Human Molecular Genetics, 2003, Vol. 12, No. 2 155-167
© 2003 Oxford University Press
Interaction of 
PIX (ARHGEF6) with ß-parvin (PARVB) suggests an involvement of PIX in integrin-mediated signaling
Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Butenfeld 42, 22529 Hamburg, Germany
Received September 19, 2002; Accepted November 16, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Members of the Rho GTPase family are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton. It is now well established that these small GTPases are also crucial for neuronal morphogenesis and connectivity. Moreover, mutations in ARHGEF6 (also known as
PIX or Cool-2 ), encoding a Rac1/Cdc42-specific guanine nucleotide exchange factor, have been implicated in X-linked mental retardation. In an attempt to get insight into the biological function of ARHGEF6 and the upstream signaling cascades leading to its activation, we used the full-length coding region of ARHGEF6 as bait in yeast-two hybrid screens and identified PARVB (ß-parvin or affixin) as a novel binding partner. The interaction was confirmed by co-immunoprecipitation and GST pull-down. We showed by immunofluorescence that ARHGEF6 and PARVB co-localize at the cell periphery to lamellipodia and ruffles in well-spread and actively spreading cells adhered to fibronectin. In addition, interaction of ARHGEF6 to ARHGEF7 (ßPIX or Cool-1), a close homolog of ARHGEF6, was confirmed. In in vivo assays, two ARHGEF6 mutations identified previously in patients with X-linked non-specific mental retardation, ARHGEF6 
aa56–83 and aa396–776, abolished interaction of ARHGEF6 to PARVB. Binding between ARHGEF6 and ARHGEF7 was not affected by ARHGEF6 aa56–83 but did not occur with ARHGEF6 aa396–776. These data suggest that both the N-terminal calponin homology (CH) and C-terminal coiled-coil domains are necessary for the ARHGEF6-PARVB binding. In contrast, it seems that only the coiled-coil domain is required for the interaction and heterodimerization of ARHGEF6 and ARHGEF7. PARVB is known to interact with integrin-linked kinase (ILK) and is involved in the early stage of cell–substrate interaction through integrins. The identification of PARVB as an ARHGEF6 interacting partner together with the co-localization of ARHGEF6 and ILK in spreading cells suggest that ARHGEF6 is involved in integrin-mediated signaling leading to activation of the GTPases Rac1 and/or Cdc42. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Small GTPases function as molecular switches, being inactive when bound to GDP and active when bound to GTP. Rho GTPases, like Cdc42, Rac and Rho, participate in the regulation of the actin cytoskeleton (1) that leads to various forms of polarized outgrowth, including formation of filopodia, lamellipodia and stress fibers (2). These signal transduction pathways are believed to be crucial for neuronal morphogenesis and connectivity (3–5). Although details of the molecular biology of Rho signaling in the central nervous system are still unknown, recent data have shed light on the connection between Rho GTPases, the cellular basis of cognition, and mental retardation (6). Mutations in ARHGEF6, encoding a Rac1/Cdc42 guanine exchange factor, were found in patients with X-linked non-specific mental retardation (MRX) (7). Remarkably, ARHGEF6 as well as two other MRX genes, OPHN1 (oligophrenin-1) (8) and PAK3 (p21-activating kinase 3) (9), encode proteins that interact with Rho GTPases. However, the molecular mechanism by which mutations in these genes result in deficiencies in neuronal morphology and/or connectivity remains to be elucidated.
Primary insights into the ARHGEF6 function came from its identification as a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), also called PIX or Cool-2, that binds tightly to PAK (10,11) and, thereby, strongly activates it (12,13). Upstream activators of ARHGEF6/PIX are the platelet-derived growth factor (PDGF) receptor and EphB2 receptor (14). Although the GEF activity of ARHGEF6/PIX is very low compared with other GEFs (11), ARHGEF6/PIX can apparently initiate morphological changes such as membrane ruffles (15). The C-terminal coiled-coil domain of ARHGEF6/PIX drives both dimerization and targeting to the cell membrane. A model has been proposed in which dimerization of PIX is necessary to form multimeric complexes with PAK, GIT1/p95PKL and associated proteins, such as paxillin, for example, all associated with the membrane (15–17).
Integrins are transmembrane receptors that represent links between the extracellular matrix and the actin cytoskeleton (18–20). Recently, it has been shown that Rac and Cdc42 are also activated after signaling through integrins and that both proteins are involved in integrin-induced cell spreading (21,22). Nevertheless, the means by which integrin-mediated adhesion activates Rac and Cdc42 is unknown. Remarkably, it was shown that ARHGEF6/PIX activates PDGF-induced spreading of Xenopus mesoderm cells on fibronectin, presumably through phosphatidylinositol 3-kinase (PI3-kinase) (14), suggesting an involvement of this Rac/Cdc42 specific-GEF in integrin signaling.
Clearly, it is important to determine which molecules downstream from integrins lead to GEF activation. In order to gain insight into ARHGEF6/PIX function and its role in neuronal development, we screened for interacting proteins and report here ß-parvin (PARVB), a novel ARHGEF6 binding partner.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of PARVB as an ARHGEF6 binding partner
We used the SOS recruitment system (SRS) for identification of ARHGEF6 protein interaction partners. The SRS system has been developed to provide an alternative approach to the two-hybrid system and extends the possibility of exploring protein–protein interactions in the cytoplasm. We performed several rounds of screens with full-length ARHGEF6 as bait and human fetal brain cDNAs cloned in the prey expression plasmid. Protein expression of all ARHGEF6 bait constructs was confirmed by immunoblotting of yeast lysates with an anti-hSos antibody (data not shown). A total of 152 clones were obtained that restored growth of cdc25H cells in an ARHGEF6-dependent manner. Protein interactions of putative positive colonies were confirmed by re-transformation of cdc25H cells using the pMyr-cDNA plasmids together with pSos-ARHGEF6 or pSos without insert. Only those clones were defined positive that showed galactose-dependent growth at the restrictive temperature of 37°C (Fig. 1A). DNA sequence analysis and BLASTN database searches at the NCBI Network Service revealed that the truly positive isolates represented 32 independent cDNA clones. As one of the putative binding partners of ARHGEF6, we identified PAK-interacting exchange factor beta, also known as ßPIX or ARHGEF7, a close homolog of ARHGEF6/
PIX. This interaction has recently been documented, indicating that ARHGEF6/7 heterodimers can be formed (15). It is of interest to note that only the C-terminal part of ARHGEF7, including residues 452–646 but lacking amino acids 526–555 encoded by a single exon, has been found to interact with full-length ARHGEF6/PIX (Fig. 1B).
