Bacterial expression vectors.

The pGEX-Ras vector was derived from GEX4T-1 as follows. The Ras^1-185 coding sequence was amplified (Vent, New England Biolabs) to include a BglII site adjacent to codon 1 and at the 3′ (adjacent to codon 185) a BamHI-EcoRI linker (GGATCCCCGAATTC). This BglII/EcoRI fragment was cloned into the pGEX-4T BamHI/EcoRI site, regenerating a BamHI site downstream of the Ras sequence (the new polylinker is identical to that in pGEX4T-3). pGEX-Ras/PIX^496-554 was constructed by cloning the PCR-amplified GIT1-binding domain from βPIX (see Fig. ​Fig.3A)3A) into the pGEX-Ras vector at BamHI-XhoI sites. pGEX-Ras/GIT1^646-770 was constructed by cloning the SmaI-NotI fragment from pGEX/GIT1 (see below) into the SmaI-NotI sites of pGEX-Ras.

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FIG. 3

βPIX binds to GIT1 via the Spa2-related sequence. (A) The structural domains of βPIX include its N-terminal SH3 domain, which binds PAK, a catalytic Dbl homology (DH) region promoting nucleotide exchange on Rac1, and a C-terminal region (bar) which was used to bind GIT1 (B) by overlay. (B) GST fusion proteins corresponding to constructs 1 to 7 of GIT1 were separated on a 9% polyacrylamide gel and overlaid with PIX^496-554 to localize the binding site. All positive binding constructs contain SHD-1 of GIT1^254-376.

The GST-GIT1 full-length expression plasmid was constructed by amplifying a cDNA fragment encoding GIT1 residues 1 to 139 into the BamHI-EcoRI site of pGEX4T-1; the HindIII-EcoRI fragment corresponding to GIT1 residues 119 to 770 was derived from pBluescript SK-GIT1. GIT1 subclones were derived from restriction fragments as follows: GIT1^1-139 and GIT1^1-376 were derived from BamHI-XhoI cDNA fragments. GIT1^254-564, GIT1^254-474, GIT1^254-423, GIT1^254-376, and GIT1^370-564 were derived from amplified cDNA using 5′ BamHI and 3′ EcoRI linkers. pFlag/FAK was constructed by moving the BamHI (filled site)-BglII fragment from pXJ-GST/FAK into the SmaI/BglII site of the pFlag.MAC vector (Kodak IBI). Full-length paxillin derived from human cDNA by PCR using 5′ HindIII and 3′ XhoI linkers for cloning. Paxillin^250-281 (LD4) was derived by PCR using 5′ BamHI and 3′ XhoI linkers.

Mammalian expression vectors.

The pXJ-HA (hemagglutinin epitope), pXJ-Flag, and pXJ-GST mammalian vectors used for transfection and microinjection are as described elsewhere (20, 41). The pXJ-GFP vector was derived from pXJ-Flag by replacing the N-terminal tag with the GFP^S65T cDNA. The pXJ-GFP-Flag derivative was constructed using a restriction-compatible BglII-EDYKDDDDK-BamHI-XhoI sequence inserted into pXJ-GFP. The pXJ-Myr vector for expression of membrane-targeted proteins was constructed using sequence corresponding to the N-terminal myristoylation signal of Fyn fused to the Flag epitope. The corresponding 18-amino-acid sequence is MGCVQCKDKEDYKDDDDK, which was cloned using a 5′ EcoRI/Kozak linker (GAATTCACC) with a 3′ BamHI site.

