Focal adhesion kinase–dependent activation of the early endocytic protein Rab5 is associated with cell migration

Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)–positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)–dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.

Because FAK phosphorylation on Tyr 397 is a central event in cell adhesion and migration, p85␣ interacts with phosphorylated FAK on Tyr 397 via its SH2 domains (3), and because sequestration of p85␣ is a mechanism that accounts for sustained Rab5 activation (18), we hypothesized that FAK is involved in Rab5 activation during integrin-mediated cell adhesion. In this work, we report that FAK stimulates Rab5 activity, leading to increased cell migration, as shown in models involving integrin-dependent activation of FAK and expression of FAK mutants in A549 cells and by reconstitution experiments in FAK-null fibroblasts. Mechanistically, FAK formed a complex and sequestered p85␣ in a phosphorylation-dependent manner, maintaining elevated Rab5-GTP levels. Accordingly, Rab5 activity was required for FAK-induced cell migration.

Inhibition of FAK decreases Rab5-GTP levels in non-stimulated and spreading cells
Studies from our group and others have shown that ligation of integrin ␤ 1 is followed by a time-dependent increase in Rab5-GTP levels (9,13). Because FAK is a central effector of downstream integrin engagement (1) and because Rab5 stimulates cellular responses that are reminiscent of those initiated by FAK, such as FA assembly and disassembly (9), Rac1 activation (14,15), persistent cell migration (9,11), and matrix metalloproteinase activation (9,23), we hypothesized that FAK is an upstream regulator of Rab5 that promotes GTP loading via an intermediate regulator yet to be identified. To address this hypothesis, A549 cells, which are known to adhere to fibronectin via integrin ␣ 5 ␤ 1 (9,13), were treated with the small molecule PF562,271, which specifically inhibits FAK autophosphorylation on Tyr 397 (24), and Rab5-GTP levels were measured by pulldown, as reported previously (9,18). As expected, PF562,271 caused a dose-dependent decrease in FAK autophosphorylation in A549 cells (Fig. S1A), with a 70% reduction at a concentration of 1 M (Fig. 1A). Interestingly, FAK inhibition with PF562,271 was associated with a substantial decrease in Rab5-GTP levels compared with control cells (Fig. 1B). These effects seemed to be selective for Rab5, because PF562,271 did not induce any fluctuations in Rab11-GTP levels (Fig. S1B), nor association of Rab4 with GTP-bound Rab5 (data not shown). Previous studies showed that Rab5-GTP loading is associated with relocalization of early endosomes (9,18). Accordingly, PF562,271-mediated reduction of Rab5-GTP levels was associated with decreased early endosome number but not size, as judged by using the effector of Rab5 and early endosome-specific marker EEA1 (Fig. 1, C and D). Importantly, these fluctuations were not associated with changes in total Rab5 or EEA1 levels ( Fig. S1C and Fig. 1B). Furthermore, PF562,271 decreased staining of Rab5-positive puncta at the cell periphery (Fig. 1, E and F), which is in agreement with previous reports showing activation-dependent relocalization of Rab5 (9,18). Intriguingly, PF562,271 did not affect the size or number of Rab5positive structures (Fig. S1D) or the localization of other related Rab proteins (Fig. S1E). These observations suggest that basal Rab5-GTP levels are partially maintained by FAK in a phosphor-ylation-dependent manner and that these events are associated with peripheral localization of early endosomes.
Because integrin ligation promotes both FAK autophosphorylation on Tyr 397 (1) and Rab5 activation (9, 13), we next evaluated whether adhesion-induced Rab5-GTP loading required FAK. To this end, A549 cells were allowed to adhere and spread onto fibronectin-coated surfaces for 2 h, and the effects of PF562,271 were analyzed. First, as anticipated, suspended cells showed minimal levels of phospho-Tyr 397 FAK, which were further inhibited by PF562,271. However, cell adhesion to fibronectin was followed by a substantial increase in phospho-Tyr 397 FAK levels, which were inhibited by PF562,271 (Fig. 2, A  and B). Then Rab5 activity was measured in suspended and adherent cells, and, in agreement with previous studies (13), Rab5-GTP levels were substantially increased during adhesion to fibronectin (Fig. 2C). Interestingly, adhesion-driven Rab5 activation depended on FAK phosphorylation because PF562,271 decreased Rab5-GTP levels in adherent but not in suspended cells (Fig. 2, C and D).
FAK inhibition experiments suggest that both basal and adhesion-induced activation of FAK are required for Rab5-GTP loading. These findings were further supported by genetic approaches because expression of WT FAK and FAK/Y180A/ M183A (open conformation, constitutively active FAK), but not FAK/Arg 454 (kinase-dead mutant of FAK), which were readily autophosphorylated on Tyr 397 (Fig. 3, A and B), increased endogenous Rab5-GTP levels in A549 cells (Fig. 3, C and D). These observations were confirmed in FAK-null mouse embryonic fibroblasts (FAK Ϫ/Ϫ MEFs), because Rab5-GTP levels were substantially decreased in FAK Ϫ/Ϫ MEFs compared with control FAK ϩ/ϩ MEFs (Fig. 3, E-H). Most importantly, reconstitution of FAK Ϫ/Ϫ MEFs with GFP-FAK, but not GFP-FAK/Arg 454 (Fig. 3, E and F), restored Rab5-GTP loading to levels comparable with those observed in FAK ϩ/ϩ MEFs (Fig. 3, G and H). Collectively, these results confirm the finding that FAK activity is necessary for Rab5 activation in nonstimulated and adhering cells and that it may constitute an unanticipated mechanism involved in FAK-driven cell migration.

