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Doudeau Michel


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tél : 02.38.25.76.05 - fax : 02.38.25.55.83

Publications

2018   Références trouvées : 1

Vallée B., Cuberos H., Doudeau M., Godin F., Gosset D., Vourc’h P., Andres C. R. and Bénédetti H.  (2018)

IMK2-1, a new isoform of Human-LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than its two homologs, LIMK2a/2b.

Biochemical Journal (2018) 475 () 3745-3761.
LIMK1 and LIMK2 (LIMKs) are kinases that play a crucial role in cytoskeleton dynamics by independently regulating both actin filament and microtubule remodeling. LIMK1, and more recently LIMK2, have been shown to be involved in cancer development and metastasis, resistance of cancer cells to microtubule targeted treatments, neurological diseases, and viral infection. LIMKs have thus recently emerged as new therapeutic targets. Databanks describe three isoforms of human LIMK2 : LIMK2a, LIMK2b, and LIMK2-1. Evidence suggests that they may not have completely overlapping functions. We biochemically characterized the three isoforms to better delineate their potential roles, focusing on LIMK2-1, which has only been described at the mRNA level in a single study. LIMK2-1 has a Protein Phosphatase 1 (PP1) inhibitory domain at its C-terminus which its two counterparts do not. We showed that the LIMK2-1 protein is indeed synthesized. LIMK2-1 does not phosphorylate cofilin, the canonical substrate of LIMKs, although it has kinase activity and promotes actin stress fiber formation. Instead, it interacts with PP1 and partially inhibits its activity towards cofilin. Our data suggest that LIMK2-1 regulates actin cytoskeleton dynamics by preventing PP1-mediated cofilin dephosphorylation, rather than by directly phosphorylating cofilin as its two counterparts, LIMK2a and LIMK2b. This specificity may allow for tight regulation of the phospho-cofilin pool, determining the fate of the cell.

LIMK1 and LIMK2 (LIMKs) are kinases that play a crucial role in cytoskeleton dynamics by independently regulating both actin filament and microtubule remodeling. LIMK1, and more recently LIMK2, have been shown to be involved in cancer development and metastasis, resistance of cancer cells to microtubule targeted treatments, neurological diseases, and viral infection. LIMKs have thus recently emerged as new therapeutic targets. Databanks describe three isoforms of human LIMK2 : LIMK2a, LIMK2b, and LIMK2-1. Evidence suggests that they may not have completely overlapping functions. We biochemically characterized the three isoforms to better delineate their potential roles, focusing on LIMK2-1, which has only been described at the mRNA level in a single study. LIMK2-1 has a Protein Phosphatase 1 (PP1) inhibitory domain at its C-terminus which its two counterparts do not. We showed that the LIMK2-1 protein is indeed synthesized. LIMK2-1 does not phosphorylate cofilin, the canonical substrate of LIMKs, although it has kinase activity and promotes actin stress fiber formation. Instead, it interacts with PP1 and partially inhibits its activity towards cofilin. Our data suggest that LIMK2-1 regulates actin cytoskeleton dynamics by preventing PP1-mediated cofilin dephosphorylation, rather than by directly phosphorylating cofilin as its two counterparts, LIMK2a and LIMK2b. This specificity may allow for tight regulation of the phospho-cofilin pool, determining the fate of the cell.


2012   Références trouvées : 1

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.


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

Amiard, S ; Doudeau, M ; Pinte, S ; Poulet, A ; Lenain, C ; Faivre-Moskalenko, C ; Angelov, D ; Hug, N ; Vindigni, A ; Bouvet, P ; Paoletti, J ; Gilson, E ; Giraud-Panis, MJ  (2007)

A topological mechanism for TRF2-enhanced strand invasion

Nature Structural & Molecular Biology 14 (2) 147-154
Telomeres can fold into t-loops that may result from the invasion of the 3' overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.

Telomeres can fold into t-loops that may result from the invasion of the 3’ overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.


Mots-clés

Signalisation cellulaire