Cell Adhesion Unit, DIBIT, S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy
*Author for correspondence (e-mail: decurtis.ivan{at}hsr.it)
Accepted September 10, 2001
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SUMMARY |
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Key words: Cell motility, Actin organization, Recycling endosomes, GTP-binding proteins
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INTRODUCTION |
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It is still unclear how ARF6-mediated vesicle recycling is incorporated into the extension process. Recent findings on proteins that share an ARF-specific GTPase activating protein (ARF-GAP) domain suggest that they are involved in the coordination between membrane trafficking and actin reorganization during cell locomotion (de Curtis, 2001). One of these proteins, p95-APP1, has been recently identified in our laboratory as part of a multi-protein complex (p95-complex) interacting with GTP-bound Rac GTPases (Di Cesare et al., 2000). P95-APP1 is a member of a recently discovered family of multi-domain proteins including GIT1 (Premont et al., 1998), p95PKL (Turner et al., 1999), and CAT2/GIT2 (Bagrodia et al., 1999). These proteins are characterized by the presence of an amino-terminal ARF-GAP domain, and by the ability to interact directly via a Spa2 homology domain (SHD) with the Rac/Cdc42 exchanging factor PIX (Zhao et al., 2000), and with the focal adhesion protein paxillin, which binds to a carboxy-terminal paxillin binding domain (Turner et al., 1999). Our previous study on p95-APP1 has shown that both wildtype and truncated p95-APP1 induce actin-rich protrusions mediated by Rac and ARF6 (Di Cesare et al., 2000). In particular, we found that p95-C, the C-terminal portion of p95-APP1 including the paxillin binding domain, localizes at sites of actin reorganization at the plasma membrane, strongly promoting the formation of actin-rich protrusions. By contrast, the N-terminal portion of p95-APP1, including the ARF-GAP domain and the three ankyrin repeats, colocalizes with N27-ARF6 in an endosomal compartment. By further dissection of this multi-domain protein, we found that the truncated p95-C2, including both PIX- and paxillin-binding domains, accumulates via PIX around large vesicles. These vesicles are distinct from the smaller endocytic structures where the N-terminal truncated polypeptides including the ARF-GAP domain and the ankyrin repeats accumulate by a PIX-independent mechanism. Together, these observations have led us to hypothesize that the p95 complex is implicated in the regulation of membrane recycling between endosomes and the plasma membrane, and it is required to organize new integrin-mediated adhesions at sites of protrusion.
In this study, we have further analyzed the requirements for the subcellular distribution of the p95-complex by using distinct p95-APP1-derived constructs, and we have characterized the endocytic compartments where distinct truncated forms of this multi-domain protein localize. Moreover, we have identified some of the mechanisms that may be involved in the regulation of the cycling of the p95 complex between the endocytic compartment and the plasma membrane. Our results show that both the ARF-GAP domain and Rac activation regulate the subcellular distribution of the p95-complex.
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MATERIALS AND METHODS |
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The pFlag-p95, pFlag-p95-C, pFlag-p95-C2, pFlag-p95-N4, pFlag-N17-Rac1B, pFlag-V12-Rac1B and pcDNA-N27-ARF6 plasmids were described elsewhere (Di Cesare et al., 2000). The cDNA fragments corresponding to p95-C3, p95-C5 and p95-N5 were cloned into the pFlag-CMV-2 vector (Kodak), to obtain the plasmids pFlag-p95-C3, p95-C5 and pFlag-p95-N5, respectively. The pFlag-p95-K39 plasmid was obtained by site-directed mutagenesis with the QuickChangeTM site-directed mutagenesis kit (Stratagene GmbH, Heidelberg, Germany), starting from the pFlag-p95 plasmid, and using the primers 5'-AGTGCTGCAGCGTGCACAAGAGCCTGGGCCGCCACAT-3' and 5'-ATGTGGCGGCCCAGGCTCTTGTGCACGCTGCAGCACT-3'. The pXJ40-HA-ßPIX plasmid coding for the HA-tagged ß-PIX polypeptide, and the pCMV6m/Pak1 plasmid coding for Myc-tagged Pak1 have been described previously (Manser et al., 1998; Bernard et al., 1999).
The Pfu DNA polymerase was from Stratagene, Klenow fragment of DNA polymerase was from Amersham Pharmacia Biotech, and restriction enzymes were from Boehringer. [-35S]dATP, 125I-anti-mouse Ig, and 125I-protein A were from Amersham Pharmacia Biotech. Other chemicals and FITC- and TRITC-conjugated phalloidin were from Sigma-Aldrich.