Rab5 is relevant for FAK-driven cell migration
Several studies have shown the relevance of Rab5 in different aspects of cell migration (reviewed in Ref. 17), and although much evidence has been focused on downstream events triggered by Rab5, a few studies are available regarding upstream regulation leading to Rab5 activation in the context of cell migration. Because FAK is a critical regulator of cell migration, and because Rab5-GTP levels were shown to be increased by FAK (Figs. 1-3), we evaluated whether FAK-induced cell migration required Rab5 activation. To this end, A549 cells were co-transfected with FAK and Rab5/S34N (the GDPbound, inactive mutant of Rab5). As expected, and in agreement with previous studies (9), expression of WT FAK promoted a significant increase in cell migration, whereas expression of Rab5/S34N decreased basal cell migration by 35% (Fig. 4, A and B). Most importantly, co-transfection of Rab5/ S34N along with WT FAK inhibited FAK-induced cell migration to levels that were below those observed in control cells (Fig. 4B). In agreement with these observations, FAK Ϫ/Ϫ MEFs FAK-driven Rab5 activation depicted decreased cell migration compared with control FAK ϩ/ϩ MEFs (Fig. S2A), whereas expression of Rab5/Q79L (the GTP-bound, active mutant of Rab5) and, to a lesser extent, WT Rab5, but not Rab5/S34N, promoted cell migration in FAK Ϫ/Ϫ MEFs (Fig. 4, C and D). The requirement of Rab5 for FAK-induced cell migration was also confirmed in Rab5 knockdown experiments because shRNA-mediated targeting of endogenous Rab5 decreased the extent of cell migration induced by FAK (Fig. S2B). Collectively, these data suggest that Rab5 is a novel downstream player in FAK-driven cell migration.

