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Originally published In Press as doi:10.1074/jbc.M505057200 on July 28, 2005

J. Biol. Chem., Vol. 280, Issue 39, 33200-33205, September 30, 2005
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AF6 Negatively Regulates Rap1-induced Cell Adhesion*

Zhongchun Zhang, Holger Rehmann, Leo S. Price, Jurgen Riedl, and Johannes L. Bos1

From the Department of Physiological Chemistry and Centre of Biomedical Genetics, University Medical Centre, Utrecht 3508 AB, The Netherlands

Received for publication, May 9, 2005 , and in revised form, July 12, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AF6 is involved in the connection of membrane-associated proteins to the actin cytoskeleton. It binds to Ras-like small GTPases and is suggested to be an effector of both Ras and Rap. Here we show that knockdown of AF6 in T cells by RNA interference enhanced Rap1-induced integrin-mediated cell adhesion, whereas overexpression of AF6 had the opposite effect. Interestingly, AF6-induced inhibition of cell adhesion correlated with an increase in RapGTP levels. Like AF6, protein KIAA1849 contains a Ras association domain and interacted with Rap1. However, KIAA1849 did not inhibit Rap1-induced cell adhesion. We concluded that AF6 is a negative regulator of Rap-induced cell adhesion. We proposed that AF6 inhibits Rap-mediated cell adhesion by sequestering RapGTP in an unproductive complex and thus prevents the interaction of Rap1 not only with effectors that mediate adhesion but also with Rap GTPase-activating proteins. Thus, AF6 may buffer RapGTP in resting T cells and maintain them in a non-adherent state.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rap proteins (Rap1a, -1b, -2a, and -2b) are small GTPases closely related to Ras. They are activated by a variety of extracellular signals through the regulation of specific guanine nucleotide exchange factors and GTPase-activating proteins (GAP2(s)) (1). Rap1 is involved in various cellular processes, most notably, the regulation of integrin-mediated cell adhesion and cadherin-mediated cell junction formation (2–4). A variety of effectors have been identified that mediate Rap1 function (5). These effectors include RapL and Riam (6–8). Both proteins interact with Rap1 through a Ras association (RA) domain and mediate Rap-induced integrin-dependent cell adhesion. RapL may function by direct binding to both Rap1 and integrins, whereas Riam may function through an interaction with the actin-regulatory proteins profilin and Ena/Vasp. Arap3 is another RA domain containing protein that interacts with Rap1. This protein is an Arf- and RhoGAP and mediates Rap1-induced inactivation of Rho (9). However, not all effectors of Rap1 have a RA domain. For instance, Vav2, a guanine nucleotide exchange factor for the small GTPase Rac, binds to Rap1 through its PH domain and mediates Rap-induced cell spreading (10).