Cell culture and transfections
Chicken embryo fibroblasts (CEFs) obtained from embryonic day 10 chicken embryos were prepared and cultured as described (Albertinazzi et al., 1998). For immunofluorescence, CEFs grown on coverslips were transfected either with the Ca2+ phosphate technique or with Dosper (Boehringer) as described (Albertinazzi et al., 1998). For biochemical analysis, transient expression of proteins was achieved by transfection of CEFs by the Ca2+ phosphate technique. Cells were used for biochemical or morphological analysis 24 hours after transfection. For stimulation with PDGF, COS7 cells were grown in DMEM with 10% serum, transfected with FuGENE (Roche) according to manufacturers procedures, starved for 3-9 hours in medium without serum, and incubated with 50 ng/ml PDGF. Treated cells were immediately fixed and analyzed for immunofluorescence as described below.
Immunoprecipitation and immunoblotting
Transfected and non-transfected cells were extracted with lysis buffer (0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 1 mM sodium-orthovanadate, 10 mM sodium fluoride and 10 µg ml1 each of antipain, chymostatin, leupeptin and pepstatin). Extracts were clarified by centrifugation. 200-300 µg of protein from lysates of transfected cells were pre-cleared by incubating them for 2 hours with rotation at 4°C with 25 µl of Protein-A Sepharose beads (Amersham Pharmacia Biotech). Beads were washed four times with 1 ml of lysis buffer. Unbound material was added to Protein A-Sepharose beads with pre-adsorbed antibodies, and incubated for 2 hours at 4°C with rotation. Pellets from immunoprecipitations were washed four times with 1 ml of lysis buffer, and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. Filters were then incubated with 0.2 µCi/ml of either 125I-protein A or 125I-anti-mouse Ig (Amersham Pharmacia Biotech), and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech).
Immunofluorescence
Transfected cells were fixed with 3% paraformaldehyde and processed for indirect immunofluorescence, as described (Cattelino et al., 1995). Fixed cells were incubated for 1 hour at room temperature with primary antibodies. Cells were subsequently incubated for 40 minutes with fluorescently-labelled secondary antibodies. Fluorescently-labelled phalloidin was added during the incubation with the secondary antibody. Samples were observed using a Zeiss-Axiophot or Zeiss-Axiovert microscope equipped with a 63x oil immersion objective, and a Hamamatsu C4742-98 camera (Hamamatsu Photonics K. K.). Fluorescent images were collected using the Image-Pro® Plus software package (Media Cybernetics, L. P.), and processed using Adobe PhotoShop 5.0. For the quantitation of the cytoplasmic protrusions present in transfected and non-transfected cells, only cytoplasmic protrusions longer than half the major axis of the cell body were considered. In each experiments, three sets of 100 cells were examined for each condition. Each value represents the average from counts obtained from three independent experiments. For each experimental condition, a total of about 900 cells were examined.
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RESULTS |
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Role of p95-APP1 in paxillin localization
We have previously reported that the expression of the p95-C polypeptide, including the C-terminal paxillin-binding domain, but not the SHD PIX-binding domain (Fig. 1A) induces protrusions in fibroblasts (Di Cesare et al., 2000). This effect is accompanied by the redistribution of paxillin away from focal adhesions, which are still detectable by using anti-integrin antibodies (Di Cesare et al., 2000). To test the requirement of paxillin-binding for enhanced protrusive activity, we have prepared the pFlag-p95-N5 plasmid coding for the p95-N5 polypeptide, in which the C-terminal paxillin-binding domain was deleted (Fig. 1A). In transfected cells, p95-N5 was often observed associated to cytoplasmic vesicles (Fig. 7Ca), not visible in cells transfected with p95-C (Fig. 7Cb). In cells transfected with p95-N5 the morphology of the Rab11 compartment was clearly affected (Fig.7Ch,i), although the colocalization of Rab11 with p95-N5-positive vesicles was only partial, and not as striking as in cells transfected with the ARF-GAP mutants. In several cases, rather than a complete colocalization of the two proteins around the vesicles (Fig. 7Cc-e), several Rab11-positive spots were observed around the large p95-N5 positive vesicles (Fig. 7Cj,k). However, staining with anti-EEA1 antibodies indicated lack of colocalization of p95-N5 with EEA1-positive vesicles (Fig. 7Cf,g). In general, cells expressing p95-N5 had less prominent protrusions compared with cells expressing p95-C (Fig. 7Ca,b, respectively). Quantitation of the percentage of cells with protrusions (Fig. 7A), and of the number of protrusions per cell (Fig. 7B) indicated a significant decrease in the protrusive activity of p95-N5-transfected cells, thus implicating the requirement of paxillin binding for the stimulation of the protrusive activity. Similar findings were observed by the expression of the p95-C5 mutant (Fig. 1A), derived from p95-C from which the paxillin-binding region had been deleted (Fig. 7Cl,m): also in this case no enhancement of protrusive activity could be observed in the transfected cells (Fig. 7A,B).