Recruitment of p85␣ by FAK prevents Rab5 inactivation
To obtain insights into the mechanisms involved in FAKmediated Rab5 activation, we searched for putative guanine nucleotide exchange factors (GEFs) and GAPs that could potentially be associated with FAK. In this group of regulators, which included the Rab5 GEFs RIN2 and ALS2 and the Rab5 GAP p85␣, only p85␣ was found to undergo relocalization within early endosomes upon FAK inhibition with PF562,271 (Fig. S3). This observation was interesting because p85␣, which is commonly described as the regulatory subunit of PI3K, is known to interact with FAK in a phosphorylation-dependent manner (3), and it is known to depict GAP activity toward Rab5 (21,22). Thus, we first confirmed the association described previously for FAK and p85␣ in A549 cells, which has been shown to be dependent on FAK phosphorylation, because reduction of phospho-Tyr 397 FAK levels with PF562,271 decreased the extent of co-immunoprecipitation between FAK and p85␣ (Fig.  5, A and B). These observations were confirmed by confocal , and whole-cell lysates were prepared for Western blot analysis (A) and R5BD pulldown assays (B). Top panels, representative Western blot images are shown for phospho-Tyr 397 -FAK (pY397-FAK), total FAK, ␤-actin, Rab5-GTP, and total Rab5. The graphs indicate the quantification of pTyr 397 -FAK versus total FAK and Rab5-GTP versus total Rab5, respectively, as obtained from scanning densitometric analysis. Data represent the average of four independent experiments (mean Ϯ S.E.; **, p Ͻ 0.01). C, EEA1-positive early endosomes were visualized by immunofluorescence and confocal microscopy using a rabbit polyclonal antibody and 4Ј,6-diamidino-2-phenylindole (DAPI) for nuclear staining in cells treated with either DMSO or 1 M PF271. Representative images are shown. Scale bar ϭ 10 m. D, early endosome number and size were quantified from data obtained as in C by using ImageJ software. Data were obtained from quantification of 40 cells per condition (mean Ϯ S.E.; ***, p Ͻ 0.001; n.s., nonsignificant). E, Rab5 localization in A549 cells treated with DMSO or 1 M PF562,271 was analyzed by immunofluorescence and confocal microscopy using a specific mAb against Rab5. Arrowheads indicate peripheral Rab5 localization. Representative images are shown. Scale bar ϭ 10 m. F, left panel, intensity profiles for Rab5 localization were obtained by tracing a line from the nuclei to the cell periphery and plotted with ImageJ software. Right panel, quantification of peripheral Rab5, obtained by measuring the relative fluorescence intensity within the proximal 5 m next to the cell periphery (inset square, left panel). Data were obtained by averaging 15 cells per condition (mean Ϯ S.E.; *, p Ͻ 0.05). A.U., arbitrary units.

FAK-driven Rab5 activation
microscopy; FAK and p85␣ co-localized at the cell periphery in a PF562,271-dependent manner (Fig. 5C). Similar results were obtained with different combinations of antibodies that recognized endogenous FAK and p85␣ (Fig. S4). These data indicate that recruitment of p85␣ by FAK requires phosphorylation on Tyr 397 , which parallels the up-regulation of Rab5-GTP levels by FAK. It is of note that, because p85␣ is a critical regulator of the PI3K/Akt pathway, we measured phospho-Thr 308 -Akt levels and observed that neither FAK inhibition nor Rab5 depletion in A549 cells affected active Akt levels, suggesting that these events are independent of activation of the conventional PI3K/ Akt pathway (Fig. S2C). Finally, by using the alternative model of FAK-null fibroblasts, we confirmed that FAK and p85␣ associate in a phosphorylation-dependent manner because FAK Ϫ/Ϫ MEFs reconstituted with WT FAK, but not FAK/Arg 454 , showed higher co-immunoprecipitation of FAK and p85␣ (Fig.  5, D and E).
Then, to evaluate the role of p85␣ in Rab5 activation by FAK, we performed pulldown assays in co-expression experiments with WT FAK and p85␣. In accordance with our observations (Fig. 3), co-transfection of GFP-FAK with an empty vector (mCherry alone) did not affect FAK-induced Rab5 activation (Fig. 6A). However, exogenous expression of p85␣ prevented FAK-induced Rab5 activation, as shown in co-transfection experiments using mCherry-p85␣ and GFP-FAK (Fig. 6A). Alternatively, the requirement of p85␣ in FAK-induced Rab5 activation was evaluated in siRNA-mediated knockdown experiments. In agreement with previous observations (Fig. 1), FAK inhibition with PF562,271 decreased Rab5-GTP levels in siRNA control cells; however, these effects were not observed upon siRNA-mediated targeting of endogenous p85␣ (Fig. 6B). In fact, sole knockdown of endogenous p85␣ caused an increase of Rab5-GTP levels, which is in agreement with earlier reports showing augmented Rab5 activity in p85␣-deficient cells (21,25). Nevertheless, under these conditions, FAK inhibition did not decrease elevated Rab5-GTP levels in siRNA-p85␣ cells (Fig. 6B). Taken together, these results indicate that p85␣ is involved in FAK-driven Rab5 activation.

