The scaffolding protein GRASP/Tamalin directly binds to Dock180 as well as to cytohesins facilitating GTPase crosstalk in epithelial cell migration
© Attar and Santy; licensee BioMed Central Ltd. 2013
Received: 27 August 2012
Accepted: 20 February 2013
Published: 26 February 2013
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 Dock180. Two scaffolding proteins that bind directly to cytohesin 2 organize this complex.
We now have found that Rac activation in response to hepatocyte growth factor (HGF) requires cytohesin 2 and Dock180. GRASP/Tamalin is one of the scaffolds that builds the complex containing cytohesin 2 and Dock180. We determine here that the Ala/Pro rich region of GRASP directly interacts with the SH3 domain of Dock180. By binding to both cytohesin 2/ARNO and Dock180, GRASP bridges the guanine nucleotide exchange factors (GEFs) that activate Arf and Rac, thereby promoting Arf-to-Rac signaling. Furthermore, we find that knockdown of GRASP impairs hepatocyte growth factor (HGF)-stimulated Rac activation and HGF-stimulated epithelial migration.
GRASP binds directly both cytohesin 2 and Dock180 to coordinate their activities, and by doing so promotes crosstalk between Arf and Rac.
KeywordsCytohesin GRASP Tamalin Dock180 Arf6 and Rac1
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-1, FGF and TGF-β [5–10]. HGF, also known as Scatter Factor (SF), is a potent motogen for numerous epithelial cells expressing the c-Met receptor . It induces scattering of multiple epithelial cell lines in 2D culture [12–14]. When epithelial cells are grown in 3D cultures, addition of HGF to the growth media initiates tubulogenesis [14, 15]. 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 Arf6 in particular has been implicated in the regulation of cell shape and motility. Initially, Arf6 was shown to regulate intracellular trafficking processes like endocytosis and recycling of membrane proteins [19, 20]. But it has subsequently been shown that Arf6 is also involved in regulating the actin cytoskeleton during migration and phagocytosis [21–26]. Arf6 is required for HGF stimulated epithelial cell motility . HGF will induce MDCK cells in culture to scatter from islands and increased Arf6 activation is observed as soon as 1 hour post HGF treatment [23, 26–28]. More recently, we found that CNK3/IPCEF, a scaffold that binds the Arf-activating cytohesin proteins, is necessary for the activation of Arf6 downstream of HGF and for HGF-stimulated migration .
While there are 6 Arf proteins in mammalian cells, a much larger number of proteins have been identified as Arf activating guanosine exchange factors (GEFs). There are 15 identified sec7 Arf GEFs divided into 5 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 4 cytohesins. Cytohesin1 and 4 are mostly hematopoetic whereas cytohesin 2/ARNO and cytohesin 3/Grp-1 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 Rac1 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 2 and Dock180 associate within a larger complex and can be co-immunoprecipitated. IPCEF/CNK3 and GRASP, two scaffold proteins that both bind the coiled-coil domain of cytohesin 2, 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 2 to the membrane in response to upstream signals, while the other acts as a bridge linking cytohesin 2 and Dock180. Our demonstration that CNK3/IPCEF is required for activation of Arf6 by HGF suggests that it is the scaffold that recruits cytohesin 2 in response to upstream signals.
Here, we test the hypothesis that GRASP binds to both Dock180 and cytohesin 2 and bridges the two GEFs. We find that GRASP interacts with Dock180 independently of its ability to bind cytohesin 2. Dock180 and GRASP interact via the SH3 domain of Dock180 and the proline rich domain of GRASP. Furthermore, in addition to physically bridging cytohesin 2 and Dock180, 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
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 Dock180 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 Dock180 with cytohesin 2/ARNO. We have found that CNK3 plays a crucial role in initiating Arf activation downstream of HGF .
The SH3 domain of Dock180 and the alanine/proline rich region of GRASP mediate the direct interaction between the two proteins
However since these proteins were immunoprecipitated out of cell lysates the interaction could be indirect. In order to determine if the interaction between Dock180 and GRASP is direct, we purified full length GRASP fused to GST and the N-terminal 80 amino acids of Dock180 fused to a Flag tag from E.coli and allowed them to interact in vitro. GRASP interacted with the N terminal 80 amino acids of Dock180 (Figure 3C). This confirms that GRASP can directly interact with Dock180 as well as with cytohesins and can act as the anchoring scaffolding protein that bridges cytohesin 2 and Dock180 and promotes therefore Arf6 to Rac1 activation.
GRASP knockdown inhibits HGF stimulated migration and Rac activation
In addition to meditating protein-protein interactions, the SH3 domain present in some Dock180 related proteins also regulates the GEF activity of these proteins. In fact, the SH3 domain has autoinhibitory properties . A Dock180 mutant that lacks the SH3 domain activates Rac1.5 fold higher than the wild type Dock180. 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 (DH-PH)-containing family of GEFs [38–40]. There are 11 mammalian Dock-related proteins divided into 4 subfamilies . The DOCK-A and DOCK-B subfamilies contain a SH3 domain at their N-termini that differentiates them from the DOCK-C and DOCK-D subfamilies. The presence of a SH3 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 Dock180, Dock2 and Dock5 for the A subfamily and Dock3 and 4 for the B subfamily.
Dock2 is present primarily in hematopoetic cells, whereas Dock180 is absent in these cells. Dock2 plays a role in lymphocyte development, homing, activation, adhesion, polarization and migration [41–44]. There are hematopoetic isoforms of cytohesins, namely Cytohesin 1 and Cytohesin 4. Cytohesins can regulate integrin function and trafficking and therefore migration and adhesion in epithelial cells [26, 45, 46] and in hematopoetic cells [47, 48]. 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.
Dock3 in conjunction with NEDD9 promotes EMT, mesenchymal migration and metastasis of cancer cells [49, 50]. 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 Arf6 has also been correlated with enhanced metastasis [27, 51]. Similarly to the effects of Dock2 on hematopoetic cell migration, it is not yet known if cytohesins regulate NEDD9/Dock3 stimulated migration.
Another area where both DOCK-A or DOCK-B proteins and cytohesins have been implicated is the neuronal development. Dock3 regulates neurite outgrowth [52, 53], while Dock 4 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 [57–59]. 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 Dock180 to promote Rac activation and cell migration as shown in Figure 5. We propose that Arf6 induced Rac1 activation depends on the assembly of a multiprotein complex containing the GEFs, cytohesin 2 and Dock180/Elmo1, as well as the necessary scaffolding proteins that bring the complex together and to its proper location in the cell (Figure 6).
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 2 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 2 and Dock180, thus is coordinating the activation of Arf6 and Rac1.
The scaffolding protein GRASP completes the picture on the large multiprotein complex that brings Arf6 and Rac1 together. The proline rich region of GRASP binds the SH3 domain of Dock180, while its leucine rich domain binds cytohesin 2. GRASP therefore helps GEFs co-localize activation of GTPases, and promotes crosstalk between Arf and Rac.
Antibodies and reagents
The mouse anti-HA (16B12) was purchased from Covance (Princeton, NJ). Goat anti-Dock180 antibodies (C-19, N-19) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse anti-Flag antibody was obtained from Sigma (St Louis, MO). DyLight™ 649 conjugated donkey anti-rabbit as well as DyLight™ 594 conjugated donkey anti mouse were purchased from Jackson ImmunoResearch laboratories (West Grove, PA). Anti phospho-cMet (Y1234/1235 clone D26) and Anti-cMet (clone 25H2) were purchased from Cell Sigaling (Danvers, MA). M2 anti-flag resin was purchased from Sigma (St. Louis, MO). And CL4B beads were obtained from Fluka (Germany) and GelCode Blue and glutathione-sepharose from Thermo Scientific (Rockford, IL). Western blots were processed by incubation with HRP-labeled secondary antibodies followed by Millipore Immobilion ECL reagent. Blots were developed using X-Ray film or with the c-Digit imaging system (LI-COR Biosciences, Lincoln, NE).
Tet-off MDCK cells were obtained from Clontech. 293H cells were obtained from Invitrogen (Carlsbad, CA). The T23 line of MDCK II as well as the 293H cells were maintained in DMEM supplemented with 10% FBS and penicillin, streptomycin and fungizone. All cells were maintained at 37°C and 5% CO2. Cell media was purchased from Mediatech (Manassas, VA) and FBS from Gemini (West Sacramento, CA).
siRNA mediated knockdown
The siRNA targeting human and dog GRASP (target sequence: GCTTTGAGATCCAGACTTA) was obtained from Dharmacon (Lafayette, CO) and BlockIt® control siRNA from Invitrogen (Carlsbad, CA). siRNAs were transfected into T23 cells using the Neon® system from Invitrogen (Carlsbad, CA). Transfections were carried out using the manufacturer’s suggested protocol: (1650 mV, 20 ms, 1 pulse) for MDCK cells.
Control or GRASP-targeting siRNAs were transfected into the cells using Neon. Cells (7×105) were seeded in the wells of the Oris migration chambers (Platypus technologies, Madison, WI) and incubated for 24 hours. Plugs were removed and media was refreshed before addition of HGF (1 ng/ml). Cells were allowed to migrate for another 18 hours. Cells were fixed with 4% paraformaldehyde and stained with a 0.1% solution of crystal violet. Images of the chambers were taken and cell migration was quantified by comparing the empty areas in the control versus GRASP knockdown on Image J. For the transwell migration assay, GRASP knockdown was performed as outlined above. The cells were allowed to migrate through the chambers as described in Attar et. al., 2012 .