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Remarkably, we obtained two independent cDNAs containing the complete open reading frame of PARVB as well as 12 bp of 5'UTR and
500 bp of 3'UTR (GenBank accession no. AF237769). PARVB encodes a 42 kDa focal adhesion protein, called ß-parvin or affixin (23,24), that contains two unconventional calponin homology (CH) domains in tandem arrangement and thus belongs to the -actinin superfamily (Fig. 1B) (reviewed in 25). To determine which domain of ARHGEF6/PIX is interacting with PARVB, we tested various ARHGEF6 bait constructs in the SRS system. Only wild-type ARHGEF6 and ARHGEF6 
aa56–83 yielded interaction with PARVB (Fig. 1C). All yeast transformants containing pMyr-PARVB together with a pSos-ARHGEF6 construct that lacks the coiled-coil domain showed no growth on galactose media at 37°C (Fig. 1C). Recently, it has been reported that the coiled-coil domains of ARHGEF6 and 7 are responsible for dimerization (15). It is tempting to speculate that dimerization of ARHGEF6 is required for binding PARVB, at least in the SRS system. Moreover, cdc25H cells expressing the ARHGEF6 aa56–83 mutant protein showed a significantly reduced growth on galactose media at 37°C (Fig. 1C), indicating that the CH domain of ARHGEF6 is also implicated in mediating binding to PARVB.
Confirmation of the ARHGEF6-PARVB interaction by in vivo binding assays
Western analysis of cell lysates has shown earlier that ARHGEF6/PIX is not present in COS-7, HeLa and NIH3T3 cells (15). However, using the same polyclonal anti-PIX antibodies, we detected both ARHGEF6 and two variants of ARHGEF7 (ß1PIX and ß2PIX) (15) in CHO-K1 cells by western analysis (Fig. 2).
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To confirm the ARHGEF6-PARVB association in vivo, we expressed full-length FLAG-PARVB in CHO-K1 cells and performed immunoprecipitation using an anti-FLAG antibody directly coupled to agarose CL-4B beads. Co-precipitation of endogeneous ARHGEF6/
PIX was detected by immunoblot using polyclonal anti-PIX antibodies. ARHGEF6 was present in immunoprecipitates of cell lysates transfected with PARVB cDNA (Fig. 2), but not in those obtained after transfection with Flag-vector (Fig. 2), suggesting that ARHGEF6 interacts with PARVB in vivo. In addition, an in vivo GST pull-down assay was performed using COS-7 cells co-transfected with GST-PARVB and full-length HA-ARHGEF6. GST-PARVB was able to trap HA-ARHGEF6, whereas GST alone was not (Fig. 3B).
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Effects of ARHGEF6 mutations on the binding to PARVB
We analyzed two ARHGEF6 mutant mRNAs that have been detected previously in patients with X-linked mental retardation. Mature transcripts were present both in skin fibroblasts and lymphoblastoid cells (7), indicating that the mRNAs were stable and that subsequent translation could lead to expression of the corresponding protein. Mutant proteins HA-ARHGEF6
aa56–83 and HA-ARHGEF6 aa396–776 were stable and overexpressed at the same level as wild-type protein in COS-7 cells (Fig. 3A). However, both proteins failed to precipitate with GST-PARVB (Fig. 3B), indicating that these ARHGEF6 variants were not able to interact efficiently with PARVB in vivo anymore. Moreover, it seems that both the C-terminal coiled-coil and the N-terminal CH domains of ARHGEF6 are necessary for the interaction with PARVB.
We also confirmed the interaction detected in the SRS system between ARHGEF6 and full-length ARHGEF7 by GST pull-down (Fig. 3B), and analyzed the binding ability of the two ARHGEF6 mutant proteins. Remarkably, ARHGEF6 lacking 28 amino acids in the CH domain (ARHGEF6 aa56–83) does still precipitate with GST-ARHGEF7, whereas HA-ARHGEF6 aa396–776 failed to interact with GST-bound ARHGEF7 (Fig. 3B). Taken together, these data suggest that in vivo interaction of PARVB with ARHGEF6 depends on the presence of the N-terminal calponin homology domain, whereas ARHGEF6/7 heterodimerization does not. Nonetheless, the latter interaction requires the coiled-coil domain of ARHGEF6 at the C-terminus.
ARHGEF6 and PARVB co-localize at the plasma membrane
To examine the subcellular distribution of wild-type and mutant ARHGEF6 proteins, COS-7 and CHO-K1 cells were transiently transfected with HA-tagged ARHGEF6 constructs, stained with anti-HA-fluorescein antibody, and observed by confocal microscopy. In CHO-K1 cells, wild-type ARHGEF6 was localized diffusely in the cytoplasm (Fig. 4A). Enhanced green fluorescence was found at the plasma membrane where lamellipodia and ruffles were formed (Fig. 4A and B). A similar distribution of HA-ARHGEF6 was observed in transiently transfected COS-7 cells (data not shown). Expression of HA-tagged ARHGEF6 stimulated slightly the formation of lamellipodia and ruffles in all cell types examined and the cells were very well spread. In marked contrast, both ARHGEF6 mutant proteins show a different subcellular localization. In CHO-K1 cells transiently expressing HA-ARHGEF6 aa56–83, green fluorescence was observed in dot-like structures in the cytoplasm (Fig. 4C). The same pattern was seen in COS-7 cells expressing this ARHGEF6 mutant (data not shown). The mutant protein HA-ARHGEF6 aa396–776 was localized mainly to the cell nuclei both in transiently expressing CHO-K1 (Fig. 4E) and COS-7 cells (data not shown). Faint fluorescence was also observed in the cytoplasm but not at the plasma membrane. These data suggest that both the N-terminal calponin homology domain and the C-terminal amino acids 396–776 are necessary for targeting ARHGEF6 to the plasma membrane. The data presented here also show that ARHGEF6 induces cell ruffling and that the C-terminal portion, including the coiled-coil domain, is important for peripheral localization of ARHGEF6, a finding consistent with those of earlier studies (15).
