SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling

Immune modulatory properties of nanoparticles on human dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells that play a potent role as a key link between innate and adaptive immunity. Given the quintessential role of DCs in orchestrating immune responses, better understanding of conditions that control DC function could provide opportunities for developing new treatment strategies for infectious and autoimmune diseases. Recently, there is a growing interest to utilize nanoparticles (NPs) with diverse chemistry, size and other physicochemical attributes in vaccination, drug delivery systems, and diagnostics. There is emerging evidence for the effect of NPs size, shape, and other physiochemical properties on cellular responses; however, the impact of NPs on the immune system, and particularly the function and phenotype of DCs, is yet to be fully understood. The overall aim of this study was investigating the influence of different properties of NPs on DC phenotype, cytokine profile, function (e.g. endocytic ability) and metabolic profile. This was done by exposing DCs to a defined concentration of NPs, particularly, PLGA NPs, Silica (SNPs) and polystyrene (PS NPs), which were successfully fabricated or commercially sourced, respectively. The findings clearly demonstrated that particle size and the choice of material can affect different aspects of DC phenotype. While spherical Silica and PLGA NPs of 100 nm and 160 nm size range respectively do not change DC phenotype and function (endocytic ability), PS NPs of similar size and PLGA NPs of 500 nm size induce significant changes in DC phenotype and function. PS NPs of 150 nm and 200 nm size significantly suppress the expression of mannose receptor (MR or CD206) on DCs by around 90%-80% without affecting their viability, maturation status or cytokine profile. In addition, PLGA NPs of 500 nm size induced DC maturation as evidenced by high levels of CD83 expression. Accordingly, the impact of NP properties on one DC key function, namely endocytic ability, was investigated. Interestingly, the data showed that PS NPs (150 nm/200 nm) and PLGA NPs (500 nm) significantly influenced this function. While PLGA NPs (160 nm) and SNPs (100 nm/500 nm) did not induce changes in DC endocytic ability. Furthermore, with regard to NP cellular uptake by antigen presenting cells, our data demonstrated that specific NP size and material (PLGA NPs 120 nm) reflect a preferential uptake by DCs compared to 500 nm PLGA NPs. By contrast, macrophages (Mϕs) showed similar trend of uptake for both PLGA NP sizes. Our data also showed the role of NP surface modification on cellular uptake. Modified SNPs to positive charge showed high percentage of uptake by DCs. Examining the impact of NP material type (PLGA NPs and PS NPs) on DC metabolic profile could provide valuable information about the impact of NPs on overall DC function in an unbiased manner. The metabolomics profile data showed a significant increase in glycolysis pathway upon stimulation by PLGA NPs and PS NPs, as indicated by lactate production. Also, significant changes were observed in DC purine and amino acid metabolism upon PLGA NPs and PS NPs treatment, respectively. In conclusion, this study gives new insights into understanding how DCs react to different properties of NPs, which can pave the way for the rational design of NPs with tuneable immune-modulatory properties for immunotherapy applications

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin–proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs

Implication de la voie MAPK ERK5 et de la famille TORC dans l'expression de Cycline D1 et le cancer du sein

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Modifications post-traductionnelles au niveau du promoteur du gène c-fos (Phosphorylations et SUMOylation)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Fonction de coactivateur in vivo et in vitro (lien entre la signalisation intracellulaire et la régulation génique)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Erk5 controls Slug expression and keratinocyte activation during wound healing.

International audienceReepithelialization during cutaneous wound healing involves numerous signals that result in basal keratinocyte activation, spreading, and migration, all linked to a loosening of cell-cell adhesion structures. The transcription factor Slug is required for this process, and EGF treatment of human keratinocytes induced activating phosphorylation of Erk5 that coincides with slug transcription. Accordingly, ectopic activation of Erk5 led to increased Slug mRNA levels and faster wound healing, whereas keratinocyte migration was totally blocked by Erk5 pathway inhibition. Expression of a shRNA specific for Erk5 strongly diminished Erk5 levels in keratinocytes and significantly decreased their motility response to EGF, along with induction of Slug expression. These Erk5-deprived keratinocytes showed an altered, more compact morphology, along with disruption of desmosome organization. Accordingly, they displayed an altered ability to form cell aggregates. These results implicate a novel EGFR/Erk5/Slug pathway in the control of cytoskeleton organization and cell motility in keratinocytes treated with EGF