AF6 (also called afadin) has a N-terminal region containing two RAs, one of which interacts with Ras-like small GTPases, including Ras and Rap (11–13). This protein was first identified as the fusion partner of ALL-1 protein in human acute myeloid leukemia (14). AF6 is a multidomain actin-binding protein that serves as a scaffold protein between cell membrane-associated proteins and the actin cytoskeleton (11). Among the proteins that interact with AF6 are the tight junction protein ZO-1, the cell-cell adherence junction molecule nectin, various Eph receptors and the actin-regulatory protein profilin (11, 15–17). AF6 was found to be an effector for Ras in the control of cell junction formation via direct interaction with ZO-1 (15). In addition, in Drosophila, the AF-6 homolog Canoe is an effector of Rap in the regulation of dorsal closure (18). Recently it was shown that AF6 can interact with Rap GAPs, such as Rap1GAP and SpaI through its PDZ domain (19). This interaction is mediated by a conserved internal {beta}-turn in the Rap GAPs. AF6 recruits Rap GAPs to negatively regulate the level of Rap1GTP and consequently Rap1-induced integrin-mediated cell adhesion. Because these functional studies were largely performed in overexpression systems, we addressed the question whether AF6 is involved in the control of cell adhesion induced by activation of endogenous Rap and depletion of endogenous AF6. Specific activation of endogenous Rap can be achieved using the cAMP analogue 8-pCPT-2'-O-Me-cAMP (007) that specifically activates Epac (exchange protein directly activated by cAMP) (20). Indeed we observed that activation of Rap1-induced adhesion is negatively regulated by AF6. Importantly, knockdown of AF6 in T cells results in an enhancement of Rap-mediated cell adhesion. Surprisingly, inhibition of Rap1-induced cell adhesion by AF6 correlated with an increase, rather than a decrease in RapGTP. We propose a mechanism of AF6-induced inhibition of Rap1-mediated cell adhesion that includes the sequestration of Rap1GTP in an unproductive complex with AF6 preventing the interaction of Rap1 with both Rap GAPs and effectors of Rap-induced cell adhesion. Thus, AF6 may buffer RapGTP in resting T cells and maintain them in a non-adherent state.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Constructs—Hemaglutamin (HA)-tagged Rap1, Rap1V12 (HA-RapV12), and Rap1GAP (HA-RapGAP I) have previously been described (21). PCR fragments containing AF6 or AF6-{Delta}RA (residues 347–1612) flanked by a KpnI site at the 5' and a NotI site at the 3' were amplified from AF6 cDNA, provided by Dr. Eli Canaani (Weizmann Institute of Science, Rehovot, Israel) and subcloned into KpnI/NotI sites of a pGEM-T vector (Promega). Subsequently, these fragments were subcloned into KpnI/NotI-digested pcDNA3-HA, and integrities of the constructs were confirmed by DNA sequencing. Myc-AF6-RA (residues 25–353) was generated by PCR amplification of a fragment flanked by a EcoRI site at the 5' and a NotI site at the 3' of AF6 from AF6 cDNA. This fragment was subcloned into EcoRI/NotI-digested pcDNA3-Myc vector. Full-length Myc tagged AF6 was provided by Dr. Kaibuchi Kozo (Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Japan). The AF6L (KIAA1849) cDNA containing the coding sequence was kindly provided by the Kazusa DNA Research Institute (22). Polymerase chain reaction fragments containing AF6L or AF6L-RA (residues 25–213) flanked by a SalI site at the 5' and a NotI site at the 3' were subcloned into the pGEM-T vector. These clones were subsequently used to generate HA-AF6L or HA-AF6L-RA by introducing the SalI/NotI fragment containing AF6L or AF6L-RA into SalI/NotI-digested pMT2-SM-HA.

Rap1 Activation Assays and Immunoblotting—Rap1 activation was assayed as described previously (23). Briefly, cells were washed with cold phosphate-buffered saline and lysed with buffer containing 1% Nonidet P-40. Lysates were cleared by centrifugation, and active Rap was precipitated with glutathione-Sepharose beads precoupled to a GST fusion protein of the Ras association domain of Ral guanine nucleotide dissociation stimulator. Precipitates were washed three times with lysis buffer and solubilized in SDS sample buffer. A portion of the cell lysate was reserved for analysis of total Rap content. HA-Rap1 was detected following Western blotting with anti-HA antibodies.

GST Pull-down Assays—For HA-AF6 and HA-AF6L pull-down assays, glutathione-agarose beads were loaded with GST-Rap1. HB6 cells were transfected with HA-AF6 or HA-AF6L, followed by cell lysis in lysis buffer containing 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% piridinium betain, 5 mM EDTA, 10 mM NaF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Cell extracts were incubated with GST-immobilized proteins for 1 h at 4°C. After 4 times washing in lysis buffer, bound proteins were resuspended in Laemmli sample buffer (Bio-Rad Laboratories) and resolved by SDS-PAGE, and HA-AF6 or HA-AF6L were detected by Western blotting using anti-HA antibody.

Western Blotting—Western blotting of all protein samples was carried out using polyvinylidene difluoride membranes. The antibodies used for protein detection are the monoclonal anti-HA (12CA5), monoclonal anti-Myc (9E10), monoclonal anti-AF6 (Transduction Laboratories), monoclonal anti-tubulin (Oncogene Science).

Cell Culture, Cell Line, and Transfection—The Epac I monoclonal Jurkat T cell line (HB6) was generated by retroviral transduction of Jurkat cells with amphotropic virus encoding Epac-IRES-GFP.3 HB6 cells were grown at 37 °C in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated (30 min at 56 °C) fetal bovine serum and 0.05% glutamine in the presence of penicillin and streptomycin. Cells were transiently transfected by electroporation using 35 µg of plasmid DNA in total. Cells (1.2 x 107 cells/ml in 0.4 ml of complete medium) were pulsed at 250 V and 960 µF with 5 µg of TK-luciferase plasmid DNA, construct plasmid as indicated in the figure legends, and added vector plasmid to keep DNA amounts constant. Subsequently, 24 h after transfection, cells were transferred to serum-free medium and used 42 h after transfection. For RNA interference experiments, cells were transferred to serum-free medium 48 h after transfection and used 72 h after transfection.

Adhesion Assay—For adhesion assays, transiently transfected Jurkat cells serum-starved overnight were harvested, washed, and resuspended in TSM buffer (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) at a concentration of 5x105 cells/ml. 24-Well Nunc Maxisorp plates (Corning) were coated with fibronectin (5 µg/ml) overnight at 4 °C, washed, and blocked for 1 h at 37°C with 1% bovine serum albumin, TSM. After washing, 200 µl of TSM was added per well with or without the indicated stimuli. 007 (BioLog) was used at 100 µM and Mn2+ was used at 4 mM. Subsequently, 200 µl of cell suspension was added per well. Cells were allowed to adhere for 1 h at 37°C, and nonadherent cells were removed with warmed 0.5% bovine serum albumin, TSM. Adherent cells were lysed and subjected to a luciferase assay as described previously (24). Expression of transfected constructs was confirmed by immunoblotting of total cell lysates. Adherent cells were calculated, and the cell numbers were corrected for transfection efficiency and nonspecific effects of constructs by measuring luciferase activity of total input cells ((counts in cells bound/counts in total input cells) x 100%).



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  		FIGURE 1.
AF6 inhibits Rap1-induced integrin-mediated cell adhesion. A, AF6 inhibits Rap1V12-induced adhesion. Jurkat cells stably expressing Epac (HB6 cells) were cotransfected with TK luciferase reporter plasmid and either empty vector or HA-AF6 with increasing amounts HA-Rap1V12 (0, 1, 2, and 10 µg) as indicated. After 42 h cells were replated and seeded in triplicate wells coated with fibronectin (FN), and after 1 h the percentage of adherent cells were measured. Results are representative of three independent experiments. The bottom panels indicate the expression of HA-AF6 and HA-RapV12 as determined by Western blotting (WB) using anti-HA as a probe. B, AF6 inhibits 007-induced adhesion. HB6 cells were cotransfected with TK luciferase reporter plasmid and either empty vector or HA-AF6 and adhesion was measured as indicated above. After replating, the cells were incubated with either 007 or Mn2+ as indicated. Results are representative of three independent experiments. The bottom panel indicates the expression of HA-AF6 as determined by Western blotting using anti-AF6 as a probe.

 
Small Interfering RNA Preparation—To make pTER-AF6, gene-specific oligonucleotides were ligated into pTER vector that had been digested with BglII and HindIII according to described protocol (25). The oligonucleotides used are, for AF6 shRNA1, 5'-gatcccgatgatcgggaaggcagatttcaagagaatctgccttcccgatcatctttttggaaa-3' and 5'-agcttttccaaaaagatgatcgggaaggcagattctcttgaaatctgccttcccgatcatcgg-3' and for AF6 shRNA2, 5'-gatcccggagagagctgacgggtctttcaagagaagacccgtcagctctctcctttttggaaa-3' and 5'-agcttttccaaaaaggagagagctgacgggtcttctcttgaaagacccgtcagctctctcc-3'.



