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Background and Purpose The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. Experimental Approach LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein–protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. Key Results LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Conclusions and Implications Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.
Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways.
METHODOLOGY/PRINCIPAL FINDINGS :
In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity : the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition : in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator.
Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases.
The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. In order to further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).
Tfs1p and Ylr179cp are yeast proteins belonging to the PEBP family. Tfs1p, but not Ylr179cp, has been shown to interact with and inhibit Ira2p, a GTPase-activating protein of Ras. Tfs1p has been shown to be a specific inhibitor of the CPY protease and the 3D structure of the complex has been resolved. To shed light on the molecular determinants of Tfs1p involved in the Tfs1/Ira2 interaction, the 3D structure of Ylr179cp has been modelled and compared to that of Tfs1p. Tfs1p point mutants and Tfs1 hybrid proteins combining regions of Tfs1p and Ylr179cp were also designed and their function was tested. Results, interpreted from a structural point of view, show that the accessibility of the surface pocket of Tfs1p, its N-terminal region and the specific electrostatic properties of a large surface region containing these two elements, play a crucial role in this interaction. (c) 2007 Elsevier Ltd. All rights reserved.
We compared deleted copies of the seven mauritiana subfamilies of mariner transposable elements in species of the Drosophilidae. All elements were detected by PCR using the inverted terminal repeats of the Mos1 element of Drosophila mauritiana as primers. A higher frequency of breakpoints in the 5’ part of the element compared to the 3’ part was observed. Of the 27 deletions, 9 (33%) occurred between short direct repeats (SDR) of 5 to 8 bp. The SDRs can be at or close to the breakpoints of the deletion. A deleted copy of D. simulans (St. Martin population) had three repeats of a motif present only once in the complete consensus sequence. The high frequency of SDRs at or near the breakpoints of the deletions strongly suggests that some of them do not occur at random. Mechanisms that might explain these deletions, such as unequal crossing-over, ectopic recombination, and abortive gap repair, are discussed.
We have performed a phylogenetic analysis of 59,mariner elements in 14 Drosophilidae species that are related to the active Drosophila mauritiana Mos1 element. This includes 38 previously described sequences and 21 new sequences amplified by PCR from 10 species. Most of the elements detected are nonfunctional due to several frameshifts and deletions. They have been subdivided into four groups according to specific signatures in the nucleotidic and amino acid sequences. The mean nucleotide diversity is 4.8 0.1% and reflects mainly the divergence of inactive elements over different periods. Although this probably gives rise to occasional homoplasies between distantly related taxa, the elements of each species remain grouped together. Horizontal transfer, reported previously between D. mauritiana and Zaprionus tuberculatus, can be extended to Z. verruca, while the Mos1-like element of Z. indianus belongs to another group. Interpretation of the phylogeny leads to a comparison of the influence of common ancestral sequences and putative horizontal transfers.
The mariner transposable elements of several natural populations of Drosophila teissieri, a rain-forest species endemic to tropical Africa, were studied. Natural populations trapped along a transect from Zimbabwe to the Ivory Coast were analyzed by Southern blotting, in situ hybridization, cloning, and sequencing of PCR products. The Brazzaville population had some full-length elements, while the remaining populations had mainly deleted elements. The main class of deleted elements lacked a 500-bp segment. A mechanism is proposed that could generate such elements rapidly. In situ hybridizations showed that there are no Mariner elements in pericentromeric heterochromatin. Finally, the phylogeny of the Mos1-like mariner full-length elements is consistent with vertical transmission from the ancestor of the melanogaster subgroup.
The distribution of the mariner transposable element among Drosophilidae species was investigated using three different techniques, i.e. squash blots, Southern blots and PCR amplification, using two sets of primers (one corresponding to the Inverted Terminal Repeats and the other to two conserved regions of the putative transposase). Our results and those of others show that the distribution of mariner is not uniform and does not follow the phylogeny of the host species. An analysis of geographical distribution, based on endemic species, shows that mariner is mainly present in Asia and Africa. At least two hypotheses may be proposed to explain the specific and geographical distributions of this element.
Assistant-ingénieur , Signalisation cellulaire