p68RacGAP Is a Novel GTPase-activating Protein That Interacts with Vascular Endothelial Zinc Finger-1 and Modulates Endothelial Cell Capillary Formation

Abstract

The endothelium is required for maintenance of vascular integrity and homeostasis during vascular development and in adulthood. However, little is known about the coordinated interplay between transcription factors and signaling molecules that regulate endothelial cell-dependent transcriptional events. Vascular endothelial zinc finger-1 (Vezf1) is a zinc finger-containing transcription factor that is specifically expressed within the endothelium during vascular development. We have previously shown that Vezf1 potently activates transcription of the endothelin-1 promoter. We now report the identification of p68RacGAP, a novel Vezf1-interacting 68-kDa RhoGAP domain-containing protein. p68RacGAP mRNA is highly expressed in vascular endothelial cells by Northern blot analysis, and immunohistochemical staining of adult mouse tissues identified p68RacGAP in endothelial cells, vascular smooth muscle cells, and epithelial cells in vivo. Rac1 and Vezf1 both bind avidly to p68RacGAP, suggesting that p68RacGAP is not only a GTPase-activating protein for Rac1 but that p68RacGAP may also be part of the protein complex that binds to and modulates Vezf1 transcriptional activity. Functionally p68RacGAP specifically activates the GTPase activity of Rac1 in vivo but not Cdc42 or RhoA. In addition, p68RacGAP potently inhibits Vezf1/DB1-mediated transcriptional activation of the human endothelin-1 promoter and modulates endothelial cell capillary tube formation. Taken together, these data suggest that p68RacGAP is a multifunctional regulatory protein that has a Rac1-specific GTPase-activating activity, regulates transcriptional activity of the endothelin-1 promoter, and is involved in the signal transduction pathway that regulates endothelial cell capillary tube formation during angiogenesis. The endothelium is required for maintenance of vascular integrity and homeostasis during vascular development and in adulthood. However, little is known about the coordinated interplay between transcription factors and signaling molecules that regulate endothelial cell-dependent transcriptional events. Vascular endothelial zinc finger-1 (Vezf1) is a zinc finger-containing transcription factor that is specifically expressed within the endothelium during vascular development. We have previously shown that Vezf1 potently activates transcription of the endothelin-1 promoter. We now report the identification of p68RacGAP, a novel Vezf1-interacting 68-kDa RhoGAP domain-containing protein. p68RacGAP mRNA is highly expressed in vascular endothelial cells by Northern blot analysis, and immunohistochemical staining of adult mouse tissues identified p68RacGAP in endothelial cells, vascular smooth muscle cells, and epithelial cells in vivo. Rac1 and Vezf1 both bind avidly to p68RacGAP, suggesting that p68RacGAP is not only a GTPase-activating protein for Rac1 but that p68RacGAP may also be part of the protein complex that binds to and modulates Vezf1 transcriptional activity. Functionally p68RacGAP specifically activates the GTPase activity of Rac1 in vivo but not Cdc42 or RhoA. In addition, p68RacGAP potently inhibits Vezf1/DB1-mediated transcriptional activation of the human endothelin-1 promoter and modulates endothelial cell capillary tube formation. Taken together, these data suggest that p68RacGAP is a multifunctional regulatory protein that has a Rac1-specific GTPase-activating activity, regulates transcriptional activity of the endothelin-1 promoter, and is involved in the signal transduction pathway that regulates endothelial cell capillary tube formation during angiogenesis. Angiogenesis, the sprouting of new blood vessels from an existing primary vascular complex, is a highly regulated process under normal conditions; it accounts for neovascularization that occurs during wound healing and during the female reproductive cycle. However, many disease processes are driven by persistent unregulated angiogenesis. These include diabetic retinopathies, inflammatory states such as rheumatoid arthritis, and tumor growth and metastasis (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7209) Google Scholar). The sprouting of new blood vessels during angiogenesis requires cell migration, a principal feature not only in embryonic development, where cell migration is required for movement of differentiating stem cells and for the coordinated movement required for proper vascular and organ formation, but also in maintenance of tissue integrity after an insult and in immune surveillance. Cell migration is usually initiated in response to extracellular cues that stimulate transmembrane receptors to initiate intracellular signaling. Cell movement requires the dynamic reorganization of the actin cytoskeleton, a process that is regulated by Ras homology (Rho) 1The abbreviations used are: Rho, Ras homology; Vezf1, vascular endothelial zinc finger-1; GAP, GTPase-activating protein; PBD, GTP-dependent binding domain for PAK1; RBD, Rhotekin binding domain; GST, glutathione S-transferase; GFP, green fluorescent protein; CMV, cytomegalovirus; MEC, myocardial endothelial cell; HUVEC, human umbilical vein endothelial cell; Ura, uracil; WT, wild type; TRITC, tetramethylrhodamine isothiocyanate; ERM, ezrin-radixin-moesin; PDGF, platelet-derived growth factor; LPA, lysophosphatidic acid; BCR, Breakpoint cluster region. 1The abbreviations used are: Rho, Ras homology; Vezf1, vascular endothelial zinc finger-1; GAP, GTPase-activating protein; PBD, GTP-dependent binding domain for PAK1; RBD, Rhotekin binding domain; GST, glutathione S-transferase; GFP, green fluorescent protein; CMV, cytomegalovirus; MEC, myocardial endothelial cell; HUVEC, human umbilical vein endothelial cell; Ura, uracil; WT, wild type; TRITC, tetramethylrhodamine isothiocyanate; ERM, ezrin-radixin-moesin; PDGF, platelet-derived growth factor; LPA, lysophosphatidic acid; BCR, Breakpoint cluster region. proteins. Rho proteins form a subgroup of the Ras superfamily of 20-30-kDa GTP-binding proteins that regulate a wide spectrum of cellular activities (2Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3840) Google Scholar). To date, 25 mammalian Rho GTPases have been identified with RhoA, Rac1, and Cdc42 being the most characterized (3Wherlock M. Mellor H. J. Cell Sci. 2002; 115: 239-240Crossref PubMed Google Scholar). These proteins act as molecular switches that cycle between an inactive, GDP-bound and an active, GTP-bound form. Among the regulatory proteins crucial for Rho GTPase function are guanine nucleotide exchange factors, which promote activation of Rho GTPase through GDP-GTP exchange, and GTPase-activating proteins (GAPs), which stimulate intrinsic GTPase activity and inactivate Rho GTPases (4Symons M. Settleman J. Trends Cell Biol. 2000; 10: 415-419Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 5Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (980) Google Scholar). The net effect of the activation and deactivation signal is a tightly regulated transduction mechanism. The RhoGAP family is defined by the presence of a 150-amino acid homology region, designated the RhoGAP domain, which is necessary and sufficient for GAP activity and shares at least 20% sequence identity among family members (6Moon S.Y. Zheng Y. Trends Cell Biol. 2003; 13: 13-22Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Specific roles for RhoGAP family members in endothelial cell events have not been clearly defined, although members of this family are likely to be crucial regulators of endothelial migratory events that are required during angiogenesis. The GTPase-induced GTP hydrolysis is a key event in intracellular signal transduction that controls numerous vital processes, including actin reorganization; gene transcription; vessel trafficking; cell proliferation, survival, and differentiation; and motility. The coordinated movement of cells depends on promotion of protrusions (lamellipodia) by Rac1 and formation of new cell adhesions to adjacent cell matrix at the leading edge. In addition, Rho-dependent actin-myosin contraction in the cell body and tail retraction are required to allow the body and rear of the cell to follow the extending front (7Ridley A.J. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar). Thus, the actin cytoskeleton provides the driving force for cell migration through its role in promoting both protrusive force toward and contraction and retraction away from a stimulus in response to extracellular cues. Connolly et al. (8Connolly J.O. Simpson N. Hewlett L. Hall A. Mol. Biol. Cell. 2002; 13: 2474-2485Crossref PubMed Scopus (147) Google Scholar) demonstrated that Rac is required for endothelial cell capillary assembly in vitro. In addition, WAVE2, a Rac effector protein crucial for Rac-induced membrane ruffling, assists in the formation of lamellipodia at the leading edge, and WAVE2 knock-out mice die at embryonic day 10 due to impaired angiogenesis that is presumably secondary to impaired migration of endothelial cells (9Yamazaki D. Suetsugu S. Miki H. Kataoka Y. Nishikawa S. Fujiwara T. Yoshida N. Takenawa T. Nature. 2003; 424: 452-456Crossref PubMed Scopus (190) Google Scholar). These observations are also consistent with studies demonstrating that Rac1-deficient embryos die before embryonic day 9.5 primarily due to deficient cell migration during and after gastrulation (10Sugihara K. Nakatsuji N. Nakamura K. Nakao K. Hashimoto R. Otani H. Sakagami H. Kondo H. Nozawa S. Aiba A. Katsuki M. Oncogene. 1998; 17: 3427-3433Crossref PubMed Scopus (276) Google Scholar). Taken together, these observations indicate that tight coupling exists between signaling events and endothelial cell migration for proper blood vessel growth, creating a need for careful regulation of Rho and related proteins. Vascular endothelial zinc finger-1 (Vezf1) was discovered by Stuhlman and colleagues (11Xiong J.W. Leahy A. Lee H.H. Stuhlmann H. Dev. Biol. 1999; 206: 123-141Crossref PubMed Scopus (61) Google Scholar) using a retroviral screen for developmentally regulated genes. Structurally Vezf1 and its human homologue, DB1 (12Koyano-Nakagawa N. Nishida J. Baldwin D. Arai K. Yokota T. Mol. Cell. Biol. 1994; 14: 5099-5107Crossref PubMed Scopus (42) Google Scholar), have six N-terminal Cys2/His2-type zinc finger motifs as well as a C-terminal proline-rich region characteristic of a transcriptional activation domain. Vezf1 mRNA expression is detected from embryonic day 7.25 to 11.5 in mouse embryos, and its expression overlaps with other endothelial (11Xiong J.W. Leahy A. Lee H.H. Stuhlmann H. Dev. Biol. 1999; 206: 123-141Crossref PubMed Scopus (61) Google Scholar). We have previously shown that endothelin-1 that is expressed in endothelial is of the that are potently and specifically by Vezf1 J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We have also shown that Vezf1 binds to the response within the endothelin-1 promoter. the endothelial expression of Vezf1, its expression with other endothelial and of Vezf1 transcriptional regulation of to Vezf1 as a to screen for other proteins that with Vezf1 and regulate blood vessel formation. In the process of for Vezf1-interacting discovered a novel RhoGAP p68RacGAP is expressed in endothelial has a Rac1-specific GTPase activity in and in and it inhibits transcriptional activation of endothelin-1 promoter activity. p68RacGAP modulates endothelial cell and angiogenesis in vivo that p68RacGAP is a between transcriptional endothelial cell and cell signaling required in adult angiogenesis. and have been (11Xiong J.W. Leahy A. Lee H.H. Stuhlmann H. Dev. Biol. 1999; 206: 123-141Crossref PubMed Scopus (61) Google Scholar, J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). expression glutathione proteins of wild and or and human Rho GTPases or the GTP-dependent binding domain for or Rhotekin or mammalian expression and have been K. K. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). p68RacGAP was the of to was by p68RacGAP was the of to an that p68RacGAP and green fluorescent protein and from and from and of myocardial endothelial cells and cells has been previously J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and endothelial vascular cells from the at at the of at from and from and was from screen was with the The six zinc finger domain of Vezf1 was with the binding domain in and used as a mouse day in which with the activation domain, was for in the of was The screen is on the between the and to 25 also from these from and To these or the binding and for activity using the and used to the and Northern from cells in was by and by J. T. Scholar). The of the was by and was was by and to a membrane by capillary The membrane was to in the with a of p68RacGAP from the adult organ blot used to tissue was from the between p68RacGAP and Vezf1 or GTPase binding with of Vezf1, the GTPase proteins or proteins as to and of from cells with or or and blot as previously Y. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). To the GAP activity of p68RacGAP of or with of with or by the cells Rac1 or Cdc42 in 10 and or cells in and 10 and with for at at for and with The was in and by to and proteins with or from the between p68RacGAP and Vezf1 in endothelial cells, cell from with to p68RacGAP as previously Y. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar) and with J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of was by as previously J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) to the that the The sequence was to with the and The sequence of the was by the for of p68RacGAP and Vezf1, cells in on with and using the after cells with of and with in for 10 was with and cells in primary in for and for with by in a an at the N-terminal region The was Cell in with or a of of in after the was with endothelial cell growth and for and cells in with and cells with as and by with or a and was with human endothelin-1 promoter and of p68RacGAP using as gene as previously J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). mouse tissues as previously Y. H. N. L. J. 2003; PubMed Scopus Google Scholar) and with a of of a by with Vezf1 in a of transcriptional regulation on the molecular between a transcription factor and regulatory proteins that to an transcription factor that binds to a promoter or for transcriptional activation or is known about regulatory proteins that with Vezf1 to its transcriptional or signaling We that such regulatory proteins likely be expressed in a and with the zinc finger of Vezf1 in a to screen of an embryonic day mouse and of the was to a with a tail that the sequence of a novel gene with the activation domain. was by of to the sequence of The a that of an and a protein of with a molecular of In transcription and using this as a a protein of the the The primary acid sequence a RhoGAP domain at its and a domain of proteins involved in membrane at its The RhoGAP domain, which is to other proteins the finger at that is characteristic of RhoGAP proteins and that is for GTPase have this protein We the of with activation of the gene by activation domain in activity was only detected the p68RacGAP activation domain was expressed in the zinc finger domain of Vezf1 with the binding domain. is that the of the p68RacGAP with its binding is that of with its well characterized protein. the binding of p68RacGAP, expressed in mammalian cells, with Vezf1, expressed as a protein in a binding shown in p68RacGAP avidly binds but not The of this that p68RacGAP is a binding for Vezf1 and the of We the that the between Vezf1 and p68RacGAP occurs in vivo in endothelial cells by cell from with to a of shown in Vezf1 is with p68RacGAP in endothelial that p68RacGAP is expressed in endothelial cells and that it with Vezf1, suggesting that p68RacGAP the transcriptional activity of this protein. of a in the of p68RacGAP, the expression of p68RacGAP mRNA in adult mouse tissues and cell by Northern blot with the C-terminal from screen the RhoGAP with mouse in of between and the of mRNA p68RacGAP mRNA was most in highly vascular and as well as in was expression of p68RacGAP in the and little or expression in muscle and The expression of p68RacGAP in highly vascular tissues that p68RacGAP be expressed in endothelial cells and endothelial to from cell that p68RacGAP was highly expressed in endothelial cell and endothelial vascular cells not expression in the cell suggesting that p68RacGAP is expressed in endothelial cells staining of and of adult mice with a of p68RacGAP that p68RacGAP is expressed in endothelial cells of myocardial tissues and epithelial cells of tissues a and We also expression of p68RacGAP in cells of the and in myocardial cells not with p68RacGAP and its binding to mouse tissues and that p68RacGAP tissues that p68RacGAP and p68RacGAP has a RhoGAP domain, to its RhoGAP activity. a the effect of on binding to RhoA, Rac1, and the characterized members of the These binding are on the that binds to the GTP-bound We or with or cell p68RacGAP Rac1 but not or Cdc42 a in these RhoA, Rac1, and Cdc42 as These clearly that p68RacGAP specifically binds Rac1, suggesting that p68RacGAP may cellular activity. To the GAP activity of p68RacGAP and its for Rac1 in cell from cells with wild Rho proteins or or and or with p68RacGAP, or shown in the presence of p68RacGAP, a of be with binding protein that only bind to the GTP-bound of Rac and Cdc42 but not suggesting that p68RacGAP to that it bind to p68RacGAP not have effect on the form of p68RacGAP GTP-bound Rac1 the well characterized that was used as a p68RacGAP not wild GTP-bound Cdc42 or RhoA, although these proteins by and and These indicate that p68RacGAP has a RhoGAP domain with a in vivo RhoGAP activity that specifically Rac1 but not Cdc42 or RhoA. The for p68RacGAP in has been that an finger is a that is required for the GTPase of proteins. The finger is an acid that to the of its GTP-binding protein through its to the the in the of the and the the intrinsic GTPase activity. The finger is within a of and is in p68RacGAP, and in other in this GAP activity binding K. A. Trends Sci. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We the of the finger of p68RacGAP by with with the activity of other known this acid the in vivo GAP activity of p68RacGAP of the p68RacGAP finger and the of Rac1 GAP activity provides that is a GTPase-activating protein for Rac1 and that its GAP function is or to that of other p68RacGAP of the of Rho GTPases is role in actin The activation of Rho, and Cdc42 by extracellular has been well characterized in a wide of cell including (2Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3840) Google Scholar). In these cells, activates Cdc42 to platelet-derived growth factor activates Rac to lamellipodia and membrane ruffling, and lysophosphatidic acid activates Rho leading to formation of actin and formation of A. 1998; PubMed Scopus Google Scholar). Thus, Rho, and Cdc42 regulate signal transduction membrane receptors to the assembly of actin To the effect of p68RacGAP on actin cells with an that p68RacGAP and that was with an of cells for 10 with 10 PDGF, or p68RacGAP to under that lamellipodia formation in the cells p68RacGAP not have effect on or formation. that p68RacGAP specifically inhibits that p68RacGAP has a Rac1-specific binding and GTPase-activating p68RacGAP with Vezf1 and of the p68RacGAP was on its with the endothelial transcription factor We have also shown that p68RacGAP avidly binds and with Vezf1 in and endothelial cells and that a of both regulatory by an for this in vivo a of between Rac signaling and transcriptional events within the We this by cells with and p68RacGAP or a related known as and by with observations J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), Vezf1 is to the in p68RacGAP and are to the and However, the expression of Vezf1 and p68RacGAP was that a of p68RacGAP with Vezf1 in the and that a of Vezf1 was in the of and Vezf1 not the intrinsic of protein The of this indicate that the of p68RacGAP and Vezf1 may protein suggesting a for of the regulatory proteins p68RacGAP and Vezf1, and that this may Rac signaling the transcriptional activity of have previously shown that Vezf1 potently the endothelin-1 promoter J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), this transcriptional activity as a to the effect of p68RacGAP on gene Vezf1 was with of p68RacGAP with endothelin-1 and the transcriptional activity of the endothelin-1 promoter was by activity. shown in p68RacGAP potently and the of the endothelin-1 promoter activity. However, p68RacGAP not have effect on the activity of or that p68RacGAP is not a of that p68RacGAP with Vezf1 and and that p68RacGAP specifically modulates transcriptional activity. p68RacGAP Cell of the during vascular development is the of endothelial cells from stem cells, the formation of and the of cells that vascular smooth muscle The migration of these cells in the and to the formation of vascular tissues and complex assembly and of the capillary is regulated by a wide of cues that to and and of actin that the that cell and has been shown previously that Rac1 regulates endothelial capillary tube assembly (8Connolly J.O. Simpson N. Hewlett L. Hall A. Mol. Biol. Cell. 2002; 13: 2474-2485Crossref PubMed Scopus (147) Google Scholar). on expression and to p68RacGAP endothelial cell assembly capillary We with p68RacGAP with or cells and on with cells a of capillary that of angiogenesis the but to formation of capillary under the that endothelial cell in cells that p68RacGAP inhibits Rac1 and the formation of protrusive lamellipodia formation necessary for the coordinated migration of endothelial cells in the presence of the extracellular We have a molecular and cellular to the function of Vezf1, a developmentally regulated endothelial transcription as part of a to the molecular that events that in of blood In to and J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), have for that may Vezf1 to angiogenesis. In the of these have discovered p68RacGAP, a GTPase that with Vezf1 in and in vivo and that also inhibits endothelial cell migration and tube formation. The RhoGAP Rho are in from to suggesting an role in cell the identification of the Breakpoint cluster region BCR, as a RhoGAP in D. S. M. N. J. Hall L. Hall A. Nature. PubMed Scopus Google Scholar), the of the 25 Rho GTPase (3Wherlock M. Mellor H. J. Cell Sci. 2002; 115: 239-240Crossref PubMed Google Scholar). that GAP proteins have a role in of The of the regulatory function of GAP proteins is by the of GAP function due to of the tumor a GAP that is a of the Ras signal transduction with in are at of in 10: Full Text PDF PubMed Scopus Google Scholar). In addition, in a RhoGAP protein that cell migration and to T. N. R. H. A. M. S. A. J. Nature. 1998; PubMed Scopus Google Scholar). studies of RhoGAP that are required for and the necessary role for these proteins in cellular Settleman J. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). it is likely that p68RacGAP regulates a of endothelial events regulated by Rac activity or that it Rac activation to other events within the that p68RacGAP endothelial cell tube formation in cells endothelial cell migration and angiogenesis are among the key cellular and processes regulated by this new the studies on cell migration in the of angiogenesis as a function of Rac within the it is to that Rac has roles within the of which may be specifically by such as Rac1 and are necessary of the vascular that an in response to and that is required for signaling of and other vascular growth factors J. J. Biol. 1999; Full Text Full Text PDF Scopus Google Scholar). cell cycle in endothelial cells is by activation of Rac A. S. M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and expression of endothelial proteins is also D. H. 2002; PubMed Scopus Google Scholar). is likely that function in cellular events in the endothelial such a has not been for family and is known about the role of in of endothelial of p68RacGAP may an to signaling to but tightly regulated events within the studies indicate that p68RacGAP is both a GAP for Rac family members and a Vezf1 these activities allow to a that form the for studies to the function of this new protein. is to that p68RacGAP provides a for the regulation of of signaling events within the In data suggest that p68RacGAP Rac signaling and transcriptional events. the function of Vezf1 within the endothelium is studies to suggest a role for this transcription factor in regulation of angiogenesis and in (11Xiong J.W. Leahy A. Lee H.H. Stuhlmann H. Dev. Biol. 1999; 206: 123-141Crossref PubMed Scopus (61) Google Scholar, J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, p68RacGAP may allow endothelial cells to at key and also to signaling and transcription during of blood between Rho signaling and transcription during vascular smooth muscle cell have been demonstrated in studies the of Rho-dependent on response factor activation M. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), a for such in vascular However, the that proteins with GAP activity are on to regulate activity, with transcription factors have not previously been in (6Moon S.Y. Zheng Y. Trends Cell Biol. 2003; 13: 13-22Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). To the of Vezf1 and p68RacGAP the in such a is where and p68RacGAP with a binding such as data indicate that p68RacGAP is within the which be consistent with its role in Rac activity. We have previously shown that Vezf1 is J. Y. T. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google data that of both regulatory proteins may to of both proteins of cellular is that p68RacGAP Vezf1 under to the of However, also a highly in expression of Vezf1 and p68RacGAP in the in the of expression is of the between Vezf1 and protein in the Rho signaling that was demonstrated by and colleagues Cell 1998; PubMed Scopus Google Scholar). with Vezf1 only in the and inhibits transcriptional Taken with the these data suggest that Vezf1 is to regulation that occurs in and adjacent to the the and a previously for activity of Rho family members are required to transcriptional activity. not the in it is that also and that activation of Vezf1 also Rac activity. These studies be and is known about Vezf1 is regulated at the cellular