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  		FIGURE 2.
Inhibition of cell adhesion by AF6 requires the RA domains of AF6. A, AF6 and Rap interaction requires the AF6 RA domains. Purified GST-Rap1 was used to precipitate HA-AF6 and HA-AF6-{Delta}RA from HB6 cells transiently transfected with either HA-AF6 or HA-AF6-{Delta}RA. The blot was probed with anti-HA antibody. Expression of each protein was determined by immunoblotting of the straight lysates. B, deletion of the RA domain abolishes AF6-induced inhibition of cell adhesion. HB6 cells were cotransfected with TK luciferase reporter plasmid and empty vector, AF6, or AF6-{Delta}RA. Adhesion was measured as indicated in legends Fig. 1. In the bottom panel expression levels of transfected proteins are shown. Results are representative of three independent experiments. FN, fibronectin. C, the AF6-RA domain blocks Rap-induced adhesion less efficient than full-length AF6. HB6 cells were cotransfected with TK luciferase reporter plasmid and either AF6 or indicated amounts of AF6-RA domain and adhesion was measured as indicated in legend Fig. 1. Results are representative of three independent experiments. Bottom panel, expression levels of transfected proteins. D, AF6 efficiently increases GTP level of overexpressed Rap1. HB6 cells were cotransfected with empty vector, HA-AF6, or HA-AF6-{Delta}RA in combination with HA-Rap1. The upper panel shows the GTP levels of the HA-Rap proteins determined using the pull-down assay (see "Materials and Methods"), the middle panel shows the level of HA-Rap1 in the total lysates, and the lower panel shows the expression of HA-AF6 and HA-AF6-{Delta}RA in the total lysates. The experiment was repeated three times with reproducible results. E, AF6 increases GTP level of endogenous Rap1. HB6 cells were cotransfected with empty vector or HA-AF6. The left panel shows the GTP levels of the Rap proteins determined using the pull-down assay (see "Materials and Methods"), the right panel shows the level of endogenous Rap1 in the total lysates and the overexpression of AF6 in the total lysates. The experiment was repeated three times with reproducible results. WB, Western blot.

 
GDI Measurements—Experiments were carried out as described previously (13). In brief, 100 nM Rap loaded with the non-hydrolyzable, fluorescent GTP analog mGppNHp were incubated in the presence of 10 µM non-labeled nucleotide and various concentrations of the RA-domain AF6L. Fluorescence was measured with a Cary Eclipse (Varian, Australia) equipped with a temperature controlled multicuvette holder.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To determine whether AF6 is involved in Rap1-induced cell adhesion, a Jurkat cell line stably expressing Epac (HB6 cells) was used. Cells were transfected with AF6 together with or without constitutive active Rap1V12 and analyzed for their ability to adhere to fibronectin. As shown in Fig. 1A, AF6 completely inhibited Rap1V12-induced cell adhesion. However, AF6 did not inhibit Mn2+-induced cell adhesion, where integrins are activated directly by divalent cations (26). Also, the stimulating effect of Rap1V12 on Mn2+-induced cell adhesion was completely inhibited by AF6. To investigate whether cell adhesion mediated by endogenous Rap1 is also inhibited by AF6, we treated HB6 cells with the Epac-specific analogue 007 to activate Epac and consequently Rap (20). Like Rap1V12-induced adhesion, 007-induced adhesion was inhibited by the overexpression of AF6 (Fig. 1B). AF6 interacts with Rap via RA domains in its N-terminal region (11, 13). To investigate whether the interaction of AF6 with Rap is required for its ability to inhibit adhesion, an N-terminal deletion mutant lacking the RA domains was made, namely AF6-{Delta}RA (deletion of residues 1–346). Pull-down experiments with immobilized Rap1 demonstrated the inability of AF6-{Delta}RA to interact with Rap1 (Fig. 2A). In contrast to full-length AF6, AF6-{Delta}RA was not able to inhibit 007-induced adhesion in HB6 cells (Fig. 2B). Conversely, the isolated RA domain of AF6 (residue 1–420) was able to inhibit 007-induced adhesion (Fig. 2C). Surprisingly, full-length AF6 was much more efficient in inhibiting 007-induced cell adhesion than the isolated RA domain. From these results we concluded that in HB6 cells, expression of AF6 inhibits Rap-induced cell adhesion. Furthermore, we concluded that although the RA domains are required for this inhibition, additional domains are required for efficient inhibition.

Previously, it was shown that AF6 binds to the RapGAP SpaI and that AF6 enhanced the SpaI-induced decrease in Rap1GTP levels (19). To investigate the effect of AF6 expression on Rap1GTP levels, HA-Rap1 and HA-AF6 were cotransfected in HB6 cells, and the levels of Rap1GTP were determined by a pull-down assay. Surprisingly, AF6 strongly increased the level of HA-RapGTP (Fig. 2D). In contrast, AF6-{Delta}RA did not influence Rap1GTP levels. We further investigated the effect of AF6 expression on the change of endogenous Rap1GTP level in cells. Expression of AF6 resulted in an increase in endogenous Rap1GTP level (Fig. 2E). From these results we concluded that AF6 binds to Rap1 through its RA domains and stabilizes Rap1 in the GTP-bound state. This result further suggests that the observed inhibition of cell adhesion by AF6 may be because of sequestration of Rap1 in an unproductive complex.