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To determine the subcellular localization of PARVB, we performed immunofluorescence with constructs transiently transfected into COS-7 and CHO-K1 cells. In general, cells expressing FLAG-PARVB show no obvious morphological changes compared to those expressing only vector (Fig. 4G and data not shown). PARVB is localized to the nucleus and throughout the cytoplasm with no significant concentration at the cell periphery. Co-localization studies in CHO-K1 cells transiently transfected with FLAG-PARVB and stained with Texas Red-conjugated phalloidin, to detect filamentous actin (Fig. 4H), yielded a yellow pseudocolor suggesting a partial co-localization of PARVB with stress fibers. Additionally, the green fluorescence of PARVB overlapped significantly with the anti-vinculin pattern in red (Fig. 4I) indicating that PARVB is present in focal adhesions.
To confirm the interaction between ARHGEF6 and PARVB, CHO-K1 and COS-7 cells were co-transfected with pHA-ARHGEF6 and pFLAG-CMV-4-PARVB and stained with anti-HA-fluorescein (Fig. 4J and N) and anti-FLAG monoclonal antibody-Cy3 conjugate (Fig. 4K and O). As shown in Figure 4L and P, the HA-tagged ARHGEF6 protein co-localizes with FLAG-PARVB at the cell periphery suggesting that ARHGEF6 and PARVB also interact within the cell.
ARHGEF6 co-localizes with PARVB and ILK during cell spreading
Spreading of cells on fibronectin involves Cdc42-dependent filopodia formation followed by Rac-activation and formation of lamellipodia and ruffles (21,22). PARVB and integrin-linked kinase (ILK) were shown to concentrate at the cell surface during early stages of cell spreading (24). The current data available for PARVB suggest an involvement in integrin-ILK signaling during the early phase of cell spreading (24,26). To determine whether ARHGEF6 co-localizes with PARVB in spreading cells plated on fibronectin, CHO-K1 cells were co-transfected with pHA-ARHGEF6 and pFLAG-CMV-4-PARVB, and observed by confocal microscopy. The spreading cells show a flattened morphology and organize actin in peripheral filopodia and lamellipodia (Fig. 4U and Y). In spreading CHO-K1 cells, ARHGEF6 was localized diffusely in the cytoplasm (Fig. 4R). Enhanced green fluorescence was found at the plasma membrane in cells that are actively forming lamellipodia and membrane ruffles (Fig. 4R). PARVB is localized throughout the cytoplasm and also in the nucleus (Fig. 4S). In contrast to well-spread CHO-K1 cells, PARVB is concentrated at the cell periphery in actively spreading cells (Fig. 4S). When the images 4R and S are overlayed, a yellow pseudocolor is yielded suggesting that PARVB and ARHGEF6 also co-localize at the leading edge of spreading cells (Fig. 4T).
Binding of PARVB to ILK (24) and ARHGEF6 suggests that the three proteins form a complex. To analyze whether ARHGEF6 co-localizes with ILK, CHO-K1 cells were transiently transfected with pHA-ARHGEF6, and stained with anti-HA-fluorescein antibody (Fig. 4V) and antibody against ILK (Fig. 4W). ILK was localized evenly in the cytoplasm whereas enhanced red fluorescence was found at the cell periphery (Fig. 4W). The green fluorescence overlapped significantly with the anti-ILK pattern in red suggesting that ARHGEF6 and ILK co-localize at the leading edge of the spreading cell (Fig. 4X).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The identification of ARHGEF6 as a gene implicated in X-linked non-specific mental retardation in human has prompted questions about the cellular function of the protein, and specifically about its role in development of the nervous system. Although different ARHGEF6/
PIX interacting proteins have been identified (11,16,17), the upstream signaling cascade necessary for ARHGEF6/PIX activation is yet to be elucidated. The identification of PARVB as an ARHGEF6/PIX binding protein provides new insights in understanding ARHGEF6/PIX function and its involvement in mental retardation through cell adhesion processes and extracellular matrix (ECM) interactions mediated by integrin signaling.
Integrin molecules are heterodimeric transmembrane receptors that mediate cell-ECM interactions at discrete sites termed ‘focal complex’ and ‘focal adhesion’. Small focal complex structures are localized at the tips of lamellipodia or filopodia of most migrating cells and are believed to be important in mediating the attachment of the extending lamellipodium to the extracellular matrix (27). Rac1 and Cdc42 are required for focal complex assembly whereas the formation of both focal adhesions and stress fibers are initiated by RhoA (1). Focal adhesions are large integrin aggregates found at the end of stress fibers. Although most studies have focused on activation of Rho GTPases by soluble factors, such as bioactive lipids, peptides and growth factors, several studies have indicated that integrin-mediated adhesion itself may also activate these GTPases (21,22,28). Indeed, a recent study has shown that, in CHO cells, ß3 integrin enhanced Rho activity and stress fiber formation, whereas overexpression of ß1 integrin increased Rac activity and lamellipodia formation (29). How might integrin-mediated adhesion activate Rho GTPases? Guanine nucleotide exchange factors, such as ARHGEF6/PIX, are obvious targets for they mediate Rho GTPase activation. In this report, we provide evidence that ARHGEF6/PIX might be implicated in integrin-mediated cell spreading by interacting with PARVB.
