (OHCs). Prestin, a membrane protein in the lateral wall of OHC plasma membrane, is required for OHC electromotility and cochlear amplifier. In addition, GLUT5, a fructose transporter, is reported to be abundant in the plasma membrane of the OHC lateral wall and has been originally proposed as the OHC motor protein. Here we provide evidence of interactions between prestin/prestin and prestin/GLUT5 in transiently transfected HEK293T cells. We used a combination of techniques: (1) membrane colocalization by confocal microscopy, (2) fluorescence resonance energy transfer (FRET) by fluorescence activated cell sorting (FACS), (3) FRET by acceptor photobleaching, (4) FRET by fluorescence lifetime imaging (FRET-FLIM), and (5) coimmunoprecipitation. Our results suggest that homomeric and heteromeric prestin interactions occur in native OHCs to facilitate its electromotile function and that GLUT5 interacts with prestin for its elusive function. 相似文献
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Tae Sik Sung Min Ji Kim Soojin Hong Jae-Pyo Jeon Byung Joo Kim Ju-Hong Jeon Seon Jeong Kim Insuk So 《Molecules and cells》2009,27(2):167-173
The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca2+-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been
assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different
from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a
muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared
not to interact with TRPC4. TPRC4 activation by GTPγS was not desensitized. TPRC4 activation by GTPγS was not inhibited by
either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK
a of 7.3. Finally, TPRC4 activation by GTPγS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5
have different properties and their own physiological roles.
These authors contributed equally to this work. 相似文献