The pXJ-GST/FAK expression plasmid was constructed by synthesizing residues 1 to 30 of chicken FAK as a BamHI-XhoI fragment in pXJ-GST then a SmaI-XhoI fragment (encoding FAK residues 30 to 1153) derived from a cDNA. Plasmids pXJ-GST/FAK-N′ (FAK^1-427), pXJ-GST/FRNK (FAK^693-1053), and pXJ-GST/FAT (FAK^916-1053) were derived from the PCR-amplified cDNA fragments with BamHI/XhoI sites in the primers. pXJ-Flag/GIT1 and pXJ-myr/GIT1 were constructed by subcloning a BamHI/NotI fragment from pGEX/GIT1. pXJ-Flag/GIT1^1-253 was then derived by deleting the BglII-BglII fragment. GIT1^253-424 represents the BglII-BglII fragment from pGEX-GIT1 cloned in the BamHI site of the vector. pXJ-Flag/GIT1^424-770 (BglII-NotI) was constructed by PCR. PXJ-Flagmyr/GIT1^1-376 was derived from the BamHI-XhoI (partial digestion) fragment from pGEX-GIT1. PXJ-myr/GIT1^1-645 was derived from a BamHI-SmaI fragment. PXJ-myr/GIT1^646-770 was derived from the SmaI-NotI fragment from pGEX/GIT1. PXJ-myr/GIT1^424-770 was constructed by PCR. The GIT1 subclones in pXJ-GST and pXJ-myr vectors were constructed in the same way. pXJ-Flag/βPIX was described previously (22).

Overlays with [γ-³²P]GTP-labeled proteins.

Proteins were separated on sodium dodecyl sulfate–10% polyacrylamide gels and transferred to polyvinylidene difluoride (NEN) membranes. The overlay protocol using radiolabeled GST/Ras-[γ-³²P]GTP fusion proteins is essentially as described previously (20).

Cell migration assays.

COS-7 cells (35-mm-diameter dish, 70% confluence) were transiently transfected with various pXJ-Flag DNAs (2 μg of each), using the calcium phosphate protocol. Controls used pXJ-Flag/GFP (soluble) or pXJ-myr, which gave the same values (~10% migration). Following overnight incubation in serum-free medium, haptotaxis was assayed using modified Boyden chambers (ChemTX; NeuroProbe Inc.) with a polycarbonate membrane (10-μm thickness, 8-μm pores). The membrane was precoated on the underside with fibronectin (20 μg/ml; Sigma) and collagen type I (10 μg/ml; Roche) in phosphate-buffered saline for 2 h at 37°C. The lower chamber contained serum-free Dulbecco modified Eagle medium with fibronectin (20 μg/ml) and collagen (10 μg/ml). Serum-starved transfected cells were suspended with 0.01% trypsin–5 mM EDTA–25 mM HEPES (pH 7.2). After two washes in serum-free culture medium, cells were resuspended at ~1.25 × 10⁵/ml. An equivalent of 2,500 cells was added on top of each well (6-mm diameter) and allowed to migrate to the underside of the membrane for 4 h at 37°C in 5% CO₂. Each assay was performed in quadruplicate. Nonmigrating cells on the upper membrane surface were removed with a rubber scraper, and migratory cells attached to the bottom surface were fixed with 3% paraformaldehyde (20 min). After immunostaining for anti-Flag (50 μg/ml), transfected cells in each well were counted. Numbers of transfected cells (total) for each plasmid transfection were determined by dilution into medium plus 10% serum and plating on 15-well slides (in triplicate). Both flattened and rounded positive cells were counted. Migration was expressed as a percentage of the level for total expressing cells. Each determination represents the average of at least three individual wells, and error bars represent the standard deviation for three experiments.

GIT1 localizes to focal adhesions and stimulates motility.

Antibodies raised against the C-terminal 125 residues of GIT1 recognized p90 in all cell lines and tissues tested, with highest levels in testis (Fig. ​(Fig.1B).1B). In cultured cells, the protein was present in FCs as previously observed with PIX (22). We noted that GFP/GIT1-expressing NIH 3T3 cells were more motile when viewed by time-lapse microscopy (data not shown). COS-7 cells contain endogenous GIT1 which colocalized to focal adhesions with paxillin (Fig. ​(Fig.1C).1C). These cells were used to assay for haptotaxis using a modified Boyden chamber under a fibronectin-collagen gradient. The individual components in the Cdc42/PAK pathway were tested, with transfection normalized for each by counting total positively stained cells for the same total number (see Materials and Methods). GIT1 promoted cell motility as effectively as Cdc42^G12V and was more potent than either PAK or PIX (Fig. ​(Fig.1D).1D). Cdc42^G12V both activates Rac and promotes loss of Rho-type FCs (20, 24).

