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Martine F Roussel

Publications and source records attributed to Martine F Roussel.

16 recordsLinked to original sources
PubMed2004

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8.

Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3- Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.

Amino Acid Sequence↗
PubMed2004

N-terminal polyubiquitination and degradation of the Arf tumor suppressor.

Unknown mechanisms govern degradation of the p19Arf tumor suppressor, an activator of p53 and inhibitor of ribosomal RNA processing. Kinetic metabolic labeling of cells with [3H]-leucine indicated that p19Arf is a relatively stable protein (half-life approximately 6 h) whose degradation depends upon the ubiquitin-proteasome pathway. Although p19Arf binds to the Mdm2 E3 ubiquitin protein ligase to activate p53, neither of these molecules regulates p19Arf turnover. In contrast, the nucleolar protein nucleophosmin/B23, which binds to p19Arf with high stoichiometry, retards its turnover, and Arf mutants that do not efficiently associate with nucleophosmin/B23 are unstable and functionally impaired. Mouse p19Arf, although highly basic (22% arginine content), contains only a single lysine residue absent from human p14ARF, and substitution of arginine for lysine in mouse p19Arf had no effect on its rate of degradation. Mouse p19Arf (either wild-type or lacking lysine) and human p14ARF undergo N-terminal polyubiquitination, a process that has not as yet been documented in naturally occurring lysine-less proteins. Re-engineering of the p19Arf N terminus to provide consensus sequences for N-acetylation limited Arf ubiquitination and decelerated its turnover.

Acetylation↗
PubMed2004

Histone deacetylase inhibitors activate INK4d gene through Sp1 site in its promoter.

Histone deacetylase (HDAC) inhibitors are known to arrest human tumor cells at the G1 phase of the cell cycle and activate the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1). However, several studies have suggested the existence of a p21(WAF1/Cip1)-independent molecular pathway. We report here that HDAC inhibitors activate a member of the INK4 family, the INK4d gene, causing G1 phase arrest, in the human T cell leukemia cell line, Jurkat. One of the major Trichostatin A (TSA)-responsive elements is a specific Sp1 binding site in the INK4d promoter. Electrophoretic mobility-shift assay revealed that Sp1 and Sp3 can specifically interact with this Sp1 binding site. Furthermore, using chromatin immunoprecipitation assay, we demonstrated that HDAC2 was present in the INK4d proximal promoter region in the absence, but not the presence, of TSA. Taken together, these results suggest that treatment with TSA transcriptionally activates INK4d by releasing HDAC2 from the histone-DNA complex at the INK4d promoter. Using a p21(WAF1/Cip1)-deleted human colorectal carcinoma cell line, HCT116 p21 (-/-), we show that upregulation of p19(INK4d) by TSA is associated with inhibition of cell proliferation. Moreover, mouse embryo fibroblasts lacking Ink4d were resistant to the growth inhibitory effects of TSA as compared to their wild-type counterpart. Our findings suggest that p19(INK4d) in addition to p21(WAF1/Cip1) is an important molecular target of HDAC inhibitors inducing growth arrest.

Animals↗
PubMed2004

Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture.

Elevated coexpression of colony-stimulating factor receptor (CSF-1R) and its ligand, CSF-1, correlates with invasiveness and poor prognosis of a variety of epithelial tumors (Kacinski, B.M. 1995. Ann. Med. 27:79-85). Apart from recruitment of macrophages to the tumor site, the mechanisms by which CSF-1 may potentiate invasion are poorly understood. We show that autocrine CSF-1R activation induces hyperproliferation and a profound, progressive disruption of junctional integrity in acinar structures formed by human mammary epithelial cells in three-dimensional culture. Acini coexpressing receptor and ligand exhibit a dramatic relocalization of E-cadherin from the plasma membrane to punctate intracellular vesicles, accompanied by its loss from the Triton-insoluble fraction. Interfering with Src kinase activity, either by pharmacological inhibition or mutation of the Y561 docking site on CSF-1R, prevents E-cadherin translocation, suggesting that CSF-1R disrupts cell adhesion by uncoupling adherens junction complexes from the cytoskeleton and promoting cadherin internalization through a Src-dependent mechanism. These findings provide a mechanistic basis whereby CSF-1R could contribute to invasive progression in epithelial cancers.

Autocrine Communication↗
PubMed2004

Monoclonal antibodies to the mouse p19(Arf) tumor suppressor protein.

The Arf tumor suppressor protein inhibits cell proliferation through both p53-dependent and -independent mechanisms. Two rat monoclonal antibodies raised against a peptide corresponding to amino acids 54-75 of the mouse p19(Arf) protein reacted with the native protein expressed in mammalian cells. Neither antibody detected human or golden hamster Arf proteins. The two antibodies, both of IgG2b isotype, are directed to adjacent epitopes contained within residues 54-62 and 62-75, respectively, of the mouse p19(Arf) protein, and both were highly efficient in detecting p19(Arf) by immunoprecipitation, immunoblotting, and immunofluorescence. One antibody proved useful for immunohistochemical staining of p19(Arf) in fixed sections of mouse testes, revealing low levels of protein expression within the nucleoli of spermatogonia. This indicates that these antibodies should be useful in detecting the endogenous p(19Arf) protein at specific stages of mouse development and during early stages of tumorigenesis.

Animals↗
PubMed2003

Arf tumor suppressor promoter monitors latent oncogenic signals in vivo.

Induction of the Arf tumor suppressor gene by elevated thresholds of mitogenic signals activates a p53-dependent transcriptional response that triggers either growth arrest or apoptosis, thereby countering abnormal cell proliferation. Conversely, Arf inactivation is associated with tumor development. Expression of Arf in tissues of adult mice is difficult to detect, possibly because its induction leads to the arrest or elimination of incipient tumor cells. We replaced coding sequences of exon 1beta of the mouse cellular Arf gene with a cDNA encoding GFP, thereby producing Arf-null animals in which GFP expression is driven by the intact Arf promoter. The Arf promoter was induced in several biologic settings previously shown to elicit mouse p19Arf expression. Inactivation of Arf in this manner led to the outgrowth of tumor cells expressing GFP, thereby providing direct evidence that the Arf promoter monitors latent oncogenic signals in vivo.

Animals↗
PubMed2003

Hemangiosarcomas, medulloblastomas, and other tumors in Ink4c/p53-null mice.

Ink4 proteins inhibit the enzymatic activities of cyclin D-dependent kinases, thereby governing transcriptional programs that depend on the activities of the retinoblastoma protein and other retinoblastoma family members (p107 and p130). Mice lacking Ink4c and p53 spontaneously develop a broad spectrum of neoplasms, usually presenting with multiple tumors of different histological types and dying of cancer by 6 months of age. Whereas thymic lymphomas or pituitary tumors predominate in mice lacking p53 or Ink4c, respectively, animals lacking both genes develop many vascular tumors and also present with medulloblastomas not observed in the parental strains. Unlike p53, loss of the Arf tumor suppressor did not contribute to the appearance of vascular or cerebellar tumors. Vascular tumors ranged in severity from angiomas to hemangiosarcomas, some of which could be transplanted into immunocompromised mice. Intriguingly, loss of Ink4c but maintenance of at least one Ink4d allele was required for formation of medulloblastomas in p53-null mice. In situ hybridization revealed that, in newborn mice, Ink4c is detected in the pia mater and in an adjacent layer of rapidly dividing cells within the cerebellar external granule layer (EGL), whereas Ink4d is primarily expressed in Purkinje neurons. Because the pia mater and Purkinje cells sandwich the cerebellar EGL from which medulloblastomas are presumed to arise, Ink4 proteins might function in a cell-autonomous manner in governing neuronal cell cycle exit as well as in a non-cell-autonomous manner in controlling the production of diffusible mitogens and chemokines that influence postnatal development of the cerebellar EGL.

Animals↗
PubMed2003

A molecular fingerprint for medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. In mice, Ptc1 haploinsufficiency and disruption of DNA repair (DNA ligase IV inactivation) or cell cycle regulation (Kip1, Ink4d, or Ink4c inactivation), in conjunction with p53 dysfunction, predispose to medulloblastoma. To identify genes important for this tumor, we evaluated gene expression profiles in medulloblastomas from these mice. Unexpectedly, medulloblastoma expression profiles were very similar among tumors and also to those of developing cerebellum. However, 21 genes were specifically up-regulated in medulloblastoma, including sFrp1, Ptc2, and Math1, members of signaling pathways that regulate cerebellar development. Coordinated deregulation of these same genes also occurred in a large subset of human medulloblastomas. These data identify a group of genes that is central to medulloblastoma tumorigenesis.

Animals↗
PubMed2003

Arf induces p53-dependent and -independent antiproliferative genes.

The tumor suppressor p19(Arf) (p14(ARF) in humans), encoded by the Ink4a/Arf locus, is mutated, deleted, or silenced in many forms of cancer. p19(Arf) induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby inducing a p53 transcriptional response. p19(Arf) can also inhibit cell cycle progression of mouse embryo fibroblasts lacking Cip1 or lacking both Mdm2 and p53, although in the absence of p53, arrest occurs more slowly. Profiling with high-density oligonucleotide GeneChips and cDNA microarrays was used to interrogate mouse genes, the expression of which was induced or suppressed by a conditionally regulated Arf gene. Cluster analysis of temporal gene expression patterns and validation of the results by RNA analysis identified Arf-responsive genes whose induction was both p53-dependent and -independent. The latter included four members of the B-cell translocation gene family (Btg1, Btg2, Btg3, and Tob1) that were demonstrated to inhibit cell proliferation in primary mouse embryo fibroblasts expressing or lacking functional p53. Together, the results indicate that p19(Arf) induces a broad spectrum of proteins that likely act in concert to arrest cell proliferation.

3T3 Cells↗
PubMed2003

Nucleolar Arf tumor suppressor inhibits ribosomal RNA processing.

The p19(Arf) tumor suppressor, a nucleolar protein, binds to Mdm2 to induce p53-dependent cell cycle arrest. Arf also prevents the proliferation of cells lacking Mdm2 and p53, albeit less efficiently. We show that p19(Arf) inhibits production of ribosomal RNA, retarding processing of 47/45S and 32S precursors. These effects correlate with but do not strictly depend upon inhibition of rRNA biosynthesis or cell cycle arrest, are not mimicked by p53, and require neither p53 nor Mdm2. Arf mutants lacking conserved amino acid residues 2-14 do not block rRNA synthesis and processing or inhibit cell proliferation. Evolution may have linked a primordial nucleolar Arf function to Mdm2 and p53, creating a more efficient checkpoint-signaling pathway for coordinating ribosomal biogenesis and cell cycle progression.

3T3 Cells↗
PubMed2003

Myc-mediated proliferation and lymphomagenesis, but not apoptosis, are compromised by E2f1 loss.

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Emu-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Emu-Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27(Kip1) and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27(Kip1).

ADP-Ribosylation Factor 1↗
PubMed2003

Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d.

Maintenance of the post-mitotic state in the post-natal mammalian brain is an active process that requires the cyclin-dependent kinase inhibitors (CKIs) p19Ink4d (Ink4d) and p27Kip1 (Kip1). In animals with targeted deletions of both Ink4d and Kip1, terminally differentiated, post-mitotic neurons are observed to re-enter the cell cycle, divide and undergo apoptosis. However, when either Ink4d or Kip1 alone are deleted, the post-mitotic state is maintained, suggesting a redundant role for these genes in mature neurons. In the organ of Corti--the auditory sensory epithelium of mammals--sensory hair cells and supporting cells become post-mitotic during embryogenesis and remain quiescent for the life of the animal. When lost as a result of environmental insult or genetic abnormality, hair cells do not regenerate, and this loss is a common cause of deafness in humans. Here, we report that targeted deletion of Ink4d alone is sufficient to disrupt the maintenance of the post-mitotic state of sensory hair cells in post-natal mice. In Ink4d-/- animals, hair cells are observed to aberrantly re-enter the cell cycle and subsequently undergo apoptosis, resulting in progressive hearing loss. Our results identify a novel mechanism underlying a non-syndromic form of progressive hearing loss in mice.

Animals↗
PubMed2002

The RING domain of Mdm2 can inhibit cell proliferation.

Mdm2 is a p53-inducible phosphoprotein that negatively regulates p53 by binding to it and promoting its ubiquitin-mediated degradation. Alternatively spliced variants of Mdm2 have been isolated from human and mouse tumors, but their roles in tumorigenesis, if any, remain elusive. We cloned six alternatively spliced variants of Mdm2 from E(mu)-Myc-induced mouse lymphomas, all of which lacked the NH(2)-terminal p53-binding domain but conserved the remainder of the Mdm2 protein. Enforced expression of full-length Mdm2 in primary mouse embryo fibroblasts or bone marrow-derived, interleukin 7-dependent pre-B cells accelerated their proliferation, whereas unexpectedly, overexpression of truncated Mdm2 isoforms inhibited their growth. Truncated variants were active as inhibitors whether they localized predominantly to the nucleus or cytoplasm. Despite the absence of the p53-binding domain, growth inhibition remained strictly p53 dependent (but not p19(Arf) dependent) and could be overcome by full-length Mdm2. The intact RING finger domain at the Mdm2 COOH terminus (amino acids 399-489) was necessary and sufficient for growth inhibition by truncated Mdm2 proteins and could physically interact with either the RING finger domain or central acidic region of full-length Mdm2. However, such interactions do not inhibit Mdm2 E3 ubiquitin ligase activity in vitro using p53 as a substrate. Expression of growth-inhibitory Mdm2 isoforms in tumors remains an enigma.

Alternative Splicing↗
PubMed2002

The cyclin-dependent kinase inhibitors p19(Ink4d) and p27(Kip1) are coexpressed in select retinal cells and act cooperatively to control cell cycle exit.

Cyclin-dependent kinase inhibitors (cdki's), including p19(Ink4d) and p27(Kip1), mediate exit from the cell cycle. To determine the function of these cdki's in regulating neurogenesis, we examined retina from wild-type, Ink4d-null, and Ink4d/Kip1-double null animals. Ink4d was expressed in progenitors and select neurons in the mature retina. Ink4d-null retina showed an extended period of proliferation, followed by apoptosis. Colabeling for p19(Ink4d) and p27(Kip1) revealed that a subpopulation of cells expressed both inhibitors. Deletion of Ink4d and Kip1 resulted in continued proliferation that was synergistic. This hyperproliferation led to an increase in number of horizontal cells and differentiated neurons reentering the cell cycle. Deletion of Ink4d and Kip1 also exacerbated the retinal dysplasia observed in Kip1-null mice, which was shown to be partly dependent on p53. These data indicate that select retinal cells express both p19(Ink4d) and p27(Kip1) and that they act cooperatively to ensure cell cycle exit.

Amacrine Cells↗
PubMed2002

p53: regular or super?

Increased p53 expression under the endogenous promoter protects "super p53" mice from tumorigenesis without the undesirable effects of premature aging.

Aging, Premature↗