The parvin family of small focal adhesion-associated proteins has been characterized by several groups (23,24,30,31). Human PARVB is identical to affixin and CLINT, and it associates in vivo with integrin-linked kinase (ILK) (24). In well-spread CHO cells cultured on fibronectin-coated coverslips, PARVB and ILK co-localize to focal adhesions and both can also be detected at the tip of the leading edge of lamellipodia, suggesting that PARVB and ILK play a role in the initial phase of focal adhesion formation (24). The data presented in this communication confirm the presence of PARVB in focal adhesions. Immunofluorescence analysis of actively spreading cells after replating on fibronectin shows that PARVB and ILK accumulate in cell surface blebs (24). These blebs are spherical out-pouchings of the plasma membrane that are considered to be transient structures resulting finally in the formation of other types of protrusions, such as membrane ruffles or small lamellipodia (32,33). Data of a more recent study suggest that the ILK/PARVB complex binds to ß3 integrin which results in ILK activation. This finding together with the observation that PARVB and ILK co-localize at the cell periphery suggests that binding of fibronectin by integrins enhances ILK activity and results in the intial phase to reorganize the actin cytoskeleton (26). Another member of the parvin protein family, -parvin, was also distributed diffusely throughout the cytoplasm with enrichment at lamellipodial ruffles in actively spreading cells, suggesting a role in matrix remodelling (23). The interaction of PARVB with ARHGEF6/PIX and the co-localization of the two proteins at the leading edge of spreading cells suggest that ARHGEF6/PIX provides the link between integrin signaling and cell spreading through activation of the Rho GTPases Rac1 and Cdc42. One may speculate that ILK functions as an adaptor protein for the assembly of proteins and, thereby, bridging the ECM to the actin cytoskeleton. Upon spreading of cells on fibronectin, integrin engagement and clustering occurs. ILK can anchor to integrins by interacting with the cytoplasmic domain of certain ß integrin subunits (34). A complex is built consisting of at least ILK, PARVB and paxillin (35) recruiting ARHGEF6/PIX to activated integrins that, in turn, leads to activation of Cdc42 and/or Rac1 inducing membrane ruffling and lamellipodia formation (Fig. 5).
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We identified endogeneous ARHGEF6/
PIX as well as two ARHGEF7/ßPIX variants, ß1PIX and ß2PIX, in immunoprecipitates from CHO-K1 cells transfected with FLAG-PARVB. Thus far, we cannot determine whether ARHGEF7/ßPIX interacts directly with PARVB or ARHGEF7/ßPIX was brought down by ARHGEF6/PIX.
Cell adhesion to fibronectin was shown to result in PAK activation, presumably via PI3-kinase and ARHGEF6/PIX (14), suggesting that this latter is the GEF most likely involved in PAK activation through Rac1/Cdc42 upon integrin-dependent adhesion. Further support for this model came from studies reporting on small integrin clusters containing calpain, calpain-cleaved integrin, and Rac binding protein(s) formed at early stages after integrin-induced adhesion (36). Calpains are Ca2+-dependent thio proteases consisting of a large catalytic subunit and a small regulatory subunit (37). By the SRS system and GST pull-down, we identified the small subunit of calpain, calpain 4, encoded by CAPNS1, to bind to ARHGEF6/PIX (unpublished data), suggesting an involvement of ARHGEF6/PIX in integrin-induced Rac activation necessary for cell adhesion and spreading.
All parvin proteins identified to date contain two calponin homology (CH) domains that are separated by a linker of 60 amino acids (23). Phylogenetic analysis of the CH domains (named type 4 and type 5 CH domains) suggests that parvins represent a separate branch within the -actinin superfamily of actin binding proteins (23,25). ARHGEF6/PIX contains an N-terminal CH domain type 3 (25) whose function is unknown. In general, any protein displaying two CH domains in tandem arrangement is supposed to bind F-actin whereas the actin binding ability of proteins containing only one CH domain is still questionable. Nonetheless, it has been postulated that this module can mediate interaction with other proteins (38–40). We demonstrated that ARHGEF6/PIX with a CH domain lacking 28 amino acids showed significantly reduced interaction with PARVB in the SOS recruitment system and failed completely to bind PARVB in GST pull-down experiments. This difference in ARHGEF6-PARVB-binding might be explained by the different organisms used for studying the interaction (yeast versus monkey COS-7 cells).
Deletion of the coiled-coil domain of ARHGEF6/PIX abrogated binding to PARVB in the SRS system, suggesting that both dimerization and the presence of an (intact) CH domain are required for interaction of ARHGEF6/PIX with PARVB and the proper localization thereof in the cell. Similarly, the N-terminal domain of Fgd1, a Cdc42 guanine nucleotide exchange factor, is necessary for the localization of the protein to the plasma membrane and Golgi complex (41). Mutations of the human orthologue, FGD1, have been implicated in faciogenital dysplasia, a syndromic form of X-linked mental retardation. It is tempting to speculate that dimerization of ARHGEF6/PIX might confer F-actin binding directly linking ARHGEF6/PIX to the actin cytoskeleton.
The mutation identified in the first intron of ARHGEF6 (IVS1-11T
C) in affected males in family MRX46 resulted in preferential skipping of exon 2 predicting a protein that lacks 28 amino acids in the CH domain. We speculated that mental retardation in family MRX46 might be due to a switch in the ratio between ARHGEF6 wild-type and the aa56–83 isotype (7). Here we show that the mutant protein remains in the cytoplasm in dot-like structures and, thus, might not be able to activate membrane-associated Rac1/Cdc42. However, it is possible that ARHGEF6 aa56–83 exerts a dominant negative effect over the wild-type protein via ARHGEF7. Given the fact that ARHGEF7 still dimerizes with ARHGEF6 aa56–83, ARHGEF6 aa56–83/ARHGEF7 heterodimers, and/or ARHGEF6 aa56–83 homodimers may act in a dominant manner in concrete cellular contexts. Further studies are required for comprehension of the molecular consequences of the mutations found in ARHGEF6.
Recent evidence has implicated Rho GTPases in neuronal morphogenesis, including migration, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation (3,4). Several GEFs are involved in these processes, and, most importantly, they are believed to play a central role in defining the spatial and temporal activation of the corresponding GTPase within this highly specialized cell type (42). Mutations in three genes, OPHN1, PAK3 and ARHGEF6, encoding proteins that participate directly in cellular signaling through Rho GTPases, have been identified in MRX patients (7–9). Very recently, the involvement of a novel Rho GTPase activating gene, MEGAP/srGAP3 on chromosome 3p25, in severe mental retardation has been proposed (43). Together, these findings suggest that MR(X) is caused by defective Rac signaling. Further support for this assumption was provided by Ng and colleagues demonstrating that axon growth, guidance, and branching is controlled by differential activation of Rac signaling pathways (44).
