Reference number : GC-2017-Q1-02474, amendment number : , agreement type : G, recipient legal name : Marignani, Paola (Dalhousie University), recipient country : CA, recipient province : nan, recipient city : Nova Scotia|Nouvelle-Écosse, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : The reorganization of cytoskeleton is a highly conserved molecular process that is required by single and multi-cell organisms to survive. The cytoskeleton is a complex structure comprised of hundreds of molecules that work synchronously to maintain the infrastructure of the cell or to move cells in response to stimuli. Although there are hundreds of molecules involved in the regulation of cell migration, our focus is on the phylogenetically conserved Vav family of guanine nucleotide exchange factors (GEFs) 1 .
 
 
In mammals there are three VAV genes, VAV1, VAV2 and VAV3 2-4 that vary in chromosomal location; human chromosomes 19, 9, and 1, respectively 2-4 . All Vav proteins contain modular domains including the prerequisite Dbl (DH) and pleckstrin homology (PH) cassettes that are necessary for the catalysis of Rho-GTPase from the inactive GDP to active GTP state. Previously, we determined that VAV2 is a GEF for Rac1 in vivo, leading to lamellapodia formation and cellular spreading 5 . In this study we determined that VAV2 was necessary for integrin-dependent activation of Rac1 in response to the fibronectin but not necessary for growth factor&ndash;dependent activation of Rac1 5,6 . In addition to DH/PH, VAV2 contains known signalling domains such as two Src-homology 3 domains (SH3) and a single SH2 domain. Both SH3 and SH2 domains have been conserved through metazoan evolution 7 . The presence of signalling domains suggest that VAV2 is capable of functions independent of GEF activity. 
 
To identify VAV2 functions independent of GEF activity towards Rac1 5 , we performed pulldown assays with the SH3-SH2-SH3 domains of VAV2, followed by mass spectrometry to identify putative binding partners. Of the putative partners identified, the Ezrin/radixin/moesin family (ERM) were found to contain proline rich regions, specifically in the C-terminus, suggestive of possible interaction with the SH3 domains in VAV2*. To build on this discovery, the objective of our study is to characterize the interaction between VAV2 and ERM family members and to understand the functional ramifications of this interaction.
 
 
*This discovery was funded by NSERC Discovery (2007-2012, extended 2013, and NSERC-USRAs)
 
 
 References 
1 Couceiro, J., Mar&iacute;a D. Mart&iacute;n-Bermudo, and Xos&eacute; R. Bustelo. Exp. Cell. Res. 308(2), 364-380 (2005).
 
2 Katzav, S., Martin-Zanca, D. &amp; Barbacid, M. Embo J 8, 2283-2290 (1989).
 
3 Trenkle, T., McClelland, M., Adlkofer, K. &amp; Welsh, J. Gene 245, 139-149 (2000).
 
4 Henske, E. P. et al. I Ann Hum Genet 59, 25-37 (1995).
 
5 Marignani, P. A. &amp; Carpenter, C. L. J Cell Biol 154, 177-186. (2001).
 
6 Arora, P. D., Marignani, P. A. &amp; McCulloch, C. A. Am J Physiol Cell Physiol 295, C130-137, (2008).
 
7 Cetkovic, H., Grebenjuk, V. A., Muller, W. E. G. &amp; Gamulin, V. Gene 342, 251-261 (2004)., program purpose french : The reorganization of cytoskeleton is a highly conserved molecular process that is required by single and multi-cell organisms to survive. The cytoskeleton is a complex structure comprised of hundreds of molecules that work synchronously to maintain the infrastructure of the cell or to move cells in response to stimuli. Although there are hundreds of molecules involved in the regulation of cell migration, our focus is on the phylogenetically conserved Vav family of guanine nucleotide exchange factors (GEFs) 1 .
 
 
In mammals there are three VAV genes, VAV1, VAV2 and VAV3 2-4 that vary in chromosomal location; human chromosomes 19, 9, and 1, respectively 2-4 . All Vav proteins contain modular domains including the prerequisite Dbl (DH) and pleckstrin homology (PH) cassettes that are necessary for the catalysis of Rho-GTPase from the inactive GDP to active GTP state. Previously, we determined that VAV2 is a GEF for Rac1 in vivo, leading to lamellapodia formation and cellular spreading 5 . In this study we determined that VAV2 was necessary for integrin-dependent activation of Rac1 in response to the fibronectin but not necessary for growth factor&ndash;dependent activation of Rac1 5,6 . In addition to DH/PH, VAV2 contains known signalling domains such as two Src-homology 3 domains (SH3) and a single SH2 domain. Both SH3 and SH2 domains have been conserved through metazoan evolution 7 . The presence of signalling domains suggest that VAV2 is capable of functions independent of GEF activity. 
 
To identify VAV2 functions independent of GEF activity towards Rac1 5 , we performed pulldown assays with the SH3-SH2-SH3 domains of VAV2, followed by mass spectrometry to identify putative binding partners. Of the putative partners identified, the Ezrin/radixin/moesin family (ERM) were found to contain proline rich regions, specifically in the C-terminus, suggestive of possible interaction with the SH3 domains in VAV2*. To build on this discovery, the objective of our study is to characterize the interaction between VAV2 and ERM family members and to understand the functional ramifications of this interaction.
 
 
*This discovery was funded by NSERC Discovery (2007-2012, extended 2013, and NSERC-USRAs)
 
 
 References 
1 Couceiro, J., Mar&iacute;a D. Mart&iacute;n-Bermudo, and Xos&eacute; R. Bustelo. Exp. Cell. Res. 308(2), 364-380 (2005).
 
2 Katzav, S., Martin-Zanca, D. &amp; Barbacid, M. Embo J 8, 2283-2290 (1989).
 
3 Trenkle, T., McClelland, M., Adlkofer, K. &amp; Welsh, J. Gene 245, 139-149 (2000).
 
4 Henske, E. P. et al. I Ann Hum Genet 59, 25-37 (1995).
 
5 Marignani, P. A. &amp; Carpenter, C. L. J Cell Biol 154, 177-186. (2001).
 
6 Arora, P. D., Marignani, P. A. &amp; McCulloch, C. A. Am J Physiol Cell Physiol 295, C130-137, (2008).
 
7 Cetkovic, H., Grebenjuk, V. A., Muller, W. E. G. &amp; Gamulin, V. Gene 342, 251-261 (2004)., agreement title english : Characterization of new VAV2-binding proteins., agreement title french : Characterization of new VAV2-binding proteins., agreement number : RGPIN, agreement value : 26000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2018-2019), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2018-2019), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada