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Vallée Méheust Béatrice


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tél : 02.38.25.76.05 - fax : 02.38.63.15.17

Publications

2015   Références trouvées : 1

Cuberos H., Vallée B., Vourc’h P., Tastet J., Andres C. R. and Bénédetti H.  (2015)

Roles of LIM kinases in central nervous system function and dysfunction

FEBS Letters (2016) 589 (24 part B) 3795-3806 - doi : 10.1016/j.febslet.2015.10.032
We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.

We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.


2012   Références trouvées : 4

Vallée B., Doudeau M., Godin F., Gombault A., Tchalikian A., de Tauzia M.L. and Bénédetti H.  (2012)

Nf1 RasGAP Inhibition of LIMK2 Mediates a New Cross-Talk between Ras and Rho Pathways.

PLoS One. 7 (10):e47283
BACKGROUND :

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. 

CONCLUSIONS/SIGNIFICANCE :

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.

BACKGROUND :
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.

CONCLUSIONS/SIGNIFICANCE :
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.

Tastet, J., Vourc’h, P., Laumonnier, F., Vallée, B., Michelle, C., Duittoz, A., Bénédetti, H. and Andres, C.R.  (2012)

LIMK2d, a truncated isoform of Lim kinase 2 regulates neurite growth in absence of the LIM kinase domain

Biochemical and Biophysical Research Communications 420 (2) 247–252
Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.

Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.

Beaufour, M, Godin, F, Vallée, B, Cadene, M. and Bénédetti, H.  (2012)

Interaction proteomics suggests a new role for the Tfs1 protein in yeast

Journal of Proteome Research (2012) 11 (6) 3211-3218
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.

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.

Godin, F., Villette, S., Vallée, B., Doudeau, M., Morisset-Lopez, S., Ardourel, M., Hevor, T., Pichon, C. and Bénédetti, B.  (2012)

A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line

Biochemical and Biophysical Research Communications 418 (4) 689-694
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).

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).


2009   Références trouvées : 2

Gombault, A., Warringer, J., Caesar, R., Godin, F., Vallee, B., Doudeau, M., Chautard, H., Blomberg, A. & Benedetti, H.  (2009)

A phenotypic study of TFS1 mutants differentially altered in the inhibition of Ira2p or CPY.

FEMS Yeast Res. 9, 867-874.

Gombault, A., Warringer, J., Caesar, R., Godin, F., Vallee, B., Doudeau, M., Chautard, H., Blomberg, A. & Benedetti, H.  (2009)

A phenotypic study of TFS1 mutants differentially altered in the inhibition of Ira2p or CPY.

FEMS Yeast Res. 9, 867-874.


2007   Références trouvées : 1

Gombault, A ; Godin, F ; Sy, D ; Legrand, B ; Chautard, H ; Vallee, B ; Vovelle, F ; Benedetti, H  (2007)

Molecular basis of the Tfs1/Ira2 interaction : A combined protein engineering and molecular modelling study

Journal of Molecular Biology 374 (3) 604-617
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.

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.


2005   Références trouvées : 1

Vallee, B ; Riezman, H  (2005)

Lip1p : a novel subunit of acyl-CoA ceramide synthase

Embo Journal 24 (4) 730-741
Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl- CoA- dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity.

Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl- CoA- dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity.


2003   Références trouvées : 2

Funato, K., Lombardi, R., Vallée, B. & Riezman, H.   (2003)

Lcb4p is a Key Regulator of Ceramide Synthesis from Exogenous Long Chain Sphingoid Base in Saccharomyces cerevisiae.

J.Biol. Chem. 278, 7325-7334.

Vallee, BS ; Coadou, G ; Labbe, H ; Sy, D ; Vovelle, F ; Schoentgen, F  (2003)

Peptides corresponding to the N- and C-terminal parts of PEBP are well-structured in solution : new insights into their possible interaction with partners in vivo

Journal of Peptide Research 61 (2) 47-57
Recently, it has been shown that mammalian PEBPs are implicated in several signalling pathways controlling the cellular cycle. In particular, during brain development, the N-terminal part of mammalian PEBP is specifically cleaved and the resulting 11 amino acid peptide stimulates the growth and activity of acetylcholinergic neurons. The crystallographic structure of bovine and human PEBPs has revealed that their N- and C-terminal parts are accessible and exposed to the solvent suggesting that they may be involved in specific interactions with cellular partners. We have chemically synthetized the two peptides corresponding to these terminal parts and studied their structure in solution by circular dichroism and NMR spectroscopies : both of them are well-structured.

Recently, it has been shown that mammalian PEBPs are implicated in several signalling pathways controlling the cellular cycle. In particular, during brain development, the N-terminal part of mammalian PEBP is specifically cleaved and the resulting 11 amino acid peptide stimulates the growth and activity of acetylcholinergic neurons. The crystallographic structure of bovine and human PEBPs has revealed that their N- and C-terminal parts are accessible and exposed to the solvent suggesting that they may be involved in specific interactions with cellular partners. We have chemically synthetized the two peptides corresponding to these terminal parts and studied their structure in solution by circular dichroism and NMR spectroscopies : both of them are well-structured.


2002   Références trouvées : 1

Funato, K ; Vallee, B ; Riezman, H  (2002)

Biosynthesis and trafficking of sphingolipids in the yeast Saccharomyces cerevisiae

Biochemistry 41 (51) 15105-15114


2001   Références trouvées : 2

Vallee, B ; Schorling, S ; Barz, WP ; Riezman, H ; Oesterhelt, D  (2001)

Lag1p and Lac1p are essential for the acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae

Molecular Biology of The Cell 12 (11) 3417-3427
Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.

Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.

Vallee, BS ; Tauc, P ; Brochon, JC ; Maget-Dana, R ; Lelievre, D ; Metz-Boutigue, MH ; Bureaud, N ; Schoentgen, F  (2001)

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes - Evidence of affinity for negatively charged membranes

European Journal of Biochemistry 268 (22) 5831-5841
The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed Of L-a -1,2-dimyristoylphosphatidylcholine, L-a -1,2-dimyristoylphosphatidylglycerol and L-a -1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing L-a -1,2-dimyristoylphosphatidylglycerol ; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed Of L-a -1,2-dimyristoylphosphatidylcholine, L-a -1,2-dimyristoylphosphatidylglycerol and L-a -1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing L-a -1,2-dimyristoylphosphatidylglycerol ; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane.


1999   Références trouvées : 1

Vallee, B ; Teyssier, C ; Maget-Dana, R ; Ramstein, J ; Bureaud, N ; Schoentgen, F  (1999)

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein

European Journal of Biochemistry 266 (1) 40-52
The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of ß-sheets, with Trp residues mostly localized in a hydrophobic environment ; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of ß-sheets, with Trp residues mostly localized in a hydrophobic environment ; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography.


1998   Références trouvées : 1

Serre, L ; Vallee, B ; Bureaud, N ; Schoentgen, F ; Zelwer, C  (1998)

Crystal structure of the phosphatidylethanolamine-binding protein from bovine brain : a novel structural class of phospholipid-binding proteins

Structure 6 (10) 1255-1265


Mots-clés

Chargé de recherche , Signalisation cellulaire