Little is known about the biological function of ARHGEF6 in neuronal processes. Remarkably, recent analysis of the Drosophila homolog dPix suggests an involvement in regulation of postsynaptic structures (45). In dPix mutant flies, dPak is not targeted to the synapse, and targeting of other proteins is partially disrupted indicating a specific and key in vivo function of dPix and dPak in the development of correct synaptic structures and assembly of postsynaptic proteins (45). Other mammalian Rho GEFs have also been implicated in the organization of the synapse, such as Kalirin-7 and collybistin (46,47). Further evidence for involvement of PAK (p21-activated protein kinase) and PIX proteins in neurite outgrowth was provided by two studies on PAK-dependent extension of neurites in PC12 cells (48,49). In PC12 cells, PAK kinases function most likely both upstream and downstream from Rac1 via interaction with PIX that tightly associated with PAK irrespective of its localization and kinase activity. Thus, it is possible that PIX is the functional link between PAK and Rac activation, at least in PC12 cells (49). Although these data were generated by analyzing the effects of ARHGEF7/ßPIX on PAK, it is tempting to speculate that, given the extensive homology between ARHGEF6 and ARHGEF7, a similar role for ARHGEF6 exists too.
Taken together, it is likely that the ARHGEF6/PIX-PAK interaction in neuronal cells is crucial for PAK-induced morphological changes and the regulation of synapse formation and/or dendrite morphology in human. A possible involvement of an integrin-mediated activation of Rac1 and/or Cdc42 through a PARVB-ARHGEF6 complex during neurite outgrowth remains to be determined. However, recent data already indicate a pivotal role for the GTPases Rac1 and Cdc42 in integrin-dependent neurite outgrowth (50–53).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Yeast two-hybrid screening
We used the yeast two-hybrid system CytoTrapTM (Stratagene), also designated Sos Recruitment System (SRS) (54), to identify ARHGEF6/
PIX interacting proteins. The SRS system is based on the recruitment of the mammalian guanine nucleotide exchange factor hSos to the plasma membrane in the temperature-sensitive cdc25H yeast mutant strain. Usually, this strain is not able to grow at the restrictive temperature of 37°C. However, translocation of hSos to the membrane via interaction between two-hybrid proteins leads to its activation and growth of the yeast strain at 37°C.
We screened a human fetal brain plasmid cDNA library with 5.3x106 primary colonies and an average insert size of 1.3 kb for ARHGEF6/PIX interacting proteins according to the manufacturer's instructions. Therefore, the N-terminal 1070 residues of human Sos were fused to full-length ARHGEF6 and used as bait. For the prey plasmids, the myristylation signal of v-Src that anchors the fusion protein to the plasma membrane was fused to cDNAs isolated from human fetal brain (Stratagene). Liquid yeast media were prepared using glucose minimal medium containing the relevant Complete Supplement MixtureTM (CSM) dropout mixes (Qbiogene), 2% glucose, 0.5% NH4SO4, and 0.17% yeast extract. Galactose medium, for induction of prey plasmid (pMyr) expression, was prepared using the relevant dropout mixes, 2% galactose, 1% raffinose, 0.5% NH4SO4, 0.17% yeast extract and 2% glycerol. For solid media, 3% bacto agar was added. Yeast transformation by the lithium acetate method was done by standard protocols. Yeast strain cdc25H was co-transformed with pSos-ARHGEF6 and pYES-mGAP to reduce isolation of Ras GTPase false positives (55). Transformants were propagated for 5 days on glucose minimal plates supplemented with dropout mix CSM-LEU-TRP and subsequently tested for galactose-independent auto-activation of growth at the restrictive temperature of 37°C. SRS screening was done by transformation of the pre-transformed cdc25H yeast strain (see above) with 3 µg pMyr-cDNA library plasmids. Resulting transformants were grown for 5 days at 22°C on selectable minimal glucose plates (CSM-LEU-URA-TRP). Colonies were replica-plated onto selectable minimal galactose plates and grown at 37°C for 7 days. Replica plating was performed with a replica-plating tool and velveteen squares (Scienceware). A total of 152 colonies out of 2 million transformants were isolated and tested for galactose-dependent growth at 37°C. Fifty-four colonies that showed growth under this condition were grown in liquid media and plasmid DNA was isolated by the Lyticase (Sigma) and alkaline lysis method. Subsequently, plasmid DNA was transformed into E.coli strain DH10B and selected on 50 ng/µl chloramphenicol for the presence of the cDNA insert-containing pMyr plasmid. From each transformation, two single E.coli transformants were grown in liquid LB medium containing chloramphenicol and plasmid DNA was isolated by standard procedures. Protein interactions of putatively positive colonies were confirmed by re-transformation of the cdc25H yeast strain with both the cDNA insert-containing pMyr-plasmid and pSos-ARHGEF6 or empty pSos. Only those clones that were able to grow on galactose media at the restrictive temperature of 37°C after 6 days were defined as positive. Co-transformation of cdc25H cells with pSos-MafB and pMyr-target, pSos and pMyr-target, pSos-MafB and pMyr-Lamin C, pSos and pMyr, pSos-ARHGEF6 and pMyr-Lamin C, and pSos-ARHGEF6 and pMyr functioned as negative controls, whereas pSos-MafB and pMyr-MafB functioned as positive control.
In subsequent yeast two-hybrid experiments, different domains of ARHGEF6/PIX were tested for interaction with PARVB, using various bait constructs (see plasmid constructs) in combination with plasmid pMyr-PARVB or empty pHyr as prey. Expression of Sos-ARHGEF6 fusion proteins was verified by standard yeast protein extraction procedures and western blot techniques using the mouse monoclonal anti-Sos antibody (BD Biosciences Transduction Laboratories).
Plasmid constructs
ARHGEF6 constructs used as bait in SRS.
The following PCR primer pairs containing SalI and NotI restriction sites, respectively, were used for PCR amplification of full-length ARHGEF6 or ARHGEF6 domains. For ARHGEF6 full-length and ARHGEF6 aa56–83, primer pairs Sal-PIX-Y2H and Not-PIX-Y2H was used. For the CH domain construct, Sal-PIX-Y2H and Not-CH-Y2H; for the CH-SH3 construct, Sal-PIX-Y2H and Not-CHSH3-Y2H; for the CH-SH3-DH construct, Sal-PIX-Y2H and Not-CHSH3DH-Y2H; for the CH-SH3-DH-PH construct, Sal-PIX-Y2H and Not-CHSH3DHPH-Y2H; for the SH3-DH-PH construct, Sal-SH3DHPH-Y2H and Not-CHSH3DHPH-Y2H; and for the CH-SH3-DH-PH-GBD construct, Sal-PIX-Y2H and Not-dLZ-Y2H were used. The KIAA0006 clone was used as template for all PCR amplifications, except for ARHGEF6 aa56–83, where ARHGEF6 aa56–83 in pMT2SM-HA (see below) served as a template. Resulting Pfu TurboTM (Stratagene) PCR products were purified, restricted with SalI and NotI and unidirectionally cloned into plasmid pSos (Stratagene).