Discussion
Several studies have shown that the small GTPase Rab5 promotes cell adhesion and migration via different mechanisms that include enhancing integrin trafficking, FA turnover, and Rac activation (9 -11, 14, 15) (reviewed in Refs. 16,26). However, leaving aside downstream signaling stemming from Rab5, upstream regulators accounting for increased Rab5 activity during cell adhesion and migration have remained largely unknown. Here we identify FAK as an unanticipated, indirect activator of Rab5 that promotes cell migration via increased Rab5-GTP loading. By using pharmacological and genetic approaches, we show that FAK activity is required for both maintenance of Rab5-GTP levels under basal conditions in resting cells and adhesion-induced Rab5-GTP loading. FAKdriven activation of Rab5 was associated with peripheral and enlarged early endosomes, which is in accordance with earlier studies indicating that Rab5 is necessary for maintenance of the number of early and late endosomes (27). A similar outcome has been documented previously, showing that Rab5-GTP levels increase upon phosphorylation of pro-Caspase-8 on Tyr 380 , leading to relocalization of Rab5 to the cell periphery, hence affecting the trafficking of cargo from early to late endosomes (18 -20).
Intriguingly, previous studies by our group and others showed that Rab5 accelerates the rates of FAK auto-and dephosphorylation on Tyr 397 , which is necessary for FA turnover and tumor cell invasion (9,28), whereas other studies showed that Rab5 expression and activity are necessary for hypoxia-driven FAK activation (29). These seemingly disparate observations could be reconciled with this study on the basis of the existence of a FAK-Rab5 circuitry that operates in a positive feedback loop that provides fine-tuning of cell adhesion and migration and whose deregulation under conditions such as hypoxia leads to sustained cell migration and invasion, as observed in tumor cells.
The observation that FAK autophosphorylation on Tyr 397 was required for Rab5-GTP loading in cells undergoing adhesion to fibronectin but not in suspended cells suggests that ␤ 1 and ␤ 3 integrins are possible initiators required to activate this signaling cascade. Our data favor the involvement of ␤ 1 integrins because, although A549 cells express both integrins (29,30), PF562,271 failed to decrease Rab5-GTP levels in cells adhered to collagen I (a ligand for both ␤ 1 and ␤ 3 -integrins) or to plates coated with anti-␤ 3 antibodies (Fig. S5). This is in addition to the fact that only ␤ 1 has been shown to associate with Rab5 in a complex, promoting its endocytic trafficking (9,12). Although further research is required to identify the subset of integrins that trigger Rab5-GTP loading via FAK, this will be relevant in the context of tumor cell migration because a preference for one specific integrin dictates different migration responses. Specifically, engagement of ␣ 5 ␤ 1 is associated with
Our data show that p85␣, which is a Rab GAP, co-immunoprecipitates with phospho-Tyr 397 FAK, allowing increased Rab5-GTP levels. This is intriguing because, among Rab5 GAPs and GEFs with reported roles in cell migration, p85␣, but not ALS2 or RIN2, were found to relocalize to/from early endosomes. However, we cannot exclude the participation of additional GAPs or GEFs yet to be identified. Our study suggests that FAK-induced Rab5 activation requires sequestration of p85␣. Importantly, our data suggest that p85␣'s GAP activity, but not side activation of the PI3K/Akt branch, is involved in FAK-driven Rab5 activation and cell migration because phospho-Thr 308 -Akt levels remained constant upon FAK inhibition or Rab5 depletion, and expression of Rab5/S34N increased phospho-Thr 308 -Akt levels (Fig. S2D), which is opposite to the effects of this mutant in cell migration. In fact, Akt inhibition did not cause any significant fluctuations in Rab5-GTP levels (Fig. S2E). Hence, we propose that FAK-dependent sequestration of p85␣ prevents Rab5 inactivation, allowing increased Rab5-GTP levels (Fig. 6C, proposed model).

