Nf1 RasGAP inhibition of LIMK2 mediates a new cross-talk between Ras and Rho pathways.
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.


RESULTS
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
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.
Centre de Biophysique Moléculaire, University of Orléans and Institut National de la Santé et de la Recherche Médicale (INSERM), Orléans, France.
Béatrice
B
Vallée
Michel
M
Doudeau
Fabienne
F
Godin
Aurélie
A
Gombault
Aurélie
A
Tchalikian
Marie-Ludivine
ML
de Tauzia
Hélène
H
Bénédetti
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D004789:Enzyme Activation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D054461:Lim Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008954:Models, Biological; D025542:Neurofibromin 1 / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D018631:ras Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D025542:Neurofibromin 1; C515892:LIMK1 protein, human; C102895:LIMK2 protein, human; D054461:Lim Kinases; D054460:rho-Associated Kinases; D018631:ras Proteins; D020741:rho GTP-Binding Proteins