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Eps15 and Eps15R are constitutive components of clathrin-coated pits that are required for clathrin-dependent endocytosis. The most striking difference between these two related proteins is that Eps15R is also found in the nucleus, whereas Eps15 is excluded from this compartment at steady state. To better understand the individual functions of these two proteins, the mechanisms responsible for their different localization were investigated. Interestingly, some mutants of Eps15 were found in the nucleus. This nuclear localization was correlated with the loss of the last approximately 100 amino acids of Eps15, suggesting the presence of a nuclear export signal (NES) within this region. As expected, the last 25 amino acids contain a leucine-rich sequence matching with classical NESs, show a leptomycin B-sensitive nuclear export activity, and bind to the exportin CRM1 in a leucine residue-dependent manner. In contrast, no NES could be found in Eps15R, a result in keeping with its constitutive nuclear localization that appears to be regulated by alternative splicing. Altogether, these results are the first characterization of nucleocytoplasmic shuttling signals for endocytic proteins. They also provide an explanation for the different nuclear localization of Eps15 and Eps15R and further evidence for a possible nuclear function for Eps15 protein family members.

We previously showed that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a manner dependent on level of expression, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and procaspase 3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacts with Hsp27, hence excluding a role of this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular relocalization was altered by changes in Hsp27 level of expression, suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizing agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspase activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway, which induces Bid intracellular redistribution, is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine-treated cells, phalloidin only partially inhibited cytochrome c release and caspase activation. Moreover, in etoposide-treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria and Bid intracellular redistribution in conditions where F-actin was not altered.

Osmotic shock stimulates the translocation of the glucose transporter Glut 4 to plasma membrane by a tyrosine kinase signaling pathway involving Gab-1 (the Grb2-associated binder-1 protein). We show here that, in response to osmotic shock, Gab-1 acts as a docking protein for phospholipase Cgamma1, the p85 subunit of the phosphoinositide 3-kinase and Crk-II. It has been shown that the adapter Crk-II is constitutively associated with C3G, a GDP to GTP exchange factor for several small GTP-binding proteins. We found that inhibition of the activity of phosphoinositide 3-kinase or phospholipase C did not prevent the stimulation of glucose transport by osmotic shock, whereas inactivation of Rho proteins by Clostridium difficile toxin B severely inhibited glucose uptake. Among the Rho family members, overexpression of dominant-interfering TC10/T31N mutant inhibited osmotic shock-mediated Glut 4 translocation suggesting that TC10 is required for this process. Further, disruption of cortical actin integrity by latrunculin B or jasplakinolide severely impaired osmotic shock-induced glucose transport. In contrast, osmotic shock increased the amount of cortical actin associated with caveolin-enriched plasma membrane domains. These data provide the first evidence that activation of TC10 and remodeling of cortical actin, which could occur through the TC10 signaling, are required for osmotic shock-mediated Glut 4 translocation and glucose uptake.

Article en ligne : http://www.jbc.org/cgi/content/full/277/7/4918; International audience; Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance. Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression. Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses. The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription. First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80. LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells. This enhancement was suppressed after re-expression of Ku80. Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA. Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells. These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression. Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

International audience; The eukaryotic MutHLS-like system plays a crucial role in both mitosis and meiosis. Until now, a number of works have focused on the function of MutS and MutL homologs during mismatch DNA repair. Nevertheless, little is known about the role of these proteins during meiosis. MSH4 is a meiosis specific protein that is necessary for meiotic recombination in Saccharomyces cerevisiae. The human MSH4 protein is only found in testis and ovary. It is involved first in synapsis and second during recombination together with MLH1 (MutL Homolog 1). Here, we report the identification of the mouse Msh4 gene that is located on chromosome 3. We examined the expression of mMsh4 in testes of increasing developmental age and in elutriated germ cells. The pattern of expression during spermatogenesis is consistent with a role for MSH4 both during zygonema and pachynema. We demonstrated a promoter activity of the mMsh4 5'-flanking region by cell transfection experiments with a luciferase reporter gene. We found several SRY/Sox binding sites in this region and co-transfection experiments showed that SRY could down regulate mMsh4 promoter transcriptional activity. We propose that the regulation of mMsh4 expression could be one of the reasons for the persistence of SRY and/or SRY-related proteins in adult testis.