FAK-driven Rab5 activation
A similar mechanism of sequestration was shown several years ago. Specifically, Src-mediated phosphorylation of procaspase-8 on Tyr 380 creates a docking site for SH2 domaincontaining proteins, including p85␣ (19,20), which, upon interaction with caspase-8, is sequestered, precluding Rab5-GTP hydrolysis and promoting relocalization of early endosomes to the cell periphery (18). It remains to be explored whether p85␣ associates with FAK via other sites and that p85␣'s SH2 domains are required for Rab5-GTP loading. Intriguingly, recent studies by our group indicated that recruitment of p85␣ within complexes containing the scaffolding protein Caveolin-1 accounts for increased Rab5-GTP levels in metastatic colon carcinoma and mouse melanoma cells, leading to sustained cell migration and invasion in a manner that depends on Src family kinases (14). This sequestration mechanism involving p85␣ as a Rab GAP and phosphorylated proteins, including pro-caspase-8 (18), Caveolin-1 (14), and FAK (this study), appears to be a more general mechanism accounting for increased Rab5-GTP levels, hence contributing to increased cell migration, which is a common target in tumor cells. In summary, this study provides mechanistic insights regarding a novel signaling axis, FAK-p85-Rab5, that is relevant for cell adhesion and migration.

FAK-driven Rab5 activation
GFP (sc-8334, Santa Cruz Biotechnology), and rabbit polyclonal anti-EEA1 (sc-33585, Santa Cruz Biotechnology). Rabbit polyclonal anti-phospho-Thr 308 -Akt (2965) and mouse monoclonal anti-Akt (2966) were from Cell Signaling Technology. Goat anti-rabbit and goat anti-mouse antibodies bound to horseradish peroxidase and anti-actin antibody (A5316) were obtained from Bio-Rad. Control (sc-37007) and p85␣-targeting (sc-36217) siRNA constructs were from Santa Cruz Biotechnology. Lipofectamine 2000 as well as Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies were from Invitrogen. Tissue culture DMEM, Opti-MEM, antibiotics, and FBS were from HyClone Laboratories (Logan, UT) and Gibco Life Technologies. GSH-Sepharose 4B was from GE Healthcare. The small molecule PF562,271, described as an inhibitor of FAK autophosphorylation on Tyr 397 (24), was from Laviana Corp. Akt inhibitor VIII was from Cayman Chemical (catalog no. 14870) and was used at 3 M concentration. The EZ-ECL chemiluminescent product and protein A/G beads were from Pierce.

Cell culture
A549 lung carcinoma cells were cultured in DMEM (highglucose) supplemented with 10% FBS and antibiotics. Downregulation of endogenous Rab5A by shRNA has been described previously, and cells expressing a nonspecific shRNA sequence (plasmid 1864, Addgene) were used as an experimental control (9). MEFs derived from FAK Ϫ/Ϫ MEFs as well as FAK Ϫ/Ϫ MEFs reconstituted with GFP, GFP-FAK, or GFP-FAK/Arg 454 were provided by Dr. David Schlaepfer and have been described previously (32). MEFs were maintained in DMEM (high-glucose) supplemented with 10% FBS and antibiotics.

Immunofluorescence
Immunofluorescence was performed as described previously (9). Briefly, cells were grown for 24 h on glass coverslips. Following each treatment, samples were fixed with 4% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 2 min, washed with PBS, and blocked with 5% BSA/

FAK-driven Rab5 activation
PBS. Samples were then incubated with primary antibodies for 1 h at 37°C, washed three times (PBS, 5 min), incubated with secondary antibodies for 1 h, washed three times, and mounted. Samples were visualized with a Nikon C2ϩ confocal microscope (model Eclipse TI), using a ϫ60 objective (Fluorite Plan, numerical aperture 0.5-1.25) unless indicated otherwise. Colocalization was analyzed with Fiji (ImageJ) software using the co-localization plugin.

Transwell migration assay
Boyden chambers were used to perform migration assays (Transwell Costar, 6.5-mm diameter and 8-m pore diameter). The outer layers of the inserts were precoated with 2 g/ml fibronectin. A549 cells (5 ϫ 10 4 ) were resuspended in serum-free medium and plated onto the top of the chambers. FBSsupplemented medium (10%) was added to the bottom chambers. After 2 h, the inserts were removed and washed. Cells that migrated to the bottom side of the inserts were stained with 0.1% crystal violet in 2% ethanol, and samples were counted in an inverted microscope.