GIT1 can stimulate FC disassembly.

To investigate further the early responses to GIT1 expression, we microinjected HeLa cells, a protocol that has previously uncovered functions of Rho p21s and their targets such as PAK, ROK, and MRCK. After plasmid injection, GIT1-expressing cells exhibited no gross morphological alteration within 2 to 4 h; however, we noted a severe reduction in paxillin staining of FCs (Fig. ​(Fig.2A,2A, top row) but not of other components including vinculin and phosphotyrosine (PY) or two other markers, talin and FAK (data not shown). To test that this was not due to epitope masking of paxillin by GIT, the paxillin-binding GIT C-terminal region was overexpressed (Fig. ​(Fig.2B,2B, left); no loss of antipaxillin FC staining was observed under these conditions.

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FIG. 2

GIT1 promotes FC disassembly. (A) GIT1 expression causes the selective loss of paxillin from FCs which are faintly visible (arrows), while vinculin and phosphotyrosine (PY) levels are unaffected. The anti-Flag immunofluorescent photomicrographs of typical HeLa cells 4 h after injection are compared with images of uninjected controls. (B) The paxillin-binding domain GIT^646-770 (left) does not affect antipaxillin staining. Coinjection of PIX and GIT1 (right) leads to focal adhesion loss, as assessed by antivinculin staining, and cell contraction. Injected cells are marked with stars. Bar, 10 μm. (C) Phase-contrast micrographs of cells showing induction of peripheral retraction. βPIX-expressing cells have small phase-dark ruffles, but coinjection of PIX plus GIT1 causes cell retraction (arrowhead). Membrane-tethered ^myrGIT1 similarly caused both cell body contraction and lamellipodia, driving neurite-like processes. Typical phenotypes were photographed 2 h after injection. (D) A C-terminal domain in GIT1 drives FC loss and cell contraction. The membrane-tethered constructs as shown were scored for cell contraction (B). The smallest myristoylated construct (646-770) was less efficient (++) in this regard. In each case, at least 15 microinjected cells were observed over a 3-h period.

In contrast to the effect of PIX or GIT1 alone, coinjection of expression plasmids led to loss of focal adhesions as assessed by antivinculin staining (Fig. ​(Fig.2B,2B, right), causing a collapse of the cells (Fig. ​(Fig.2C).2C). This phenotype superficially resembles that induced by active forms of PAK (20) or dominant inhibitory ROK (17), but we noted that PIX plus GIT1 did not cause any actin stress fiber loss. At early time points we also observed reduction in paxillin staining in FCs, suggesting that this is a primary event that then leads to FC loss. Targeting GIT1 to the membrane using an N-terminal myristoylation signal from Fyn (^myrGIT1) led to similar cell contraction (independent of PIX expression). Time-lapse analysis also revealed a counteracting activity which often produced cells with neurite-like outgrowths (Fig. ​(Fig.2C,2C, right). By comparison, ^myrβPIX-expressing cells were phenotypically normal (not shown). The ^myrGIT1 effects cannot be ascribed to the membrane anchor or indirectly to PIX recruitment to the plasma membrane.

Various regions of GIT1 were cloned into the pXJ-myr vector and tested for the ability to cause FC loss and cell body collapse, as summarized in Fig. ​Fig.2D.2D. The FC disassembly activity, typified by loss of vinculin staining with ^myrGIT1, was localized to the C-terminal 125 residues of GIT1. It seemed that PIX potentiated the activity of this C-terminal region when introduced with full-length GIT1, thereby promoting FC loss and cell contraction.