Pax3 is an evolutionarily conserved transcription factor that plays a major role in a variety of developmental processes. Mutations in Pax3 lead to severe malformations as seen in human Waardenburg syndrome and in the Splotch mutant mice. The transcriptional activity of Pax3 was recently shown to be repressed by Daxx whereas the oncogenic fusion protein Pax3-FKHR is unresponsive to this repressive action. Here we demonstrate that Daxx-mediated repression of Pax3 can be inhibited by the nuclear body (NB)-associated protein PML. Interestingly, this suppression of Daxx properties correlates with its recruitment to the NBs. Factors such as arsenicals and interferons that enhance NB formation, trigger both the targeting of Daxx to these nuclear structures and the relief of the repressive activity of Daxx. Conversely, lack of structurally intact NBs profoundly impairs Pax3 transcriptional activity, likely by increasing the pool of available nucleoplasmic Daxx. Moreover, a PML mutant that can not be modified by the ubiquitin-related SUMO-1 modifier is no more able to interact with Daxx. Consistently, such a mutant fails both to inhibit the Daxx repressing effect on Pax3 and to induce its accumulation into the NBs. Taken together, these results argue that SUMO-1 modified PML can derepress Pax3 transcriptional activity through sequestration of the Daxx repressor into the NBs and suggest a role for these nuclear structures in the transcriptional control by Pax proteins. Oncogene (2001) 20, 1 - 9.

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.

International audience; Transcription factors of the basic Helix-Loop-Helix (bHLH) protein family play key roles in several developmental processes. Mist1 belongs to this group of proteins and shares several properties with the other family members. For example, Mist1 is capable of dimerization with the ubiquitously expressed E2A bHLH proteins and exhibits a strong DNA-binding activity to the core E-box sequence. Using in-situ hybridization and Northern blot hybridization, Mist1 mRNA has been detected in a variety of embryonic and adult rodent tissues. To understand the molecular mechanisms involved in the expression of the gene, we have cloned the rat Mist1 gene and analyzed 2.5 kb of its 5' flanking region. The Mist1 gene spans over 5 kilobases and is composed of two exons separated by a unique intron. The entire coding region is localized in the second exon. Sequence analysis of the promoter region indicated an absence of TATA-box or CAAT-box sequence, but several consensus Sp1-binding sites were present near the transcription start site. Deletion analysis of the promoter region identified a 272 bp proximal fragment to be sufficient to drive expression of a reporter gene in NIH3T3 fibroblasts. Subsequent deletion of potential Sp1 sites results in a marked decrease in promoter activity. Electrophoretic mobility shift assays revealed that Sp1 binds to two different regions in the proximal promoter, a typical Sp1 site located at (-38; -33) and a G/C-rich region between (-67; -62). These data suggest that the basal expression of this TATA-less gene might be driven by general transcription factors, such as Sp1.

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-kappaB in macrophages from PARP-1(+/+) but not from PARP-1(-/-) mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process.

In the search for the existence of adrenergic regulation of the autocrine/paracrine function of the white adipose tissue, it was observed that conditioned media from isolated adipocytes or dialysates obtained by in situ microdialysis of human subcutaneous adipose tissue increased spreading and proliferation of 3T3F442A preadipocytes. These effects were amplified when an alpha2-adrenergic agonist was present during the obtention of conditioned media and microdialysates. This alpha2-adrenergic-dependent trophic activity was completely abolished by pretreatment of the conditioned media or microdialysates with the lysophospholipase, phospholipase B. Among the different lysophospholipids tested only lysophosphatidic acid (LPA) was able to induce spreading and proliferation of 3T3F442A preadipocytes. Moreover, previous chronic treatment of 3T3F442A preadipocytes with LPA which led to a specific desensitization of LPA responsiveness, abolished the alpha2-adrenergic-dependent trophic activities of the conditioned media and microdialysates. Finally, alpha2-adrenergic stimulation led to a rapid, sustained, and pertussis toxin-dependent release of [32P]LPA from [32P]-labeled adipocytes. Based upon these results it was proposed that in vitro and in situ stimulation of adipocyte alpha2-adrenergic receptors provokes the extracellular release of LPA leading, in turn, to regulation of preadipocyte growth.