Generation of N-terminal HA-tagged ARHGEF6 constructs.
To generate HA-tagged wild-type ARHGEF6, we performed PCR amplification using KIAA0006 clone as template and primers Not-KIA6-Nter and Eco-KIA6-Cter. ARHGEF6 aa56–83 was generated by PCR-mediated mutagenesis (56). Two overlapping PCR products lacking exon 2 were generated with primer pair Not-KIA6-Nter and KIA6-Ex1/3-R and primer pair KIA6-Ex1/3-F and Eco-KIA6-Cter. Both PCR products were purified and applied to megaprime PCR. Three PCR cycles with the two PCR products and without any primer were performed before adding primer Not-KIA6-Nter and Eco-KIA6-Cter to the reaction. Subsequently, 20 cycles of a standard PCR were performed. For ARHGEF6 aa396–776, we used the KIAA0006 clone as template and primers Not-KIA6-Nter and EcoRI-KIA6-exon10. All amplifications were done with Pfu TurboTM Polymerase, and PCR products were purified, restricted with NotI and EcoRI and cloned unidirectionally into plasmid pMT2SM-HA (a gift from Rheza Mohammad Ahmadian).
Generation of N-terminal FLAG-tagged PARVB construct.
Plasmid pFLAG-CMV-4 (Sigma) was digested with NotI and XbaI, purified, and subsequent filling of recessed 3' termini was done by Klenow fragment (Invitrogen) to obtain blunt ends. After a second purification step, the plasmid was ligated to the GATEWAYTM Reading Frame Cassette A (RfA) of the GATEWAYTM Vector Conversion system according to the manufacturer's instructions. The clones obtained represent GATEWAYTM-compatible ‘destination vectors’ (pFLAG-CMV-4-cassetteA) that were sequenced for integrity and correct orientation of the cassette with primers PFLAG-CMV-N and PFLAG-CMV-C. These vectors were propagated in E.coli host DB3.1TM (Invitrogen) to compensate for the expression of the ccdB gene present in the cassette. Construction of FLAG-fusion plasmids with the GATEWAYTM system was carried out in three steps. First, we amplified the open reading frame of PARVB to generate a product compatible with the GATEWAYTM cloning system. Therefore, we used full-length PARVB in pMyr, identified by the Sos recruitment system, as template with primers bParvin-ATTB1 and bParvin-ATTB2 and cDNA Advantage polymerase (BD Biosciences Clontech) in a PCR reaction. Second, the purified PCR product of the PARVB coding region was cloned into ‘donor vector’ pDONR201 (Invitrogen) via BP reaction according to the protocol provided. Three independent clones were picked, grown in 2 ml of LB with kanamycin (25 ng/µl) and DNA mini preparations were done by standard protocol. The DNA insert was sequenced with primers SeqL-A, SeqL-B, Parvin1 and Parvin2. Third, verified clones were used for cloning into pFLAG-CMV-4-cassetteA via LR reaction according to the manufacturer's protocol. After transformation into DH10B (Invitrogen), the respective destination clones were selected by plating on ampicillin (100 ng/µl) containing agar plates.
Generation of N-terminal GST-tagged PARVB construct.
A verified clone of pDONR201-PARVB was used for cloning the coding region of PARVB into GATEWAYTM pDEST27 vector (Invitrogen) via LR reaction following the manufacturer's instructions. After transformation into E.coli DH10B, the cells were plated on ampicillin (100 ng/µl) containing agar plates to select for the presence of the destination vector.
Generation of N-terminal GST-tagged ARHGEF7 construct.
The open reading frame of ARHGEF7 was amplified to generate a product compatible for the GATEWAYTM cloning system. Therefore, we used the KIAA0142 clone as template with primers bPIX-ATTB1 and bPIX-ATTB2 and cDNA Advantage polymerase (BD Biosciences Clontech) in a PCR reaction. The purified PCR product of the ARHGEF7 coding region was cloned into ‘donor vector’ pDONR201 (Invitrogen) via BP reaction according to the protocol provided. Three independent clones were picked, grown in 2 ml of LB with kanamycin (25 ng/µl) and DNA mini preparations were done by standard protocol. The DNA insert was sequenced with primers SeqL-A, SeqL-B, bPIX-1, bPIX-2, bPIX-3 and bPIX-4 and a verified clone was used for cloning into pDEST27 (Invitrogen) via LR reaction according to the manufacturer's protocol. After transformation into DH10B (Invitrogen), the respective destination clones were selected by plating on ampicillin (100 ng/µl) containing agar plates.
Large and pure amounts of plasmid DNA were prepared by using a plasmid midi or maxi kit (Qiagen). All primer sequences are shown in Table 1. The described constructs were sequenced for integrity.
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Immunoblotting
Protein lysates were separated by 10 or 12.5% SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Roche) via semi-dry blotting method. The membrane was blocked with 4% low-fat milk in Tris-buffered saline and then incubated with the appropriate antibody solution. Detection of bound antibodies was carried out using ECLTM detection kit (Amersham Pharmacia Biotech). Horseradish peroxidase-conjugated (HRP) goat polyclonal anti-GST antibody (Amersham Pharmacia Biotech), HRP-conjugated murine monoclonal anti-FLAG antibody (Sigma) and HRP-conjugated rat monoclonal anti-HA antibody, high-affinity 3F10 (Roche) were used at 1:4000 dilutions. Mouse monoclonal anti-Sos1 antibody (BD Biosciences Transduction Laboratories) and rabbit anti-PIX antibody (11) were used at 1:400 dilutions. HRP-conjugated anti-Mouse Ig (Amersham Pharmacia Biotech) and HRP- conjugated anti-Rabbit Ig (Amersham Pharmacia Biotech) were used at 1:5000 dilutions.