The GIT1 SHD-1 binds PIX.

To determine if these effects of GIT1 on FCs in vivo could be understood in terms of PIX regulation of the GIT C terminus, we localized the site of PIX binding. PIX^496-554 (Fig. ​(Fig.3A)3A) was used in overlay assays on various GIT1 fragments expressed as GST fusion proteins. Figure ​Figure3B3B shows that the fragments containing the SHD-1 bound PIX, but neither the ankyrin repeat nor central coiled-coil region (Fig. ​(Fig.1A)1A) was essential. Sequences between positions 376 and 423 apparently enhanced this binding (Fig. ​(Fig.3B,3B, compare lanes 5 and 6). Interestingly, only GIT1 and Spa2 family members contain SHD-1. The yeast SHD-1 covering ~150 residues at the N terminus has recently been shown to bind to Mkk1p, Mkk2p, and Ste7p (34). Comparison of Mkk1p(1-197) with Mkk2p revealed a single region of similarity which is weakly related to the PIX GIT1-binding domain (not shown).

We concluded that the ability of GIT1 to cause FC disassembly (through residues 646 to 770) is not due to recruitment of PIX-PAK complexes. The coinjection experiments indicated, however, that PIX was able to modulate this activity, either through localization of GIT1 or by altering its conformation.

Regulated binding of GIT1 to paxillin.

It has recently been reported that a region of paxillin (LD4 motif) binds to GIT1-like proteins, through a C-terminal region of PKL (37). By cotransfection of fragments of GIT1 (Fig. ​(Fig.4C)4C) with paxillin, we localized the C-terminal paxillin-binding domain of GIT1 further (Fig. ​(Fig.4A).4A). The C-terminal 125-residue GIT1^646-770 bound paxillin as efficiently as the larger GIT1^376-770 fragment; functional constructs smaller than this could not be derived (data not shown). Significantly, full-length GIT1 is a poor paxillin binder, but upon cotransfection with βPIX there was a dramatic increase in paxillin binding (Fig. ​(Fig.4B,4B, compare lanes 1 and 2). Thus, the paxillin-binding domain of GIT1 appears masked and is regulated by PIX. When GIT1 and PIX were cotransfected into cells, the potentiation of FC loss appeared to result from an increased affinity for paxillin.

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FIG. 4

GIT1 association with paxillin LD4 is regulated by PIX binding. (A) Paxillin is immunoprecipitated with C-terminal constructs of GIT1. COS-7 cells were transfected with expression vectors containing Flag-tagged ΔGIT1 constructs depicted in panel C and GFP-paxillin. Western blots (WB) of anti-Flag immunoprecipitates or total lysates (50 μg per lane) were probed by antipaxillin. (The smallest Δ3 GIT1 [residues 646 to 770] is detected in separate gels but not shown here.) (B) Coexpression of PIX potentiates the ability of GIT1 to associate with paxillin. GIT1 or Δ4 and Δ3 represent cotransfection. A significant increase of paxillin (top) associated with Flag-GIT1 immunoprecipitates (IP) (middle) was observed when PIX was also present. Total GFP-paxillin expression is shown at the bottom. (C) Summary of constructs used and their ability to bind paxillin via the conserved C-terminal region (black).

GIT1 can interact directly with FAK.

