Rôle et régulation des isoformes de la MAPKinase Kinase Kinase B-Raf

La sérine/thréonine kinase B-Raf est impliquée dans de nombreux processus cellulaires chez les Métazoaires. Le gène B-raf code plusieurs isoformes résultant d'épissage alternatif. La présence des séquences codées par les exons alternatifs 8b et 9b, situées entre le domaine régulateur et le domaine kinase de B-Raf, module les propriétés biochimiques et oncogéniques de B-Raf : l'exon 9b augmente l'activité kinase et l'interaction avec MEK, l'exon 8b les diminue. Mon travail de thèse a eu pour objectif à la fois de comprendre les bases moléculaires du mécanisme de régulation différentielle des isoformes de B-Raf et également d'analyser leur importance fonctionnelle. J'ai ainsi montré que la présence des séquences codées par les exons 8b et 9b interférent avec l'autoinhibition du domaine régulateur sur le domaine kinase. Ces séquences interfèrent également avec la phosphorylation des résidus S365 et S429 situés dans la même région charnière, indépendamment des interactions intramoléculaires. Ainsi, alors que le niveau de phosphorylation de la S365 est modulé par la présence des exons, la phosphorylation de la S429 n'est pas modulée par les séquences alternatives mais aurait des conséquences différentes en fonction de l'isoforme. D'autre part, afin de comprendre le rôle physiologique des isoformes de B-Raf contenant les séquences codées par les exons 8b et 9b, nous avons généré des lignées de souris pour lesquelles la délétion conditionnelle de ces exons est rendue possible grâce à l'approche Cre/Lox. L'obtention de souris invalidées pour les exons 8b et 9b permettra de mieux comprendre le rôle spécifique des isoformes de B-Raf, en particulier au cours du développement.The B-Raf serine/threonine kinase is involved in many cellular processes in Metazoans. The B-raf gene encodes several isoforms resulting from alternative splicing. The presence of the sequences encoded by exons 8b and 9b, located between the regulation domain and the kinase domain of B-Raf, modulates the biochemical and oncogenic properties of B-Raf: the exon 9b increases the kinase activity and the interaction with MEK, the exon 8b decreases them. My work aimed at the same time to understand the molecular basis of the mechanism of differential regulation of B-Raf isoforms and also to analyze their functional roles. I thus showed that the presence of the sequences encoded by exons 8b and 9b interferes with the autoinhibition of the regulation domain on the kinase domain. These sequences also interfere with phosphorylation of the residues S365 and S429 located in the same hinge region, independently of the intramolecular interactions. Thus, whereas the phosphorylation level of S365 is modulated by the presence of exons, the phosphorylation of S429 is not modulated by the alternative sequences but would have different consequences according to the isoform. In addition, in order to understand the physiological role of the B-Raf isoforms containing the sequences encoded by exons 8b and 9b, we generated mice strain lines in which the conditional deletion of these exons is possible by a Cre/Lox strategy. Obtaining mice invalidated for exons 8b and 9b will render possible to better understand the specific role of B-Raf isoforms, especially during development.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells

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Differential Regulation of B-Raf Isoforms by Phosphorylation and Autoinhibitory Mechanisms

The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf(8b) isoform and exon 9b in the B3-Raf(9b) isoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities, respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We show that exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf(8b) was more pronounced. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf(9b) isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but the S429A mutation activated B2-Raf(8b), whereas it inhibited B3-Raf(9b). These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing

B-raf alternative splicing is dispensable for development but required for learning and memory associated with the hippocampus in the adult mouse.

The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species

Dual function of MyD88 in RAS signaling and inflammation, leading to mouse and human cell transformation

Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation

B-raf Alternative Splicing Is Dispensable for Development but Required for Learning and Memory Associated with the Hippocampus in the Adult Mouse

The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species

Safety of CD34+ Hematopoietic Stem Cells and CD4+ T Lymphocytes Transduced with LVsh5/C46 in HIV-1 Infected Patients with High-Risk Lymphoma

International audienceAlthough the risk of developing lymphoma has decreased in the highly active antiretroviral therapy era, this cancer remains the major cause of mortality in HIV-infected patients. Autologous hematopoietic stem cell transplantation (ASCT) outcome does not differ for HIV-infected versus HIV-uninfected patients. We propose to develop a new treatment for HIV-associated high-risk lymphoma based on autologous transplantation of two genetically modified products: CD4+ T lymphocytes and CD34+ hematopoietic stem cells (HSPCs). The cells will be transduced ex vivo with the Cal-1 lentiviral vector encoding for both a short hairpin RNA (shRNA) against CCR5 (sh5) and the HIV-1 fusion inhibitor C46. The transduced cells will be resistant to HIV infection by two complementary mechanisms: impaired binding of the virus to the cellular CCR5 co-receptor and decreased fusion of the virus as C46 interacts with gp41 and inhibits HIV infection. This phase I/II pilot study, also entitled GENHIV, will involve two French participating centers: Saint Louis Hospital and Necker Hospital in Paris. We plan to enroll five HIV-1-infected patients presenting with high-risk lymphoma and require a treatment with ASCT. The primary objective of this study is to evaluate the safety, feasibility, and success of engraftment of Cal-1 gene-transduced CD4+ T lymphocytes and CD34+ HSPCs