Co-immunoprecipitations
CHO-K1 cells were cultured in 100 mm culture dishes in Nutrient Mixture F-12 (HAM) containing 10% FCS and penicillin-streptomycin (Invitrogen). CHO-K1 cells, 1x106 were transfected with pFLAG-CMV-4-PARVB (5 µg DNA) using LipofectAMINETM 2000 Reagent (Invitrogen) according to the manufacturer's protocol. pFLAG-CMV-4-cassetteA transfected cells were used as negative control. After 24 h incubation, cells were lysed with ice-cold cell lysis buffer [150 mM Tris–HCl, pH 8.0; 50 mM NaCl; 1 mM EDTA; 0.5% Nonidet P40; one tablet CompleteTM Mini protein inhibitor cocktail/10 ml (Roche); 0.7 µg/ml Pepstatin]. Lysates were clarified by centrifugation at 20 000g for 10 min at 4°C, and the supernatants were applied to 50 µl EZviewTM Red anti FLAGTM M2 Agarose (Sigma) and incubated overnight at 4°C. Subsequently, the beads were washed three times with washing buffer (50 mM Tris–HCl, pH 7.5; 250 mM NaCl). All centrifugation steps were carried out at 8000g for 1 min at 4°C. Protein complexes were dissociated from beads by heating to 95°C for 3 min. The immunoprecipitates were separated by SDS–PAGE, and immunoblotting was carried out using mouse anti-FLAG and rabbit anti-PIX antibodies.
GST fusion affinity precipitations
COS-7 cells were cultured in 100 mm culture dishes in Dulbecco modified Eagle medium containing 10% FCS and penicillin-streptomycin (Invitrogen). After 24 h, cells co-transfected with pDEST27-PARVB (7.5 µg DNA) and pMT2SM-HA-ARHGEF6 (7.5 µg DNA) were rinsed once with phosphate-buffered saline (PBS) and lysed with ice-cold cell lysis buffer [150 mM Tris–HCl, pH 8.0; 50 mM NaCl; 1 mM EDTA; 0.5% Nonidet P40; 1 tablet CompleteTM Mini protein inhibitor cocktail/10 ml (Roche)]. Cell lysates were incubated on ice for 15 min and then centrifuged for 10 min at 20 000g at 4°C. The supernatants were incubated overnight at 4°C with 70 µl of GST
Bind resin (CN Biosciences Novagen) and subsequently washed three times with ice-cold TBS (50 mM Tris–HCl, pH 7.4; 150 mM NaCl). All centrifugation steps were done at 8000 g and 4°C. Proteins bound to the beads were eluted by boiling in Lämmli buffer and the samples were subjected to SDS–PAGE. Immunoblotting was carried out using anti-GST and anti-HA antibodies.
Immunofluorescence
Sterile cover slips (13 mm) were placed into 24-well dishes and seeded with 2.5x104 cells. Twenty-four hours later, cells were transfected with pMT2SM-HA-ARHGEF6 (0.5 µg DNA) and pFLAG-CMV-4-PARVB (0.5 µg DNA) constructs using LipofectAMINETM 2000 Reagent (Invitrogen) according to the manufacturer's protocol. The pMT2SM-HA and pFLAG-CMV-4-cassetteA vector co-transfected cells were used as negative control. Twenty-four hours after transfection, the coverslips were rinsed with PBS and fixed in 4% paraformaldehyde in PBS for 10 min. After residual formaldehyde had been quenched with PBS for 10 min, cells were incubated with 2% BSA, 3% goat serum, and 0.5% Nonidet P40 in PBS for 60 min to permeabilize cells and block non-specific antibody binding. For the detection of cells expressing HA-tagged ARHGEF6, cells were incubated either with monoclonal antibody anti-HA-fluorescein 3F10 (1.5 µg/ml; Roche) or with mouse monoclonal antibody HA.11 (1.0 µg/ml; Eurogentec) followed by incubation with Alexa Fluor 488 goat anti-mouse IgG (4 µg/ml; Mobitec). FLAG-tagged PARVB was detected either by anti-FLAG-M2 monoclonal antibody-Cy3 conjugate (5 µg/µl; Sigma) or by anti-FLAG-M2 rabbit monoclonal antibody (15 µg/µl; Sigma) followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (4 µg/ml; Mobitec). For the detection of F-actin, the cells were treated with Texas Red-conjugated phalloidin (1 U/ml; Mobitec). To detect vinculin, cells were stained with monoclonal anti-human vinculin hVIN-1 (1:200 dilution; Sigma), followed by incubation with Alexa Fluor 546 goat anti-mouse IgG (4 µg/ml; Mobitec). After washing twice with high salt PBS (650 mM NaCl) and three times with PBS, cells on coverslips were mounted in glycerol gelatin containing 1% phenol (Sigma).
For immunofluorescence staining of spreading cells, CHO-K1 cells were transfected in six-well dishes with pMT2SM-HA-ARHGEF6 (1.6 µg DNA) or pMT2SM-HA-ARHGEF6 (0.8 µg DNA) together with pFLAG-CMV-4-PARVB (0.8 µg DNA). Eighteen hours after transfection, cells were washed twice with PBS, trypsinized, resuspended in Nutrient Mixture F-12 (HAM) containing 5% FCS and penicillin–streptomycin and seeded onto fibronectin-coated coverslips (BD Biosciences). After cells were allowed to attach and spread for 4 h, immunofluorescence staining was done as described using monoclonal antibody anti-HA-fluorescein 3F10 (1.5 µg/ml), anti-FLAG-M2 monoclonal antibody-Cy3 conjugate (5 µg/µl) and rabbit polyclonal antibody anti-ILK (10 µg/ml; Upstate).
Cells were examined with a Leica TCS-NT confocal microscope equipped with an Apo 40-by-1.0 oil immersion objective lens.
Database searches and accession numbers
Database searches were done at the NCBI BLAST Network Service. Deduced protein sequences were searched for functional domains using PROSITE and SMART. GenBank accession numbers are AF207831 and D25304 for the ARHGEF6 sequence, AF237769 for PARVB, and D63476 for ARHGEF7.
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ACKNOWLEDGEMENTS |
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The results summarized here are part of the doctoral thesis of Georg Rosenberger at the University of Hamburg. We are grateful to Reza Mohammad Ahmadian for helpful suggestions and comments. We thank Nobuo Nomura (Kazusa DNA Research Institute, Japan) for providing us with cDNA clones KIAA0006 and KIAA0142, Ed Manser (Institute of Molecular and Cell Biology, Singapore) for polyclonal anti-PIX antibody, and Michaela Schweizer for support in confocal microscopy. This work was supported by grants of the Deutsche Forschungsgemeinschaft (SFB444) and the BMBF (NGFN-FKZ 01GS0119).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +49 40428034597; Fax: +49 40428035138; Email: kkutsche{at}uke.uni-hamburg.de
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REFERENCES |
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 B. Ho, G. Hou, J. G. Pickering, G. Hannigan, B. L. Langille, and M. P. Bendeck Integrin-Linked Kinase in the Vascular Smooth Muscle Cell Response to Injury Am. J. Pathol., July 1, 2008; 173(1): 278 - 288. [Abstract] [Full Text] [PDF]
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 K. Missy, B. Hu, K. Schilling, A. Harenberg, V. Sakk, K. Kuchenbecker, K. Kutsche, and K.-D. Fischer {alpha}PIX Rho GTPase Guanine Nucleotide Exchange Factor Regulates Lymphocyte Functions and Antigen Receptor Signaling Mol. Cell. Biol., June 1, 2008; 28(11): 3776 - 3789. [Abstract] [Full Text] [PDF]
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C. N. Johnstone, P. S. Mongroo, A. S. Rich, M. Schupp, M. J. Bowser, A. S. deLemos, J. W. Tobias, Y. Liu, G. E. Hannigan, and A. K. Rustgi Parvin- Inhibits Breast Cancer Tumorigenicity and Promotes CDK9-Mediated Peroxisome Proliferator-Activated Receptor Gamma 1 Phosphorylation Mol. Cell. Biol., January 15, 2008; 28(2): 687 - 704. [Abstract] [Full Text] [PDF]
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 J. Liu, S. D. Fraser, P. W. Faloon, E. L. Rollins, J. Vom Berg, O. Starovic-Subota, A. L. Laliberte, J.-N. Chen, F. C. Serluca, and S. J. Childs A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish PNAS, August 28, 2007; 104(35): 13990 - 13995. [Abstract] [Full Text] [PDF]
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 G. E. Hannigan, J. G. Coles, and S. Dedhar Integrin-Linked Kinase at the Heart of Cardiac Contractility, Repair, and Disease Circ. Res., May 25, 2007; 100(10): 1408 - 1414. [Abstract] [Full Text] [PDF]
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 E. Boulter, D. Grall, S. Cagnol, and E. Van Obberghen-Schilling Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase FASEB J, July 1, 2006; 20(9): 1489 - 1491. [Abstract] [Full Text] [PDF]
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H. Chu, I. Thievessen, M. Sixt, T. Lammermann, A. Waisman, A. Braun, A. A. Noegel, and R. Fassler {gamma}-Parvin Is Dispensable for Hematopoiesis, Leukocyte Trafficking, and T-Cell-Dependent Antibody Response. Mol. Cell. Biol., March 1, 2006; 26(5): 1817 - 1825. [Abstract] [Full Text] [PDF]
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E. Liu, S. Sinha, C. Williams, M. Cyrille, E. Heller, S. B. Snapper, K. Georgopoulos, R. St-Arnaud, T. Force, S. Dedhar, et al. Targeted Deletion of Integrin-Linked Kinase Reveals a Role in T-Cell Chemotaxis and Survival Mol. Cell. Biol., December 15, 2005; 25(24): 11145 - 11155. [Abstract] [Full Text] [PDF]
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 D. P. LaLonde, M. C. Brown, B. P. Bouverat, and C. E. Turner Actopaxin Interacts with TESK1 to Regulate Cell Spreading on Fibronectin J. Biol. Chem., June 3, 2005; 280(22): 21680 - 21688. [Abstract] [Full Text] [PDF]
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H. Zhou and R. H. Kramer Integrin Engagement Differentially Modulates Epithelial Cell Motility by RhoA/ROCK and PAK1 J. Biol. Chem., March 18, 2005; 280(11): 10624 - 10635. [Abstract] [Full Text] [PDF]
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G. Rosenberger, A. Gal, and K. Kutsche {alpha}PIX Associates with Calpain 4, the Small Subunit of Calpain, and Has a Dual Role in Integrin-mediated Cell Spreading J. Biol. Chem., February 25, 2005; 280(8): 6879 - 6889. [Abstract] [Full Text] [PDF]
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 E.-E. Govek, S. E. Newey, and L. Van Aelst The role of the Rho GTPases in neuronal development Genes & Dev., January 1, 2005; 19(1): 1 - 49. [Abstract] [Full Text] [PDF]
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 M. C. Brown and C. E. Turner Paxillin: Adapting to Change Physiol Rev, October 1, 2004; 84(4): 1315 - 1339. [Abstract] [Full Text] [PDF]
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Y. Zhang, K. Chen, Y. Tu, and C. Wu Distinct Roles of Two Structurally Closely Related Focal Adhesion Proteins, {alpha}-Parvins and {beta}-Parvins, in Regulation of Cell Morphology and Survival J. Biol. Chem., October 1, 2004; 279(40): 41695 - 41705. [Abstract] [Full Text] [PDF]
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D. M. Clarke, M. C. Brown, D. P. LaLonde, and C. E. Turner Phosphorylation of actopaxin regulates cell spreading and migration J. Cell Biol., September 13, 2004; 166(6): 901 - 912. [Abstract] [Full Text] [PDF]
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S. Yamaji, A. Suzuki, H. Kanamori, W. Mishima, R. Yoshimi, H. Takasaki, M. Takabayashi, K. Fujimaki, S. Fujisawa, S. Ohno, et al. Affixin interacts with {alpha}-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction J. Cell Biol., May 24, 2004; 165(4): 539 - 551. [Abstract] [Full Text] [PDF]
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 W. Mishima, A. Suzuki, S. Yamaji, R. Yoshimi, A. Ueda, T. Kaneko, J. Tanaka, Y. Miwa, S. Ohno, and Y. Ishigatsubo The first CH domain of affixin activates Cdc42 and Rac1 through {alpha}PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor Genes Cells, March 1, 2004; 9(3): 193 - 204. [Abstract] [Full Text] [PDF]
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T. Sakai, S. Li, D. Docheva, C. Grashoff, K. Sakai, G. Kostka, A. Braun, A. Pfeifer, P. D. Yurchenco, and R. Fassler Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation Genes & Dev., April 1, 2003; 17(7): 926 - 940. [Abstract] [Full Text] [PDF]
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