Mutation of Vav1 adaptor region reveals a new oncogenic activation

International audienceVav family members function as remarkable scaffold proteins that exhibit both GDP/GTP exchange activity for Rho/Rac GTPases and numerous protein-protein interactions via three adaptor Src-homology domains. The exchange activity is under the unique regulation by phosphorylation of tyrosine residues hidden by intra-molecular interactions. Deletion of the autoinhibitory N-terminal region results in an oncogenic protein, onco-Vav, leading to a potent activation of Rac GTPases whereas the proto-oncogene barely leads to transformation. Substitution of conserved residues of the SH2-SH3 adaptor region in onco-Vav reverses oncogenicity. While a unique substitution D797N did not affect transformation induced by onco-Vav, we demonstrate that this single substitution leads to transformation in the Vav1 proto-oncogene highlighting the pivotal role of the adaptor region. Moreover, we identified the cell junction protein β-catenin as a new Vav1 interacting partner. We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of β-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts. In addition, a similar interaction is evidenced in epithelial lung cancer cells expressing ectopically Vav1. In these cells, Vav1 is also involved in the modulation of β-catenin phosphorylation. Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity. INTRODUCTION The Vav1 proto-oncogene has a restricted hematopoietic expression and exhibits both GTP/GDP exchange activities (GEF) for Rho family GTPases and adaptor functions within signalling complexes [1, 2]. Two other genes, Vav2 and Vav3 belong to the same family of signalling effectors and share high structural similarities and properties with Vav1. Unlike Vav1, Vav2 and Vav3 have an ubiquitous expression [3, 4]. Vav proteins display a number of characteristic structural domains with homology for: Calponin (CH), Dbl (DH), Pleckstrin (PH) and Src (SH2 and SH3) altogether with acidic residues-rich (AcR) and cysteine-rich (CR) motives. These domains mediate interactions with membrane receptors

A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes.</p> <p>Results</p> <p>New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1.</p> <p>Conclusions</p> <p>Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate protein/DNA interactions. The finding of a STAT3-specific hpdODN establishes the first rational basis for designing STAT3 DBD-specific inhibitors.</p

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

<p>Abstract</p> <p>Background</p> <p>The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear.</p> <p>Results</p> <p>The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death.</p> <p>Conclusions</p> <p>The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.</p

Heterogeneous secretory profile in the progression of CLL

International audienc

Analyse structurale et fonctionnelle de recepteurs pour la partie Fc des immunoglobulines G et E

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78399 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Rôle fonctionnel de la protéine adaptatrice Lnk dans le système hématopoïetique

L'invalidation de Lnk in vivo a mis en évidence le rôle de régulateur négatif de cet adaptateur sur la prolifération des progéniteurs hématopoïétiques. Mon travail a montré que le récepteur Kit est un partenaire physiologique important de Lnk. Leur association implique le domaine SH2 de Lnk et une tyrosine régulatrice de Kit. J'ai également montré que suite à cette association, Lnk régule des voies de signalisation dépendantes de Kit permettant le contrôle de la prolifération, la survie et la migration des mastocytes (voies MAPK, PI3K/Akt et Rac/JNK). Le phénotype des souris Lnk-/- ressemble à celui observé dans certains syndromes myéloprolifératifs. Il est donc probable que Lnk joue un rôle dans ces pathologies. J'ai identifié d'autres partenaires de Lnk essentiels au développement des progéniteurs hématopoïétiques. L'analyse en cours de ces interactions permettra de clarifier le rôle de Lnk dans l'hématopoïèse et son implication dans des pathologies hématologiques.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Régulation des complexes d'ubiquitinylation et de sumoylation par la ligase E3 hSIAH2

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

The strange case of nurse cells: Dr. Jekyll or Mr. Hyde?

International audienceIn developmental biology and physiology of reproduction the term nurse cells refers to cells which provide feeding, support, and stability to their neighboring cells. The notion of nurse cell is used in several unrelated ways in various scientific fields, such as parasitology, mycology, invertebrate and vertebrate development. In this review, we focus on “nurse cells” described to support hemopoietic cell differentiation which can act as a double-edged sword in normal versus pathological conditions. Particularly, the nurturing capacity of stromal cells to assist the differentiation and survival of normal cells within both the bone marrow or the synovium can turn to detrimental effects. In the later cases, nurse cells induce and maintain inflammation and rheumatological disease. Similarly, the anti-tumoral potential of myeloid cells might be switched to tumor promoting function in most cancers and leukemia, including chronic lymphocytic leukemia. A better understanding of the interactions between pathological cells and supporting cells could inspire new drug combination strategies, translational studies, and novel therapeutic options in clinical trial

Étude de l'adressage et de l'implication nucléaire du proto-oncogène Vav-1

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Rôle du domaine SH3 C-terminal dans l'adressage fonctionnel au noyau du proto-oncogène Vav1

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF