BACKGROUND
epithelial cells form barriers that can selectively regulate transport between different compartments.
an extensive network of junctions joins the cells into sheets and limits their mobility under normal circumstances.
however these cells do become migratory under both normal and pathological conditions.
epithelial cells must migrate during normal development and during the repair of damage.
in addition cancerous epithelial cells aberrantly activate pro-migratory pathways during metastasis.
epithelial migration involves a remodeling of the cell’s structure and behavior that starts by redirecting polarity in the direction of migration.
at the leading edge, actin rich protrusions and new cell-matrix adhesions anchor the cell to help propel the cell forward and the trailing edge retracts
<cit> .
epithelial cells can adopt several different types of migration depending on the biological circumstances at hand
<cit> .
during tissue morphogenesis, development and wound healing, epithelial cells move in sheets.
in this case, they maintain their cell-cell junctions
<cit> .
epithelial cells can also detach from each other and migrate individually during development or cancer metastasis
<cit> .
epithelial cell motility is initiated by various growth factors, such as hgf, egf, pdgf, vegf, csf- <dig>  fgf and tgf-β
<cit> .
hgf, also known as scatter factor , is a potent motogen for numerous epithelial cells expressing the c-met receptor
<cit> .
it induces scattering of multiple epithelial cell lines in 2d culture
<cit> .
when epithelial cells are grown in 3d cultures, addition of hgf to the growth media initiates tubulogenesis
<cit> .
hgf production by mesenchymal cells
<cit>  is increased in the event of injury to epithelia
<cit> .
in addition, hgf is involved in the invasive behaviors of some cancers
<cit> .
a number small gtpases, including members of the ras, rho and arf families regulate the cell shape changes that underlie motility.
there are six arf proteins, and arf <dig> in particular has been implicated in the regulation of cell shape and motility.
initially, arf <dig> was shown to regulate intracellular trafficking processes like endocytosis and recycling of membrane proteins
<cit> .
but it has subsequently been shown that arf <dig> is also involved in regulating the actin cytoskeleton during migration and phagocytosis
<cit> .
arf <dig> is required for hgf stimulated epithelial cell motility
<cit> .
hgf will induce mdck cells in culture to scatter from islands and increased arf <dig> activation is observed as soon as  <dig> hour post hgf treatment
<cit> .
more recently, we found that cnk3/ipcef, a scaffold that binds the arf-activating cytohesin proteins, is necessary for the activation of arf <dig> downstream of hgf and for hgf-stimulated migration
<cit> .
while there are  <dig> arf proteins in mammalian cells, a much larger number of proteins have been identified as arf activating guanosine exchange factors .
there are  <dig> identified sec <dig> arf gefs divided into  <dig> subfamilies.
it is thought that the various arf-gefs activate arfs at different subcellular locations and in response to different signals.
one class of arf-gefs, the cytohesins, has been extensively implicated in the regulation of cell shape and migration.
there are  <dig> cytohesins.
cytohesin <dig> and  <dig> are mostly hematopoetic whereas cytohesin 2/arno and cytohesin 3/grp- <dig> are ubiquitously expressed
<cit> .
overexpression of cytohesin 2/arno enhances cell motility in mdck cells
<cit> , and the phenotype is strikingly reminiscent of the response of these cells to hgf.
cytohesin 2-induced scattering of mdck cells requires the activation of rac <dig> by the rac-gef, dock180
<cit> .
cytohesin 2-dependent rac activation also depends on the coiled-coil domain in cytohesin 2
<cit> .
we previously found that cytohesin  <dig> and dock <dig> associate within a larger complex and can be co-immunoprecipitated.
ipcef/cnk <dig> and grasp, two scaffold proteins that both bind the coiled-coil domain of cytohesin  <dig>  are necessary for the assembly of this complex, and for cytohesin dependent rac activation
<cit> .
these data led us to propose a model where one scaffold recruits cytohesin  <dig> to the membrane in response to upstream signals, while the other acts as a bridge linking cytohesin  <dig> and dock <dig>  our demonstration that cnk3/ipcef is required for activation of arf <dig> by hgf suggests that it is the scaffold that recruits cytohesin  <dig> in response to upstream signals.
here, we test the hypothesis that grasp binds to both dock <dig> and cytohesin  <dig> and bridges the two gefs.
we find that grasp interacts with dock <dig> independently of its ability to bind cytohesin  <dig>  dock <dig> and grasp interact via the sh <dig> domain of dock <dig> and the proline rich domain of grasp.
furthermore, in addition to physically bridging cytohesin  <dig> and dock <dig>  grasp affects cell migration directly.
knockdown of grasp inhibits hgf-induced migration in mdck cells.
RESULTS
hgf-induced cell migration and rac activation are mediated by cytohesins and dock180
our previous work has shown that cytohesin  <dig> can promote epithelial migration by stimulating the activation of arf <dig> and the subsequent activation of rac1
<cit> .
cytohesin  <dig> induces rac activation by associating with the rac-gef dock <dig>  this interaction is mediated by the scaffolding proteins cnk <dig> and grasp, and knockdown of grasp or cnk <dig> inhibits cytohesin 2-induced rac activation
<cit> .
we have also found that knockdown of cnk <dig> inhibits hgf-stimulated arf <dig> activation and migration
<cit> .
these data suggest that the cytohesin-dependent arf-to-rac signaling module may act during hgf-stimulated motility.
in order to test this hypothesis we tested the effect of inhibition of cytohesins and dock <dig> on hgf-stimulated rac activation.
we incubated mdck cells with hgf for six hours and added secinh <dig>  a specific, small-molecule cytohesin inhibitor, or adenovirus encoding a catalytically inactive and dominant negative dock <dig> mutant  to selected samples.
we then evaluated the effect of inhibiting either gef on rac activation using a gst-pbd pulldown assay.
when the gef activity of either cytohesins or dock <dig> was impaired, rac activation by hgf was significantly impaired .
to further confirm that hgf-induced migration is dependent on the gef activities of cytohesins and dock <dig>  we evaluated wound healing in the presence of secinh <dig> and dockisp.
mdck cells were grown to form a monolayer around plugs in the platypus migration chambers.
the plugs were removed and the cells were allowed to migrate into the wound in the presence or absence of 1ng/ml of hgf.
the suppression of cytohesin or dock <dig> activity reduced the amount of wound filled in hgf-stimulated samples, but had no significant effect on basal migration .
both results are consistent with the conclusion that cytohesin dependent arf-to-rac signaling promotes epithelial cell migration downstream of hgf.
the scaffolding protein grasp binds independently to both cytohesin 2/arno and dock180
we know that cytohesin dependent arf-to-rac activation depends on the assembly of cytohesins and dock <dig> and scaffolding proteins into a multiprotein complex
<cit> .
we proposed that one of the scaffolds recruits cytohesin 2/arno in response to upstream signals while the other coordinates the association of dock <dig> with cytohesin 2/arno.
we have found that cnk <dig> plays a crucial role in initiating arf activation downstream of hgf
<cit> .
grasp on the other hand, seemed to be a good candidate for bridging dock <dig> and cytohesin  <dig>  grasp, also known as tamalin, consists of multiple protein-protein interaction domains , and binds to cytohesin 2/arno via its leucine rich region
<cit> .
if grasp acts to links together cytohesin- <dig> and dock <dig>  then it should bind directly to both gefs.
grasp binds to cytohesins via the direct interaction of its leucine rich domain with the cytohesin coiled-coil domain
<cit> .
we have previously shown that dock <dig> and grasp can be co-immunoprecipitated when co-expressed in mdck cells
<cit> .
therefore, we produced a series of truncations of grasp  in order to narrow down the domain that interacts with dock <dig> and to test if grasp can interact with dock <dig> independently of cytohesin 2/arno.
cells expressing wild type flag-tagged dock <dig> and the various truncations of ha-tagged grasp were lysed and the post-nuclear supernatant was subjected to immunoprecipitation .
dock <dig> co-immunoprecipitated with grasp regardless of the presence or absence of its cytohesin-binding domain .
by interacting with both dock <dig> and cytohesins via separate domains, grasp can act as a bridge to co-localize the gefs for arf <dig> and rac <dig>
the sh <dig> domain of dock <dig> and the alanine/proline rich region of grasp mediate the direct interaction between the two proteins
we previously demonstrated that the n-terminal  <dig> amino acids of dock <dig> were responsible for the interaction with cytohesin 2/arno
<cit> .
furthermore knockdown of grasp impairs interaction of dock <dig> and cytohesin 2/arno as well as cytohesin 2/arno induced rac <dig> activation.
if grasp is linking cytohesin 2/arno to dock <dig> then this same region should be required for the interaction of grasp and dock <dig>  we found that the n-terminus of dock <dig> does indeed mediate the interaction with grasp  supporting the hypothesis that grasp is bridging dock <dig> and cytohesin 2/arno.
furthermore, we further refined our localization of the region of dock <dig> that interacts with grasp by producing a truncation of the first  <dig> amino acids of dock <dig>  this portion of dock <dig> encodes a sh <dig> domain.
this dock <dig> truncation or full-length dock <dig> were transfected along with ha-tagged wild type grasp into hek  <dig> cells.
the ability of the dock <dig> truncation to interact with grasp was tested by co-immunoprecipitation.
wt grasp fails to interact with the truncation of dock <dig> that lacks the sh <dig> domain.
similarly we further defined the region of grasp required for this interaction by producing a truncation of grasp lacking the n-terminal  <dig> amino acids, which includes the proline-rich domain.
del82-grasp did not interact with dock <dig> .
therefore we conclude that the sh <dig> domain of dock <dig> preferentially binds to the proline-rich region of grasp.
however since these proteins were immunoprecipitated out of cell lysates the interaction could be indirect.
in order to determine if the interaction between dock <dig> and grasp is direct, we purified full length grasp fused to gst and the n-terminal  <dig> amino acids of dock <dig> fused to a flag tag from e.coli and allowed them to interact in vitro.
grasp interacted with the n terminal  <dig> amino acids of dock <dig> .
this confirms that grasp can directly interact with dock <dig> as well as with cytohesins and can act as the anchoring scaffolding protein that bridges cytohesin  <dig> and dock <dig> and promotes therefore arf <dig> to rac <dig> activation.
the co-immunoprecipitation experiments demonstrate that the dock <dig> sh <dig> domain can interact with grasp’s proline-rich domain.
we wanted to confirm that these proteins interact within intact cells.
the bi-molecular fluorescent complementation assay, or bifc for short, is based on the assembly of the n and c terminal fragments of venus into a fluorescent protein.
the individual fragments vn and vc are not fluorescent individually.
the detection of fluorescence depends on in vivo assembly of the fragments into a functional yfp molecule, which only occurs if they are fused to proteins that interact
<cit> .
we designed vn-dock <dig> and vc-grasp and co-transfected them into mdck cells.
cells were then fixed and stained as described in experimental methods.
grasp and cytohesin 2/arno are known to interact via the coiled-coil domain of cytohesin 2/arno and the leucine rich region in grasp
<cit> .
we used the interaction of vn-cytohesin  <dig> and vc-grasp as a positive control for the detection of yfp .
vn-dock <dig> and vc-grasp also produce yfp fluorescence when co-expressed in mdck cells .
vn-del80-dock <dig> fails to produce yfp when co-expressed with vc-grasp , confirming the co-immunoprecipitation data shown in figure
<dig>  when mdck cells were transfected with a plasmid containing the truncation of vc-grasp lacking the first  <dig> amino acids of grasp, and full length vn-dock <dig>  the two proteins also failed to show interaction by detection of yfp .
these results confirm that the sh <dig> domain of dock <dig>  located in the n-terminal  <dig> amino acids, and the ala/pro rich region of grasp mediate the interaction of the two proteins, thus confirming the observations made in the co-ip studies shown above.
grasp knockdown inhibits hgf stimulated migration and rac activation
our data suggest that cytohesin 2/arno and dock <dig> interact indirectly via grasp and that hgf stimulated rac activation and migration require both cytohesins and dock <dig>  therefore, we hypothesized that both hgf-induced migration and hgf-stimulated rac activation would be impaired by knockdown of grasp.
mdck cells were transfected with control or grasp targeting sirnas .
the hgf-stimulated and basal levels of migration of these cells were tested using both a wound healing assay and a transwell migration assay .
hgf-induced migration, but not basal migration, is significantly decreased after reducing grasp expression .
we hypothesize that this reduced migration is due reduced activation of rac in response to hgf.
we determined the amount of active rac in mdck cells transfected with control or grasp-targeting sirnas in the presence and absence of hgf stimulation.
the grasp knockdown cells had reduced levels of hgf-stimulated rac activation .
this reduced level of rac activation was not due to impaired cmet activation in response to hgf.
grasp knockdown cells had a similar pattern of cmet activation to that seen in control cells with a peak of cmet phosphorylation at  <dig> hour of hgf treatment .
we conclude that the grasp mediated association of cytohesins and dock <dig> is required for rac activation and migration in response to hgf.
discussion
in this study we demonstrated that grasp/tamalin, in addition to binding cytohesins, also interacts with dock <dig>  we have previously shown that both grasp and ipcef/cnk <dig> are required for the interaction of cytohesin 2/arno and dock <dig> and for cytohesin induced rac activation.
the need for two scaffolding proteins to bring about this pro-migratory protein complex was surprising.
we hypothesized that one scaffold recruits cytohesin 2/arno in response to upstream signals, while the other bridges cytohesin 2/arno and dock <dig>  further studies in our lab showed that the cnk3/ipcef acts downstream of hgf and c-met to activate arf6
<cit> .
therefore, we hypothesized that grasp is the scaffolding protein that brings the gefs of arf <dig> and rac <dig> together by binding to both.
we have shown here that grasp binds directly to dock <dig>  independently of its cytohesin binding domain.
these data provide evidence to support the model that grasp acts to bridge dock <dig> and cytohesin 2/arno.
truncations of dock <dig> and grasp allowed us to determine that the sh <dig> domain at the n-terminus of dock <dig> and the proline rich region at the n-terminus of grasp are the regions that interact.
furthermore, knockdown of grasp impairs hgf-stimulated migration.
these studies identify grasp as a lynchpin molecule that links cytohesin-dependent arf activation to dock <dig> dependent rac activation .
in addition to meditating protein-protein interactions, the sh <dig> domain present in some dock <dig> related proteins also regulates the gef activity of these proteins.
in fact, the sh <dig> domain has autoinhibitory properties
<cit> .
a dock <dig> mutant that lacks the sh <dig> domain activates rac <dig>  fold higher than the wild type dock <dig>  interaction of this domain with grasp might have the further effect of relieving this auto-inhibition.
dock-related proteins are atypical gefs for rho gtpases.
they differ from the bigger class of rho gefs known as the -containing family of gefs
<cit> .
there are  <dig> mammalian dock-related proteins divided into  <dig> subfamilies
<cit> .
the dock-a and dock-b subfamilies contain a sh <dig> domain at their n-termini that differentiates them from the dock-c and dock-d subfamilies.
the presence of a sh <dig> domain suggests that other members of the dock-a and dock-b families may be able to bind grasp and coordinate with cytohesin dependent arf activation.
the members of these sub-families include dock <dig>  dock <dig> and dock <dig> for the a subfamily and dock <dig> and  <dig> for the b subfamily.
dock <dig> is present primarily in hematopoetic cells, whereas dock <dig> is absent in these cells.
dock <dig> plays a role in lymphocyte development, homing, activation, adhesion, polarization and migration
<cit> .
there are hematopoetic isoforms of cytohesins, namely cytohesin  <dig> and cytohesin  <dig>  cytohesins can regulate integrin function and trafficking and therefore migration and adhesion in epithelial cells
<cit>  and in hematopoetic cells
<cit> .
no studies have yet investigated coordination between hematopoetic dock-related proteins and cytohesins, so it remains to be seen if similar mechanisms are at work in these cells.
dock <dig> in conjunction with nedd <dig> promotes emt, mesenchymal migration and metastasis of cancer cells
<cit> .
mesenchymal migration is characterized by the activation of rac and the production of lamellipodia.
this type of migration is consistent with hgf and cytohesin-induced motility.
furthermore activation of arf <dig> has also been correlated with enhanced metastasis
<cit> .
similarly to the effects of dock <dig> on hematopoetic cell migration, it is not yet known if cytohesins regulate nedd9/dock <dig> stimulated migration.
another area where both dock-a or dock-b proteins and cytohesins have been implicated is the neuronal development.
dock <dig> regulates neurite outgrowth
<cit> , while dock  <dig> has been implicated in the regulation of dendritic development
<cit> , rac-dependent cell migration
<cit>  and tumorigenesis
<cit> .
cytohesins have also been shown to regulate neurite outgrowth and dendritic branching in hippocampal neuron cultures
<cit> .
all of these functions involve cytohesins and the sh3-containing dock family members, and therefore might involve coordination by grasp.
CONCLUSIONS
the results reported here in conjunction with our earlier studies allow us to build a detailed model to explain how cytohesins coordinate with dock <dig> to promote rac activation and cell migration as shown in figure
<dig>  we propose that arf <dig> induced rac <dig> activation depends on the assembly of a multiprotein complex containing the gefs, cytohesin  <dig> and dock180/elmo <dig>  as well as the necessary scaffolding proteins that bring the complex together and to its proper location in the cell .
an external stimulus, in this case hgf, activates a cascade of events.
this leads to the recruitment of cytohesin 2/arno where it’s needed for the activation of arf6
<cit> .
the proper functioning of cytohesin 2/arno is not only dependent on its catalytic ability to facilitate the gdp to gtp exchange on arfs, but also on its interactions with scaffolding proteins
<cit> .
we demonstrated that cnk3/ipcef is the scaffolding protein necessary for activation of arf6
<cit> .
however, cytohesin  <dig> also binds another scaffolding protein, grasp/tamalin
<cit> .
and as we previously described, knockdown of the expression of either cnk3/ipcef or grasp/tamalin impairs the assembly and function the multiprotein complex described above and the ability of cytohesin 2/arno to stimulate rac
<cit> .
the data shown in this paper defines the role of grasp as providing a physical bridge between cytohesin  <dig> and dock <dig>  thus is coordinating the activation of arf <dig> and rac <dig>
the scaffolding protein grasp completes the picture on the large multiprotein complex that brings arf <dig> and rac <dig> together.
the proline rich region of grasp binds the sh <dig> domain of dock <dig>  while its leucine rich domain binds cytohesin  <dig>  grasp therefore helps gefs co-localize activation of gtpases, and promotes crosstalk between arf and rac.

 epithelial cells form barriers that can selectively regulate transport between different compartments. an extensive network of junctions joins the cells into sheets and limits their mobility under normal circumstances. however these cells do become migratory under both normal and pathological conditions. epithelial cells must migrate during normal development and during the repair of damage. in addition cancerous epithelial cells aberrantly activate pro-migratory pathways during metastasis. epithelial migration involves a remodeling of the cell’s structure and behavior that starts by redirecting polarity in the direction of migration. at the leading edge, actin rich protrusions and new cell-matrix adhesions anchor the cell to help propel the cell forward and the trailing edge retracts. epithelial cells can adopt several different types of migration depending on the biological circumstances at hand. during tissue morphogenesis, development and wound healing, epithelial cells move in sheets. in this case, they maintain their cell-cell junctions. epithelial cells can also detach from each other and migrate individually during development or cancer metastasis. epithelial cell motility is initiated by various growth factors, such as hgf, egf, pdgf, vegf, csf- fgf and tgf-β. hgf, also known as scatter factor, is a potent motogen for numerous epithelial cells expressing the c-met receptor. it induces scattering of multiple epithelial cell lines in 2d culture. when epithelial cells are grown in 3d cultures, addition of hgf to the growth media initiates tubulogenesis. hgf production by mesenchymal cells is increased in the event of injury to epithelia. in addition, hgf is involved in the invasive behaviors of some cancers. a number small gtpases, including members of the ras, rho and arf families regulate the cell shape changes that underlie motility. there are six arf proteins, and arf in particular has been implicated in the regulation of cell shape and motility. initially, arf was shown to regulate intracellular trafficking processes like endocytosis and recycling of membrane proteins. but it has subsequently been shown that arf is also involved in regulating the actin cytoskeleton during migration and phagocytosis. arf is required for hgf stimulated epithelial cell motility. hgf will induce mdck cells in culture to scatter from islands and increased arf activation is observed as soon as hour post hgf treatment. more recently, we found that cnk3/ipcef, a scaffold that binds the arf-activating cytohesin proteins, is necessary for the activation of arf downstream of hgf and for hgf-stimulated migration. while there are arf proteins in mammalian cells, a much larger number of proteins have been identified as arf activating guanosine exchange factors. there are identified sec arf gefs divided into subfamilies. it is thought that the various arf-gefs activate arfs at different subcellular locations and in response to different signals. one class of arf-gefs, the cytohesins, has been extensively implicated in the regulation of cell shape and migration. there are cytohesins. cytohesin and are mostly hematopoetic whereas cytohesin 2/arno and cytohesin 3/grp- are ubiquitously expressed. overexpression of cytohesin 2/arno enhances cell motility in mdck cells, and the phenotype is strikingly reminiscent of the response of these cells to hgf. cytohesin 2-induced scattering of mdck cells requires the activation of rac by the rac-gef, dock180. cytohesin 2-dependent rac activation also depends on the coiled-coil domain in cytohesin 2. we previously found that cytohesin and dock associate within a larger complex and can be co-immunoprecipitated. ipcef/cnk and grasp, two scaffold proteins that both bind the coiled-coil domain of cytohesin are necessary for the assembly of this complex, and for cytohesin dependent rac activation. these data led us to propose a model where one scaffold recruits cytohesin to the membrane in response to upstream signals, while the other acts as a bridge linking cytohesin and dock our demonstration that cnk3/ipcef is required for activation of arf by hgf suggests that it is the scaffold that recruits cytohesin in response to upstream signals. here, we test the hypothesis that grasp binds to both dock and cytohesin and bridges the two gefs. we find that grasp interacts with dock independently of its ability to bind cytohesin dock and grasp interact via the sh domain of dock and the proline rich domain of grasp. furthermore, in addition to physically bridging cytohesin and dock grasp affects cell migration directly. knockdown of grasp inhibits hgf-induced migration in mdck cells.  hgf-induced cell migration and rac activation are mediated by cytohesins and dock180 our previous work has shown that cytohesin can promote epithelial migration by stimulating the activation of arf and the subsequent activation of rac1. cytohesin induces rac activation by associating with the rac-gef dock this interaction is mediated by the scaffolding proteins cnk and grasp, and knockdown of grasp or cnk inhibits cytohesin 2-induced rac activation. we have also found that knockdown of cnk inhibits hgf-stimulated arf activation and migration. these data suggest that the cytohesin-dependent arf-to-rac signaling module may act during hgf-stimulated motility. in order to test this hypothesis we tested the effect of inhibition of cytohesins and dock on hgf-stimulated rac activation. we incubated mdck cells with hgf for six hours and added secinh a specific, small-molecule cytohesin inhibitor, or adenovirus encoding a catalytically inactive and dominant negative dock mutant to selected samples. we then evaluated the effect of inhibiting either gef on rac activation using a gst-pbd pulldown assay. when the gef activity of either cytohesins or dock was impaired, rac activation by hgf was significantly impaired. to further confirm that hgf-induced migration is dependent on the gef activities of cytohesins and dock we evaluated wound healing in the presence of secinh and dockisp. mdck cells were grown to form a monolayer around plugs in the platypus migration chambers. the plugs were removed and the cells were allowed to migrate into the wound in the presence or absence of 1ng/ml of hgf. the suppression of cytohesin or dock activity reduced the amount of wound filled in hgf-stimulated samples, but had no significant effect on basal migration. both results are consistent with the conclusion that cytohesin dependent arf-to-rac signaling promotes epithelial cell migration downstream of hgf. the scaffolding protein grasp binds independently to both cytohesin 2/arno and dock180 we know that cytohesin dependent arf-to-rac activation depends on the assembly of cytohesins and dock and scaffolding proteins into a multiprotein complex. we proposed that one of the scaffolds recruits cytohesin 2/arno in response to upstream signals while the other coordinates the association of dock with cytohesin 2/arno. we have found that cnk plays a crucial role in initiating arf activation downstream of hgf. grasp on the other hand, seemed to be a good candidate for bridging dock and cytohesin grasp, also known as tamalin, consists of multiple protein-protein interaction domains, and binds to cytohesin 2/arno via its leucine rich region. if grasp acts to links together cytohesin- and dock then it should bind directly to both gefs. grasp binds to cytohesins via the direct interaction of its leucine rich domain with the cytohesin coiled-coil domain. we have previously shown that dock and grasp can be co-immunoprecipitated when co-expressed in mdck cells. therefore, we produced a series of truncations of grasp in order to narrow down the domain that interacts with dock and to test if grasp can interact with dock independently of cytohesin 2/arno. cells expressing wild type flag-tagged dock and the various truncations of ha-tagged grasp were lysed and the post-nuclear supernatant was subjected to immunoprecipitation. dock co-immunoprecipitated with grasp regardless of the presence or absence of its cytohesin-binding domain. by interacting with both dock and cytohesins via separate domains, grasp can act as a bridge to co-localize the gefs for arf and rac the sh domain of dock and the alanine/proline rich region of grasp mediate the direct interaction between the two proteins we previously demonstrated that the n-terminal amino acids of dock were responsible for the interaction with cytohesin 2/arno. furthermore knockdown of grasp impairs interaction of dock and cytohesin 2/arno as well as cytohesin 2/arno induced rac activation. if grasp is linking cytohesin 2/arno to dock then this same region should be required for the interaction of grasp and dock we found that the n-terminus of dock does indeed mediate the interaction with grasp supporting the hypothesis that grasp is bridging dock and cytohesin 2/arno. furthermore, we further refined our localization of the region of dock that interacts with grasp by producing a truncation of the first amino acids of dock this portion of dock encodes a sh domain. this dock truncation or full-length dock were transfected along with ha-tagged wild type grasp into hek cells. the ability of the dock truncation to interact with grasp was tested by co-immunoprecipitation. wt grasp fails to interact with the truncation of dock that lacks the sh domain. similarly we further defined the region of grasp required for this interaction by producing a truncation of grasp lacking the n-terminal amino acids, which includes the proline-rich domain. del82-grasp did not interact with dock. therefore we conclude that the sh domain of dock preferentially binds to the proline-rich region of grasp. however since these proteins were immunoprecipitated out of cell lysates the interaction could be indirect. in order to determine if the interaction between dock and grasp is direct, we purified full length grasp fused to gst and the n-terminal amino acids of dock fused to a flag tag from e.coli and allowed them to interact in vitro. grasp interacted with the n terminal amino acids of dock. this confirms that grasp can directly interact with dock as well as with cytohesins and can act as the anchoring scaffolding protein that bridges cytohesin and dock and promotes therefore arf to rac activation. the co-immunoprecipitation experiments demonstrate that the dock sh domain can interact with grasp’s proline-rich domain. we wanted to confirm that these proteins interact within intact cells. the bi-molecular fluorescent complementation assay, or bifc for short, is based on the assembly of the n and c terminal fragments of venus into a fluorescent protein. the individual fragments vn and vc are not fluorescent individually. the detection of fluorescence depends on in vivo assembly of the fragments into a functional yfp molecule, which only occurs if they are fused to proteins that interact. we designed vn-dock and vc-grasp and co-transfected them into mdck cells. cells were then fixed and stained as described in experimental methods. grasp and cytohesin 2/arno are known to interact via the coiled-coil domain of cytohesin 2/arno and the leucine rich region in grasp. we used the interaction of vn-cytohesin and vc-grasp as a positive control for the detection of yfp. vn-dock and vc-grasp also produce yfp fluorescence when co-expressed in mdck cells. vn-del80-dock fails to produce yfp when co-expressed with vc-grasp, confirming the co-immunoprecipitation data shown in figure  when mdck cells were transfected with a plasmid containing the truncation of vc-grasp lacking the first amino acids of grasp, and full length vn-dock the two proteins also failed to show interaction by detection of yfp. these results confirm that the sh domain of dock located in the n-terminal amino acids, and the ala/pro rich region of grasp mediate the interaction of the two proteins, thus confirming the observations made in the co-ip studies shown above. grasp knockdown inhibits hgf stimulated migration and rac activation our data suggest that cytohesin 2/arno and dock interact indirectly via grasp and that hgf stimulated rac activation and migration require both cytohesins and dock therefore, we hypothesized that both hgf-induced migration and hgf-stimulated rac activation would be impaired by knockdown of grasp. mdck cells were transfected with control or grasp targeting sirnas. the hgf-stimulated and basal levels of migration of these cells were tested using both a wound healing assay and a transwell migration assay. hgf-induced migration, but not basal migration, is significantly decreased after reducing grasp expression. we hypothesize that this reduced migration is due reduced activation of rac in response to hgf. we determined the amount of active rac in mdck cells transfected with control or grasp-targeting sirnas in the presence and absence of hgf stimulation. the grasp knockdown cells had reduced levels of hgf-stimulated rac activation. this reduced level of rac activation was not due to impaired cmet activation in response to hgf. grasp knockdown cells had a similar pattern of cmet activation to that seen in control cells with a peak of cmet phosphorylation at hour of hgf treatment. we conclude that the grasp mediated association of cytohesins and dock is required for rac activation and migration in response to hgf. discussion in this study we demonstrated that grasp/tamalin, in addition to binding cytohesins, also interacts with dock we have previously shown that both grasp and ipcef/cnk are required for the interaction of cytohesin 2/arno and dock and for cytohesin induced rac activation. the need for two scaffolding proteins to bring about this pro-migratory protein complex was surprising. we hypothesized that one scaffold recruits cytohesin 2/arno in response to upstream signals, while the other bridges cytohesin 2/arno and dock further studies in our lab showed that the cnk3/ipcef acts downstream of hgf and c-met to activate arf6. therefore, we hypothesized that grasp is the scaffolding protein that brings the gefs of arf and rac together by binding to both. we have shown here that grasp binds directly to dock independently of its cytohesin binding domain. these data provide evidence to support the model that grasp acts to bridge dock and cytohesin 2/arno. truncations of dock and grasp allowed us to determine that the sh domain at the n-terminus of dock and the proline rich region at the n-terminus of grasp are the regions that interact. furthermore, knockdown of grasp impairs hgf-stimulated migration. these studies identify grasp as a lynchpin molecule that links cytohesin-dependent arf activation to dock dependent rac activation. in addition to meditating protein-protein interactions, the sh domain present in some dock related proteins also regulates the gef activity of these proteins. in fact, the sh domain has autoinhibitory properties. a dock mutant that lacks the sh domain activates rac fold higher than the wild type dock interaction of this domain with grasp might have the further effect of relieving this auto-inhibition. dock-related proteins are atypical gefs for rho gtpases. they differ from the bigger class of rho gefs known as the -containing family of gefs. there are mammalian dock-related proteins divided into subfamilies. the dock-a and dock-b subfamilies contain a sh domain at their n-termini that differentiates them from the dock-c and dock-d subfamilies. the presence of a sh domain suggests that other members of the dock-a and dock-b families may be able to bind grasp and coordinate with cytohesin dependent arf activation. the members of these sub-families include dock dock and dock for the a subfamily and dock and for the b subfamily. dock is present primarily in hematopoetic cells, whereas dock is absent in these cells. dock plays a role in lymphocyte development, homing, activation, adhesion, polarization and migration. there are hematopoetic isoforms of cytohesins, namely cytohesin and cytohesin cytohesins can regulate integrin function and trafficking and therefore migration and adhesion in epithelial cells and in hematopoetic cells. no studies have yet investigated coordination between hematopoetic dock-related proteins and cytohesins, so it remains to be seen if similar mechanisms are at work in these cells. dock in conjunction with nedd promotes emt, mesenchymal migration and metastasis of cancer cells. mesenchymal migration is characterized by the activation of rac and the production of lamellipodia. this type of migration is consistent with hgf and cytohesin-induced motility. furthermore activation of arf has also been correlated with enhanced metastasis. similarly to the effects of dock on hematopoetic cell migration, it is not yet known if cytohesins regulate nedd9/dock stimulated migration. another area where both dock-a or dock-b proteins and cytohesins have been implicated is the neuronal development. dock regulates neurite outgrowth, while dock has been implicated in the regulation of dendritic development, rac-dependent cell migration and tumorigenesis. cytohesins have also been shown to regulate neurite outgrowth and dendritic branching in hippocampal neuron cultures. all of these functions involve cytohesins and the sh3-containing dock family members, and therefore might involve coordination by grasp.  the results reported here in conjunction with our earlier studies allow us to build a detailed model to explain how cytohesins coordinate with dock to promote rac activation and cell migration as shown in figure  we propose that arf induced rac activation depends on the assembly of a multiprotein complex containing the gefs, cytohesin and dock180/elmo as well as the necessary scaffolding proteins that bring the complex together and to its proper location in the cell. an external stimulus, in this case hgf, activates a cascade of events. this leads to the recruitment of cytohesin 2/arno where it’s needed for the activation of arf6. the proper functioning of cytohesin 2/arno is not only dependent on its catalytic ability to facilitate the gdp to gtp exchange on arfs, but also on its interactions with scaffolding proteins. we demonstrated that cnk3/ipcef is the scaffolding protein necessary for activation of arf6. however, cytohesin also binds another scaffolding protein, grasp/tamalin. and as we previously described, knockdown of the expression of either cnk3/ipcef or grasp/tamalin impairs the assembly and function the multiprotein complex described above and the ability of cytohesin 2/arno to stimulate rac. the data shown in this paper defines the role of grasp as providing a physical bridge between cytohesin and dock thus is coordinating the activation of arf and rac the scaffolding protein grasp completes the picture on the large multiprotein complex that brings arf and rac together. the proline rich region of grasp binds the sh domain of dock while its leucine rich domain binds cytohesin grasp therefore helps gefs co-localize activation of gtpases, and promotes crosstalk between arf and rac. 
we now have found that rac activation in response to hepatocyte growth factor requires cytohesin and dock grasp/tamalin is one of the scaffolds that builds the complex containing cytohesin and dock we determine here that the ala/pro rich region of grasp directly interacts with the sh domain of dock by binding to both cytohesin 2/arno and dock grasp bridges the guanine nucleotide exchange factors that activate arf and rac, thereby promoting arf-to-rac signaling. grasp binds directly both cytohesin and dock to coordinate their activities, and by doing so promotes crosstalk between arf and rac. the transition of epithelial cells from their normal non-motile state to a motile one requires the coordinated action of a number of small gtpases. furthermore, we find that knockdown of grasp impairs hepatocyte growth factor -stimulated rac activation and hgf-stimulated epithelial migration. this crosstalk depends upon the assembly of a multi-protein complex that contains the arf-activating protein cytohesin 2/arno and the rac activating protein dock two scaffolding proteins that bind directly to cytohesin organize this complex.
 the transition of epithelial cells from their normal non-motile state to a motile one requires the coordinated action of a number of small gtpases. we have previously shown that epithelial cell migration is stimulated by the coordinated activation of arf and rac gtpases. this crosstalk depends upon the assembly of a multi-protein complex that contains the arf-activating protein cytohesin 2/arno and the rac activating protein dock two scaffolding proteins that bind directly to cytohesin organize this complex.  we now have found that rac activation in response to hepatocyte growth factor requires cytohesin and dock grasp/tamalin is one of the scaffolds that builds the complex containing cytohesin and dock we determine here that the ala/pro rich region of grasp directly interacts with the sh domain of dock by binding to both cytohesin 2/arno and dock grasp bridges the guanine nucleotide exchange factors that activate arf and rac, thereby promoting arf-to-rac signaling. furthermore, we find that knockdown of grasp impairs hepatocyte growth factor -stimulated rac activation and hgf-stimulated epithelial migration.  grasp binds directly both cytohesin and dock to coordinate their activities, and by doing so promotes crosstalk between arf and rac. cytohesingrasptamalindock180arf and rac1 