Del82-GRASP forward: 5′-CAC GCTCGAGACCATGGCATACCCATACGATG TCCTGACTATGCAGGCTCAGGATTCCGTTG-3′ and Del80-Dock180: 5′-CGCGCGGCCGCATGGACTACAAAGACGATGAAGATAAAGTCATCCCGGGTGACCTCCCCC-3′.
HEK 293 cells were transfected (calcium phosphate) with plasmids encoding various forms of Dock180 and GRASP. They were allowed to express overnight. Cells were lysed in 50mM Tris, pH 7.5, 150mM NaCl, 10 mM NaF, 1 mM NaVO4, 10 mM sodium pyrophosphate, 1% Triton X-100 and 0.1 mM PMSF and 1 mg/ml each pepstatin, leupeptin, and antipain. Lysates were clarified with Sepharose CL-4B beads and unclarified materials removed after 10 minutes at 12,000 × g. Some of the cleared cell lysate was saved (3% was used for protein expression) and the remainder was then incubated rotating overnight 4°C with acid-washed M2-anti Flag resin. The process involved washing the resin three times with 1X TBS followed by 2 washes with 0.1M glycine pH 3.5 and finished with 3 more washes with TBS. IPs were washed three times with lysis buffer and once with TBS. Precipitated proteins were eluted into SDS-PAGE sample buffer and the samples were boiled for 3 minutes then analyzed by Western blot.
MDCK cells were transfected with siRNAs as described for migration assays. 48 hours later the cells were treated with 20 ng/ml HGF for the indicated times and harvested as described for immunoprecipitations. Unsolubilized material was removed by centrifugation and the supernatant Western blotted with anti-phospho-cMet and anti-cMet antibodies.
MDCK were plated on fibronectin coated glass coverslips (40 μg/ml). They were fixed and stained as previously described . Cells were observed and photographed using an Olympus IX81 equipped with SlideBook5 software for image processing.
Total RNA was isolated using the RNeasy kit (Qiagen). Custom primers to amplify bases 153–511 of canine GRASP, and ReadyMade primers to amplify GAPDH were obtained from Integrated DNA Technologies. RT-PCR was performed with 0.5 μg total RNA as template for GAPDH and 2 μg as template for GRASP using the Qiagen One-Step RT-PCR kit.
In Vitro binding assay
The coding sequence of GRASP was inserted to the pGEX2T plasmid to produce an in-frame fusion of GRASP to GST. This plasmid was transfected into BL21 E. coli. Cultures were grown to OD600 = 0.4 and then expression of GST-GRASP was induced by addition of IPTG for 4 hours. Cells were then lysed in PBS + 0.01% Triton X-100 for 10 minutes. The lysate was cleared by centrifugation and added to Glutathione-Sepharose beads. After rotation for 2 hours, beads were washed and GST-GRASP was eluted using a 10 mM solution of glutathione.
The coding sequence of the N-terminal 80 amino acids of Dock180 was fused in frame to a Flag tag using the bacterial expression plasmid pST50  and expression was induced as indicated above. Cells were lysed in 50 mM tris pH 7.5, 20% sucrose and 10% glycerol. The cleared lysate was added to M2-Flag gel and incubated rotating for 2 hours then washed with lysis buffer to remove unbound proteins.
Eluted GST-GRASP was added to either the Dock180 conjugated M2-Flag resin or to just the M2-Flag gel as a control. TBS was added and samples were rotated at 4°C for 2 hours washed and the proteins were then eluted in SDS-PAGE sample buffer. Proteins were detected by GelCode staining and Western blot using the goat anti-GRASP and the N-term goat anti-Dock180 antibodies.
Arf nucleotide binding site opener
Connector enhancer of KSR 3
Colony stimulating factor 1
Dbl homology–pleckstrin homology
Epithelial growth factor
Fibroblast growth factor
GTPase activating protein
Glyceraldehyde 3-phosphate dehydrogenase
Guanine nucleotide exchange factor
Grp1 associated scaffold protein
General receptor for phosphoinositides-1
Hepatocyte growth factor
Interacting protein for cytohesin exchange factor
Madin-Darby canine kidney
Neural precursor cell expressed developmentally down-regulated protein 9
Phosphate buffered saline
Platelet derived growth factor
Src homology 3
Transforming growth factor beta
Vascular endothelial growth factor.
The authors thank David T. White for conducting preliminary experiments and Song Tan for the bacterial expression plasmid. This work was supported by grants from the National Institutes of Health (R01 DK093729), American Cancer Society (RSG-09-168-01-CSM) and American Heart Association (SDG 0730229N) to L.C.S.
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