To investigate whether the negative effect of AF6 on cell adhesion is specific, we used shRNA to knock-down AF6. For that purpose HB6 cells were transiently transfected with two different shRNA constructs. Both resulted in a significant decrease of the endogenous AF6 levels (Fig. 3A). In these experiments the transfection efficiency was between 50 and 70% as measured by green fluorescent protein cotransfection, indicating that the two shRNA constructs function rather efficiently in knocking down the endogenous AF6. Importantly, both AF6 shRNAs resulted in an increased integrin-mediated adhesion (Fig. 3B). To investigate whether AF6 shRNA-induced cell adhesion is Rap-dependent, we introduced Rap1GAP to inhibit endogenous Rap (27). Indeed, Rap1GAP completely inhibited AF6 shRNA-induced cell adhesion (Fig. 3C). Moreover, knockdown of AF6 further enhanced 007-induced adhesion (Fig. 3D). Although overexpression of AF6 resulted in an increased level of Rap1GTP, it could well be that the increased adhesion by AF6 knockdown is caused by increased levels of Rap1GTP. We therefore tested the level of Rap1GTP in the presence of AF6 shRNA. HB6 cells were transiently transfected with AF6 shRNA and Rap1, stimulated with or without 007 and the level of RapGTP was determined by the pull-down assay. Knockdown of AF6 did not increase the level of Rap1GTP but slightly decreased particularly the basal level of Rap1GTP (Fig. 3E). From these results we concluded that endogenous AF6 is a negative regulator of Rap-induced cell adhesion.



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  		FIGURE 3.
AF6 depletion increases adhesion. A, AF6 shRNA decreases protein expression. HB6 cells were transfected with 30µg of indicated shRNA constructs and grown for 72 h. In control experiments using GFP we observed more than 50% transfection efficiency using this protocol. Cell lysates were analyzed by Western blotting with anti-AF6 antibody. Anti-tubulin was used to demonstrate equal loading. WB, Western blot. B, AF6 shRNA induces cell adhesion. HB6 cells were cotransfected with TK luciferase reporter plasmid and the indicated shRNA constructs. After 72 h cells were replated and seeded in triplicate wells coated with fibronectin (FN), and after 1 h the percentage of adherent cells were measured. Results are representative of three independent experiments. C, AF6 shRNA-induced cell adhesion is Rap-dependent. HB6 cells were transfected with TK luciferase reporter plasmid, together with empty vector, Rap1GAP, AF6 shRNA2, or Rap1GAP and AF6 shRNA2. Adhesion was measured as indicated above. Results are representative of three independent experiments. In the bottom panel expression of transfected Rap1GAP is shown. D, AF6 shRNA enhances 007-induced cell adhesion. HB6 cells were transfected with TK luciferase reporter plasmid, together with either empty vector or AF6 shRNA2, and adhesion was measured in the presence of 007 (concentration of 007 as indicated in the figure) as indicated above. Results are representative of three independent experiments. E, AF6 depletion slightly decreases the Rap1GTP levels. HB6 cells were transfected with HA-Rap1 and either empty vector or AF6 shRNA2. After 72 h the level of Rap1GTP was measured by the pull-down assays. The experiment was repeated three times with reproducible results.

 
In search for effectors of the Rap family we identified a protein (KIAA1849) in the Kazusa DNA data base with similarities to AF6 (Fig. 4A). KIAA1849 is characterized by an N-terminal RA domain and a C-terminal PDZ domain. Because of its similarity to AF6 on domain structure, we called this protein AF6-like (AF6L) (Fig. 4A). We then investigated whether the AF6L can interact with Rap1. Indeed, like AF6, AF6L can be recovered from cell lysate using GST-Rap1 (Fig. 4B). This interaction was further characterized with recombinant AF6L-RA (residues 25–213). The binding of effector proteins to small G-protein often results in a reduced exchange of nucleotide bound to the G-protein (GDI-effect) (28). The dissociation of a fluorescently labeled nucleotide bound to Rap can be detected as a decrease of fluorescence intensity in the presence of an excess unlabeled nucleotide. Indeed, increasing concentration of AF6L-RA reduced the exchange rate of nucleotide bound to Rap1 (Fig. 4C). From this measurement an affinity for the interaction of AF6L-RA and Rap1 of 0.9 µM was determined. This is similar to the affinity of AF6-RA for Rap1 (0.25 µM) (13). From these results we conclude that AF6L can interact with Rap1.