International audience; BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

International audience; Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor alpha, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.

International audience; Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

International audience; We have previously generated primary embryonic fibroblasts lacking either the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR) or the cation-dependent MPR, two trans-membrane proteins that bind the mannose 6-phosphate (Man-6-P) recognition marker on soluble lysosomal enzymes (Ludwig, T., Munier-Lehmann, H., Bauer, U., Hollinshead, M., Ovitt, C., Lobel, P., and Hoflack, B.(1994) EMBO J. 13, 3430-3437). These two cell types partially missort phosphorylated lysosomal enzymes. Using two-dimensional gel electrophoresis, we show here that they secrete, in a large part, different phosphorylated ligands. In order to better understand the sorting function of the MPRs, we have re-expressed each MPR in MPR-negative fibroblasts. We show that the MPRs have similar capacities for transporting the bulk of the newly synthesized lysosomal enzymes and that they target individual ligands with various efficiencies. However, high levels of one MPR do not fully compensate for the absence of the other, demonstrating that the two MPRs have complementary targeting functions, perhaps by recognizing different features on lysosomal enzymes. The analysis of the phosphorylated oligosaccharides shows that the ligands missorted in the absence of the cation-dependent MPR are slightly but significantly depleted in oligosaccharides with two Man-6-P residues, when compared with those missorted in the absence of the cation-independent MPR. While these results could explain some differences between the structure and the sorting function of the two MPRs, they strongly suggest that the reason why cells express two different but related MPRs is to maintain an efficient Man-6-P-dependent targeting process that could be potentially regulated by MPR expression.

International audience; We have designed several retroviral constructs to transduce the PML-RA Ralpha fusion product of human acute promyelocytic leukemia. Our aim to generate high-titer stable vector-producing cell lines was hindered by a toxic effect of PML-RARalpha expression on packaging cells. To circumvent this, we tested retroviral vectors expressing the transgene from several internal promoters including inducible and myelospecific promoters. To compare efficiency of these constructs in their ability to generate protein expression in the appropriate target cells and optimal viral titers, we used the BOSC23 transient packaging cell line. We found that the direct-oriented vector did not ensure tissue-specificity of PML-RARalpha expression, while the reverse-oriented retroviral vector did. The latter construct, however, failed to generate high-titer recombinant virus. This study exemplifies the unpredictable behavior of retroviral constructs and the superiority of transient systems for transduction of a toxic product.

International audience; Mouse biliary glycoprotein (Bgp) is a member of the carcinoembryonic antigen gene family and is highly expressed in the epithelial cells of normal hepatic biliary ducts and intestine. Nine mouse Bgp isoforms have been identified through molecular cloning and shown to be splice and allelic variants of one Bgp gene. These glycoproteins function in vitro as intercellular adhesion molecules and serve as the mouse hepatitis virus receptors. Since human carcinoembryonic antigen is overexpressed in gastrointestinal tumors, we have investigated the expression of mouse Bgp in primary tumors and carcinoma cell lines. Our results demonstrate that the expression of the major mouse Bgp isoforms is down-regulated in tumors at the transcriptional and the posttranscriptional levels. This decrease in expression is corroborated by immunostaining of primary colonic tumors with anti-mouse Bgp antibodies. In addition, Bgp expression is influenced by transcriptional control mechanisms involving DNA methylation of the Bgp gene upstream regulatory region. Our results demonstrate that mouse Bgp protein expression is decreased upon malignant transformation and further suggest that Bgp proteins may be involved in the maintenance of the differentiated cellular phenotype.

International audience; In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.