The localization of GIT1 to focal adhesions suggested that FAK might be the tyrosine kinase that copurifies with PIX-GIT1 complexes (22). It has been reported that GIT1 is a FAK substrate whose phosphorylation is up-regulated upon integrin ligation (2). To test whether FAK might be indirectly recruited to PIX-GIT1 complexes by paxillin, we examined GIT1 or paxillin immunoprecipitates for the presence FAK (Fig. ​(Fig.5A).5A). Surprisingly, GIT1 could bind a far greater proportion of FAK than paxillin (compare bottom and top panels), suggesting that FAK directly interacts with GIT1. This was confirmed using GST-GIT1^1-376 and recombinant (Escherichia coli-expressed) FAK (Fig. ​(Fig.5B).5B). The FAK-binding region was further determined by cotransfecting FAK separately with three fragments of GIT1 in COS-7 cells (Fig. ​(Fig.5C).5C). Only GIT1^253-424 (containing SHD-1) was capable of binding FAK (in the absence of the paxillin-binding domain). To test whether a trimeric complex of PIX, GIT1, and FAK could be formed, Flag-PIX was used to immunoprecipitate FAK in the presence or absence of HA-GIT1 (Fig. ​(Fig.5C,5C, right panel). Since GIT1 homodimers were not detected when we cotransfected different tagged forms of full-length GIT1 (data not shown), we conclude that the SHD-1 region can simultaneously bind FAK and PIX.

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FIG. 5

GIT1 directly binds FAK through SHD-1. (A) Flag-GIT1 or Flag-paxillin was coexpressed with GST-FAK, and the anti-Flag immunoprecipitates (IP) were analyzed for the presence of FAK. Little FAK was detected in paxillin immunoprecipitates compared to the amount in the total lysate. (B) Full-length FAK was expressed from pFlag-FAK vector. E. coli cell lysates (300 μl, 8 mg/ml) were passed through a 30-μl column of glutathione-Sepharose containing 2 mg of GST or GST/GIT1^1-376 per ml. rFak, recombinant FAK. (C) FAK and PIX can both associate with the GIT1 SHD-1. Flag-GIT1 constructs which were coexpressed with GST-FAK in COS-7 cells cover the entire GIT1 protein as shown schematically (domains as in Fig. ​Fig.1).1). The Flag-tagged immunoprecipitates (lower panel) were used to detect the presence of FAK. The right panels show that Flag-PIX was able to immunoprecipitate FAK only in the presence of HA-GIT1. (D) FAK or Src was cotransfected with GIT1 and/or paxillin and analyzed for phosphotyrosine (PY) content. The amount of phosphotyrosine relative to anti-Flag signal indicates the efficiency with which GIT1 and paxillin are substrates for FAK (right lane).

GIT1, which is phosphorylated on three sites (data not shown), also served as a more efficient FAK substrate than paxillin, as judged from the relative levels of tyrosine phosphorylation after appropriate cotransfection (Fig. ​(Fig.5D).5D). GIT1 was also found to be a substrate for the Src kinase.

GIT and FAK cause FC loss without affecting actin stress fibers.

The paxillin LD4 motif that is targeted by GIT1 also interacts with FAK but not with other paxillin binders such as vinculin or talin which associate with different regions (4, 5). Since our experiments suggested that the function of GIT1 interaction with the paxillin LD4 motif is to promote paxillin loss from FCs, we reckoned that the paxillin-binding domain of FAK would exhibit a similar effect. Indeed, microinjection of an expression plasmid encoding FAK^916-1053 (the so-called FAT [focal adhesion targeting] region) caused FC disassembly as assessed by vinculin staining, leaving smaller residual structures (Fig. ​(Fig.6).6). The larger FAK-related nonkinase (FRNK) construct could also promote FC disassembly and eventual cell contraction as efficiently as the FAT construct. Mutant FRNK (L343S), which cannot bind paxillin (reference 36 and our unpublished data), caused no alteration in either vinculin or paxillin distribution in FCs. As with ^myrGIT1 expression, neither FRNK nor FAT domains induced significant actin stress fiber disassembly (Fig. ​(Fig.6,6, bottom row); in contrast, ^myrPAK, like other activated versions of PAK, caused complete stress fiber loss.