We next investigated whether AF6L inhibits cell adhesion. AF6L did not inhibit 007-induced adhesion to fibronectin, although it was expressed to a much higher level than AF6 (Fig. 4D). Also the level of Rap1GTP was not affected by AF6L (data not shown). Furthermore, AF6L did not rescue the inhibitory effect of AF6 (data not shown). This failure of AF6L to inhibit Rap-induced cell adhesion was not due to a failure of AF6L-RA to interact with Rap, because this domain, like the AF6-RA domain, inhibited 007-induced cell adhesion (Fig. 4E). Although the domain structures of AF6 and AF6L are quite similar, there are differences between the proteins in amino acid composition. From these results we concluded that the difference between AF6 and AF6L in the inhibition of Rap1-induced cell adhesion is an intrinsic property of the proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this paper we show that AF6 negatively regulates Rap-dependent adhesion in T cells. Expression of AF6 results in the inhibition of Rap-induced cell adhesion to fibronectin. This inhibition is observed both when cell adhesion is induced by expression of constitutively active Rap1V12 and when endogenous Rap is activated. This inhibition requires the RA domain-containing region of AF6 that interacts with the GTP-bound form of Rap1 (13). AF6-induced inhibition correlates with an increase in the levels of the GTP-bound form of Rap suggesting that AF6 binds to Rap and protects it from hydrolysis by Rap GAPs. One simple explanation for this result would be that AF6 inhibits Rap artificially due to overexpression as was previously shown for the RA-domain of Ral guanine nucleotide dissociation stimulator (29). However, several observations indicate that the inhibitory effect of AF6 on cell adhesion is specific. First, the isolated region of AF6 that efficiently interacts with Rap (13) was much less efficient in inhibiting Rap-induced cell adhesion than full-length AF6. Secondly, knockdown of AF6 by using two independent shRNA constructs resulted in enhanced adhesion. Furthermore, the enhancing effect of AF6 shRNA on cell adhesion was still Rap-dependent, which is compatible with the notion that endogenous AF6 inhibits endogenous Rap function. Thus, cell adhesion induced by activation of endogenous Rap1 is enhanced by knockdown of endogenous AF6. Thirdly, although the RA domain of the related AF6L protein can inhibit Rap-induced cell adhesion, the full-length AF6L protein fails to do so. These combined observations suggested to us that the inhibitory effect of AF6 on Rap-induced cell adhesion is specific. Apparently, AF6 forms a complex with RapGTP and thereby competes for effectors of Rap that regulate Rap-induced cell adhesion. A number of these effectors have been identified, most notably RapL and Riam. Both proteins contain RA domains involved in the binding to Rap, and both proteins are directly implicated in Rap1-mediated cell adhesion (6, 8). Previously, it was shown that AF6 through distinct domains binds to both active Rap1 and Rap GAPs (19). These authors did not observe a significant effect of AF6 alone on the levels of Rap1GTP and Rap-induced cell adhesion. However, both SpaI-induced inhibition of Rap1GTP and inhibition of Rap1-induced adhesion were augmented by AF6. This led to the proposal that AF6 is a negative regulator of Rap-induced cell adhesion by recruiting Rap in a complex with RapGAP. As a consequence RapGTP levels are decreased resulting in the inhibition of Rap effects. These results are at variance with our observations in that AF6 by itself inhibits Rap1-induced cell adhesion and in that AF6 increases the levels of Rap1GTP. Some of the differences may be because of difference in experimental set up, i.e. the level of expression of the various proteins, and the cell lines used. However, it may also point to the intriguing possibility of a dual negative control of Rap1-induced cell adhesion by AF6. First, as shown by our results, AF6 is recruited by RapGTP and prevents RapGTP from RapGAP-induced hydrolysis as well as interaction with effectors. Secondly, Rap GAPs are recruited in the complex and inhibit free Rap1. In this way Rap is efficiently inhibited. Indeed, full-length AF6 is much more efficient in inhibiting Rap1-induced cell adhesion than the isolated AF6 RA domain, supporting the notion that additional domains of AF6 are required for the efficient inhibition of Rap1. Effectors may compete with AF6 for binding to Rap1. Interestingly, when we knocked down AF6 we observed a slight decrease in Rap1GTP levels but an increase in Rap1 function. We interpreted this result that in the absence of AF6, RapGTP is released and therefore exposed to GAP activity but also free to interact with effectors.

