Melanoma AntiGEn D2 : a new nucleolar protein undergoing delocalization during cell cycle and after cellular stress

MAGED2 belongs to the Melanoma AntiGEn (MAGE) family of proteins. Actually, little is known on MAGED2 function. It contributes to the initiation of melanoma when its overexpression is associated with the mutation of BRAF and it increases apoptosis induced by TRAIL in a p53-dependent manner. MAGED2 was also shown to be a negative regulator of p53, bur we did not confirm this properties. Moreover, proteomic analyses detected numerous phosphorylated or acetylated residues in response to stess and within cell cycle suggesting its involvement in cellular signal transduction. We investigated the intra-cellular re-localization of MAGED2 during cell cycle and after genotixc stress. Both nucleolar and nuclear signals were identifed

Melanoma AntiGEn D2 (MAGED2): a new nucleolar protein undergoing re-localization after cellular stress

MAGED2 belongs to the Melanoma AntiGEn (MAGE) family of proteins. Actually, the only known function of this protein is its involvement in the p53 pathway. Indeed, MAGED2 could be a negative regulator of p53. It contributes to the initiation of melanoma when its overexpression is associated with the mutation of BRAF and it increases apoptosis induced by TRAIL in a p53 dependent manner. Moreover, phosphoproteomic experiments have shown that this protein is likely phosphorylated by kinases implicated in the DNA damage response (DDR). We decided to investigate the intra-cellular localization of MAGED2 in order to find new functions of this protein. In resting cell, MAGE D2 is detected is the nucleus, the nucleolus and the cytoplasm. We observed that MAGED2 localization change during cell cycle and genotoxic stress. Nuclear and nucleolar localization signals were identified. Though present in the nucleolus, the depletion of MAGED2 does not affect the structure of this organel

A unifying model of self-tolerance and autoimmunity to the neuroendocrine system

THYDI

Promoter specific regulation of NF-kappaB by RelA phosphorylation on Ser547

NF-KB (p50/RelA) controls the expression of numerous genes involved in inflammation, survival, proliferation, and cancer initiation and progression. Both classical NF-kB activation by pro-inflammatory cytokines and ATM-dependant activation by DNA damage require IKK activation and IkBa degradation. Stimuli dependant phosphorylation of p65 controls its transcriptional potential often in a gene specific manner. Previously, we have reported a direct interaction between RelA and ATM, and, demonstrated the in vitro phosphorylation of Ser547 by this kinase. A comparative transcriptomic analysis performed in HEK cells expressing either p65WT or p65S547A identified several differentially transcribed genes after an etoposide treatment. Substitution of S547 to alanine does not affect p65 binding on the kB site of the modulated promoters but it reduces p65 interaction with HDAC1. The resulting enhanced histone H3 acetylation increases gene transcription at some specific promoters. Our data indicate that ATM regulates a sub-set of NF-kB dependent genes after a genotoxic stress by direct phosphorylation of p65. Presently, we are investigating the impact of p65S547A/D mutations after the addition of TNFa in Mefs p65 KO complemented with HA-p65WT or S547A/D. No differences are observed in the degradation of IkBb or the nuclear translocation of p50/p65. However both basal and TNFa-induced transcription levels of some kB dependent genes are elevated in Mefs expressing p65S547D. The role of ATM in NF-kB activation by TNFa is analyzed

Melanoma antigen-D2 controls cell cycle progression and modulates the DNA damage response.

Overexpression of the ubiquitous type II melanoma antigen-D2 (MAGED2) in numerous types of cancer suggests that this protein contributes to carcinogenesis, a well-documented characteristic of other MAGE proteins. Modification of MAGED2 intracellular localization during cell cycle phases and following treatment with camptothecin (CPT) and phosphorylation by ATM/ATR following ionizing irradiation led us to investigate the molecular functions of MAGED2 in the cellular response to DNA damage. Cell cycle regulators, cell cycle progression, and bromodeoxyuridine (BrdU) incorporation were compared between MAGED2-sufficient and -depleted U2OS cells following exposure to CPT. At 24 h post-CPT removal, MAGED2-depleted cells had lower levels of p21 and p27, and there was an increase in S phase BrdU-positive cells with a concurrent decrease in cells in G2. These cell cycle modifications were p21-independent, but ATR-, SKP2-, and CDC20-dependent. Importantly, while MAGED2 depletion reduced CHK2 phosphorylation after 8 h of CPT treatment, it enhanced and prolonged CHK1 phosphorylation after a 24 h recovery period, indicating sustained ATR activation. MAGED2 depletion had no impact on cell survival under our experimental conditions. In summary, our data indicate that MAGED2 reduced CPT-related replicative stress, suggesting a role for this protein in genomic stability

The phosphorylation of RelA on Ser547 does not modulate NF-kB activation after TNFa treatment like after a genotoxic stress

NF-kB controls the expression of hundred of genes involved in inflammatory and innate responses, proliferation, survival, cancer initiation and progression. Numerous post-translational modifications of p65 modulating NF-kB transcriptional activity are known. We identified Ser547 as a new site of p65 phosphorylation targeted by ATM kinase, which coordinates the “DNA Damage Response” pathway in the event of DNA double-strand breaks. We demonstrated that the phosphorylation of Ser547 regulates the transcription of a sub-set of NF-κB dependent genes after genotoxic stress by modifying HDAC1 recruitment(1). Presently, we are investigating the role of this specific phosphorylation in an inflammatory context. We observe that the mutations of p65 (S547A or S547D) also affect the transcriptional potential of the NF-κB in a promoter specific manner after an exposition to TNFα and H2O2. The study of the molecular mechanism of this regulation after TNFα and H2O2 exposition are both in progress