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FIG. 6

The FAK C-terminal domain can cause FC disassembly. (Top) FAK^916-1053 and FRNK result in marked loss of FCs from HeLa cells as visualized by vinculin staining, but the mutant FRNK(L343S) is ineffective. Expression of each construct was by anti-Flag (middle). Typical cells are shown 2 h after injection with expression plasmid (100 μg/ml). FAK^916-1053-expressing cells begin to narrow and undergo cell body retraction. Similar effects were noted with full-length FAK. (Bottom) Distribution of polymerized actin as visualized by phalloidin-fluorescein isothiocyanate in typical cells expressing ^myrαPAK, ^myrGIT1, or FRNK. Only in the first case is there loss of actin stress fibers. Arrowheads indicate injected cells. Bar, 10 μm.

Both GIT1 and FAK can promote Rac activities.

Full-length FAK showed similar abilities to cause FC loss in NIH 3T3 and HeLa cells stained for vinculin. However, while the FRNK construct showed only peripheral collapse, FAK (full length) produced a counteracting membrane protrusive activity characterized by phase-dark dynamic lamellipodia (Fig. ​(Fig.7).7). For time-lapse experiments, HeLa cells were chosen because they are relatively static; FAK-expressing HeLa cells became very motile. Similarly, ^myrGIT1-injected cells are characterized by cell body rounding but concomitantly exhibit peripheral extension by active lamellipodial production. In both cases, the protrusive activity was blocked by coexpression of Rac1^N17 (not shown). The paxillin-binding ^myrGIT1^646-770 domain did not induce a similar lamellipodial formation, and once cells began to show signs of collapse, we noted that the process is extremely rapid. This might be attributable to internal tension generated by the intact actin-myosin system. Thus, full-length FAK and GIT1 promote two distinct activities: a decrease in cell adhesion (FC loss) and a Rac1-induced increase in peripheral membrane extensions, both facilitating migration.

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FIG. 7

FAK and GIT1 can drive lamellipodial formation. Phase-contrast micrographs of HeLa cells microinjected with expression plasmids (0.1 μg/μl) as shown. FAK-expressing cells typically extend many phase-dark membrane ruffles (arrowheads), while ^myrGIT1 cells become bipolar with lamellipodia forming at the ends (arrows). Uninjected cells (U) do not show movement over this time frame. These new structures were completely blocked by coexpression of Rac^N17. The FRNK and ^myrGIT^646-770 cells were captured at an intermediate stage of collapse: photomicrographs were taken with a 10-min interval approximately 1 h after injection.

Events downstream of Cdc42 and PAK involve GIT1 and paxillin.

We have recently shown that PAK (which is not usually detected in the RhoA-type FCs of cultured cells) becomes concentrated in such FCs when we introduce its autoinhibitory region PAK^83-149 (41). Thus, the kinase does play a role in these complexes but interacts only transiently, due to a negative regulation of its association with PIX (42). We tested whether increased levels of FC-localized PAK would lead to additional recruitment of GIT1, therefore indicating that PAK does recruit GIT1. Indeed, endogenous GIT1 was significantly concentrated in the FCs of HeLa cells expressing the PAK inhibitor (Fig. ​(Fig.8A)8A) relative to vinculin levels, which were unaffected. Thus, PAK is probably a limiting factor for GIT1 localization to FCs, which explains the rather weak FC staining pattern obtained with GIT1 antibodies.

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FIG. 8

PAK can recruit GIT1 to FCs. (A) Increased FC-localized GIT1 in cells expressing the kinase inhibitor PAK^83-149. The photomicrograph shows that in HeLa cells expressing the inhibitor (star), endogenous GIT1 staining is more concentrated in FCs relative to vinculin (B).

In the microinjection experiments, ^myrGIT1^646-770 or the FAK^916-1053 caused FC disassembly (via the LD4 region of paxillin), though these constructs might be predicted to merely drive the association of paxillin with the plasma membrane or FCs, respectively. Could these effects be attributable simply to such targeting of paxillin to juxtamembrane regions? Rather than causing FC loss, ^myrpaxillin expression increased FC numbers, particularly at peripheral locations (not shown). This suggested that ^myrpaxillin might stabilize newly emergent peripheral FCs (under the influence of Cdc42/Rac1). We then examined the effects of coexpressing ^myrpaxillin with Cdc42^G12V (Fig. ​(Fig.9A).9A). Typical vinculin staining patterns are shown: the coexpressing cells consistently exhibited much denser, brush-like peripheral FCs than cells expressing Cdc42^G12V alone (Fig. ​(Fig.9A).9A). We interpret this as ^myrpaxillin stabilizing these FCs by countering the effects of GIT1 (and FAK) which normally drive paxillin loss from FCs. In any case, paxillin recruitment to the membrane certainly does not result in FC disassembly.

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FIG. 9

PAK can recruit GIT1 to FCs. (A) Cdc42-induced peripheral FCs are stabilized by coexpression of membrane-targeted ^myrpaxillin. The typical rounded Cdc42^G12V phenotype is seen 4 h after injection with or without coexpressed ^myrpaxillin. Note that with ^myrpaxillin the peripheral FCs are much denser. (B) COS-7 cells were transfected with Flag expression plasmids as shown. Motility of positively stained cells was assessed as for Fig. ​Fig.11 (4 h after plating onto transwells). The top panel shows that ^myrpaxillin and the paxillin LD4 constructs effectively reduce Cdc42^G12V-driven cell motility. KID, the PAK inhibitor αPAK^83-149. (C) Smaller GIT1 and FAK constructs also promote motility. FAK^1-427 corresponds to the band 4.1-like domain, and FAK^916-1053 corresponds to the FAT domain.

Since our experiments indicated a Cdc42/PAK/PIX/GIT1/FAK pathway acting on FC-bound paxillin to promote FC turnover (thereby facilitating motility), we tested the effects of ^myrpaxillin and paxillin^250-281 (an LD4 peptide derived independently of Turner et al. [37] that binds to GIT and FAK) on Cdc42^G12V-induced cell motility. Both constructs attenuated the motility driven by Cdc42^G12V (Fig. ​(Fig.9B),9B), though LD4 had little effect on basal migration. This was also partially blocked by the kinase-inhibitory PAK^83-149 construct, consistent with PAK activity contributing to FC disassembly. By masking the GIT1 and FAK C-terminal regions, the LD4 peptide can block FC turnover.

GIT1 a multifunctional protein linking PAK and FAK pathways.

The direct repeat SHD-1 mediates GIT1 association with both PIX and FAK. This domain is found uniquely in GIT1 family and yeast Spa2-like proteins. Both are indirectly linked to Cdc42: Spa2p colocalizes with Cdc42 to the emerging bud site and is thought to bind directly to Ste11p and Ste7p (34), which lie downstream of the PAK-like Ste20p; GIT proteins bind PIX and thereby PAK. The direct association of FAK with GIT1 as well as its integrin-dependent tyrosine phosphorylation in vivo (2) suggest that GIT1 is a bona fide FAK substrate. Although the C-terminal domains of both FAK and GIT1 (138 and 125 residues, respectively) form a paxillin-binding domain (targeting LD4), the sequences are essentially unrelated. However, both contain a region with a high potential to form a coiled-coil structure (Fig. ​(Fig.11A).

Interestingly, GIT1 was first identified as being involved in heterotrimeric G-protein signaling (28), but how GIT1 binds to GRKs and whether this binding affects the various activities of GIT1 with respect to the cytoskeleton remains to be investigated. Two related Arf GAPs have recently been shown to associate with FCs: ASAP/centaurin β4 binds Src and phosphatidylinositol-4,5-bisphosphate and is localized to FCs (29); PAG3 contains a paxillin-binding domain and is suggested to regulate transport of paxillin to FCs (16). It has also been suggested that Arf1 mediates paxillin recruitment to FCs and potentiates RhoA function (25).

This work is supported by the Glaxo Singapore Research Fund.