In the absence of an immune challenge, T cells circulate the body in a non-adhesive state. The maintenance of this non-adhesive condition prevents inappropriate immune responses from occurring. An increase in Rap activation is sufficient to rapidly up-regulate T cell adhesiveness, demonstrating that Rap signaling must be tightly controlled in unstimulated T cells (30, 31). We observed that a reduction of endogenous AF6 expression by RNA interference was sufficient to induce T cell adhesion in the absence of stimulation. This suggests that endogenous AF6 may function to buffer GTP-Rap in resting cells, maintaining it in a nonproductive complex. Loss of this function of AF6 may therefore result in immunological disorders.



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  		FIGURE 4.
AF6L is not a negative regulator of Rap1 in cell adhesion. A, schematic comparison of AF6 and AF6L. AF6L consists of a RA domain in the N terminus and a PDZ domain in the C terminus. B, AF6L binds to Rap1 in a pull-down assay. HA-AF6 and HA-AF6L were expressed in HB6 cells and recovered by GST-Rap1. The blot was probed with anti-HA (12CA5) antibody. C, dissociation rate constant of mGppNHp from Rap1A in dependence of AF6L-RA concentration. The rate constant were measured at 37 °C and plotted against the concentration of the RA domain. From this data an affinity of 0.9 µM was calculated. D, AF6L does not block Rap1-induced adhesion. HB6 cells were cotransfected with TK luciferase reporter plasmid and either AF6 or AF6L, and adhesion was measured as indicated in legend Fig. 1. Results are representative of three independent experiments. Bottom panel, expression of AF6 and AF6L proteins. E, AF6L-RA domain inhibits Rap-induced adhesion. HB6 cells were cotransfected with TK luciferase reporter plasmid and either AF6L or indicated amounts of AF6L-RA domain, and adhesion was measured as indicated in legend to Fig. 1. Results are representative of three independent experiments. Bottom panel, expression levels of transfected proteins. FN, fibronectin; WB, Western blot.

 
In Drosophila the AF6 homolog Canoe is an effector of Rap1 in the regulation of dorsal closure, presumably by regulating adherens junction formation (18). This implies that AF6 may be a mediator of Rap1 functions other than integrin-mediated cell adhesion. Moreover, in the regulation of cell junction formation AF6 was shown to be an effector of Ras, rather than Rap (15). This suggests a possible model in which AF6 is an effector of Ras but a negative regulator of Rap1, which is consistent with the opposing functions of these proteins in cell adhesion.

Finally our results show that a protein closely related to AF6, AF6L, is able to interact with Rap1. However, this protein may have a different function than AF6 in that it does not function as a negative regulator of Rap1 in integrin-mediated cell adhesion. Further research is required to establish the real function of this protein and the possible connection with small GTPases.


   FOOTNOTES
 
* This work is supported by the Netherlands Genomics Initiative through the Cancer Genomics Center (to Z. Z.), the Dutch Cancer Society (KWF) (to L. S. P.), and Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW) (to H. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Physiological Chemistry and Centre of Biomedical Genetics, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Tel.: 31-30-2538988; Fax: 31-30-2539035; E-mail: J.L.Bos{at}med.uu.nl.

2 The abbreviations used are: GAP, GTPase-activating protein; RA, Ras association; RapL, regulator of adhesion and polarization enriched in lymphoid tissues; Riam, Rap1-GTP-interacting adaptor molecule; 007, 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate; HA, hemagglutinin; GST, glutathione S-transferase; mGppNHp, 2'-/3'-O-(N'-methylanthraniloyl)-guanyl-5'-yl-imidodiphosphate; shRNA, short hairpin RNA; AF6L, AF6-like; TK, thymidine kinase. Back

3 L. S. Price and J. L. Bos, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Eli Canaani for providing AF6 cDNA. We thank Dr. Kaibuchi Kozo for providing Myc-AF6 construct. We are grateful to Dr. Fried J. T. Zwartkruis and Dr. Karen S. Lyle for helpful discussion. Marc van de Wetering is acknowledged for providing pTER vector.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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