Rab5-GTP pulldown assay
Spread cells underwent a 2-h spreading assay, and suspended cells were incubated for 2 h on ice. Spread and suspended A549 cells were lysed in a buffer containing 25 mM HEPES (pH 7.4), 5 mM MgCl 2 , 100 mM NaCl, 1% NP-40, 1 mM DTT, 10% glycerol, and protease inhibitors. Lysates were incubated for 5 min on ice and clarified by centrifugation (10,000 ϫ g, 1 min, 4°C). Super- Figure 6. The RabGAP p85␣ prevents Rab5-GTP loading induced by FAK. A, A549 cells were co-transfected with plasmids encoding for mCherry, mCherry-p85␣, GFP, and GFP-FAK for 24 h. Cell extracts were prepared and used for subsequent whole-cell lysate analysis or R5BD pulldown assays. Representative Western blot images are shown for p85␣, FAK, ␤-actin, Rab5-GTP, and total Rab5. The graph shows a quantification of Rab5-GTP with respect to total Rab5, as obtained from scanning densitometric analysis. Data represent the average of four independent experiments (mean Ϯ S.E.; *, p Ͻ 0.05; n.s., nonsignificant). B, A549 cells were transfected with a pool of three siRNAs targeting endogenous p85␣ (for details, see "Experimental procedures"; a scrambled-sequence siRNA was used as a control) and treated with DMSO (Ctrl) or 1 M PF271 (PF) for 2 h. Cell extracts were prepared and used for subsequent whole-cell lysate analysis or R5BD pulldown assays. Representative Western blot images are shown for p85␣, FAK, phospho-Tyr 397 -FAK, ␤-actin, Rab5-GTP, and total Rab5. Rab5-GTP levels were quantified by scanning densitometric analysis of Western blots and normalized to total Rab5. The graph shows quantification of Rab5-GTP with respect to total Rab5, as obtained from scanning densitometric analysis. Data represent the average of four independent experiments (mean Ϯ S.E.; *, p Ͻ 0.05; **, p Ͻ 0.01). C, proposed model. Rab5 is known to promote cell migration by increasing focal adhesion disassembly (9), although the upstream regulators accounting for both Rab5 activation and cell migration remain poorly understood. Here we show that adhesion-dependent phosphorylation of FAK on Tyr 397 promotes the recruitment of p85␣ (step 1), precluding its Rab-GAP activity toward Rab5 and leading to augmented Rab5-GTP levels (step 2). These events contribute to understanding upstream regulation of Rab5 in the context of cell migration (step 3), which has been shown previously to be associated with Calpain2-dependent proteolysis of Talin (10) and sustained focal adhesion turnover (9). A.U., arbitrary units; endo, endogenous.

FAK-driven Rab5 activation
natants were used for pulldown assays with 30 g of GST-R5BD-precoated GSH beads for each condition. R5BD beads were incubated with recovered supernatant for 15 min at 4°C in a rotating shaker. Rab5-GTP bound beads were recovered and washed three times with lysis buffer containing 0.01% NP-40. Samples were analyzed by Western blotting. For Rab5-GTP, Rab11-GTP levels were measured in pulldown assays using the GST-FIP3 fusion protein.

Western blotting
Cells were washed twice with ice-cold PBS and lysed in 0.2 mM HEPES (pH 7.4), 0.1% SDS, and phosphatase (1 mM Na 3 VO 4 ) and protease inhibitors for whole-cell lysate analysis. Protein extracts were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed by blocking with 5% gelatin in 0.1% Tween/PBS, followed by the specific antibodies.

Immunoprecipitation
Immunoprecipitations were performed as described previously (29). Briefly, cell extracts were prepared in a buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, and protease inhibitors by incubating samples on ice (5 min). Samples were centrifuged at 13,000 ϫ g for 1 min at 4°C, and postnuclear supernatants (500 g of total protein) were immunoprecipitated with protein A/G bead-immobilized antibodies for 30 min. FAK was immunoprecipitated with 10 g of polyclonal antibody. Immunoprecipitated samples were solubilized in Laemmli buffer, boiled, separated by SDS-PAGE, and analyzed by Western blotting as indicated above.