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Rac activation affects p95-C2 localization
The p95-APP1 protein was first identified by us as an indirect interactor of activated Rac GTPases. To test the hypothesis that activation of Rac at the cell surface may regulate the distribution of p95-APP1 within the cell, we coexpressed the truncated p95-C2 protein with constitutively activated Rac. In contrast to the strong concentration of p95-C2 at large recycling vesicles observed in cells transfected with pFlag-P95-C2 only (Fig. 9A), p95-C2 was found redistributed to the cell periphery in cotransfected cells (Fig. 9B). The cotransfected cells showed the appearance of large lamellipodia (Fig. 9B,C,E) when compared with cells expressing p95-C2 alone, or with cells coexpressing p95-C2 and an inactive form of the GTPase (Fig. 9D,F). P95-C2 was often found concentrated with V12-Rac at the edge of lamellipodia (Fig. 9C,E). Interestingly, N17Rac colocalized with p95-C2 at the large intracellular vesicles (Fig. 9D,F). Similar findings were obtained by coexpressing p95-C3 and V12Rac. In contrast to the concentration of the p95-C3 protein at large intracellular vesicles (Fig. 6A), in the cotransfected cells, which had evident, large lamellipodia, the localization of p95-C3 was often diffuse, with some concentration at peripheral areas together with V12Rac (Fig. 9G,H). Fewer large vesicles were still evident only in some cotransfected cells (Fig. 9I). In these cases, colocalization at the large vesicles of p95-C3 and V12Rac was obvious. Interestingly, paxillin also co-distributed with p95-C2 and p95-C3 at the cell periphery in cells co-expressing V12Rac (Fig. 9L-O). Similarly, both PAK (Fig. 9R,S) and PIX (Fig. 9T,U) co-distributed with p95-C3 and p95-C2 at the cell edge in cells expressing V12Rac. Under these conditions, Rab11 showed a diffuse punctuate pattern (Fig. 9P,Q).
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To test whether the activation of endogenous Rac is able to induce the recruitment of ARF-GAP-deficient p95 polypeptides at the cell periphery, we have looked at the distribution of p95-C2 in COS7 cells treated with PDGF. In transfected cells we found that p95-C2 localized at large vesicles (Fig. 10A). In cells treated with PDGF (Fig. 10B-E), as well as in a fraction of untreated cells, in which ruffles were still evident (not shown), p95-C2 co-localized with actin at ruffles and lamellipodia. This result indicates that p95-C2 may localize to sites of endogenous Rac activation.
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DISCUSSION |
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The C-terminal p95-C fragment of p95-APP1 induces the formation of protrusions, while this effect is not observed in p95-C2-expressing cells (Di Cesare et al., 2000). While p95-C is evidently localized at sites of protrusion, p95-C2 is predominantly concentrated at large intracellular vesicles. These striking differences originate from the presence (in p95-C2) or absence in (p95-C) of the SHD domain. As for GIT1 (Zhao et al., 2000), we found that the SHD domain is required for the interaction of p95-APP1 with PIX, a putative exchanging factor for Rac and Cdc42 (Oh et al., 1997; Bagrodia et al., 1998; Manser et al., 1998). Our findings support a role of PIX in the localization of the p95-complex to Rab11-positive large vesicles. Rab11 is a small GTP-binding protein required for transferrin recycling through the pericentriolar recycling endosomes (Ullrich et al., 1996; Ren et al., 1998), and represents a functional marker for this compartment. The large Rab11-positive vesicles seem to form specifically as a consequence of PIX-mediated accumulation of p95-APP1 in the endosomal compartment. The specificity is supported by the observation that only markers for the endosomal recycling compartment accumulate at these vesicles, while earlier and later endosomal compartments are not affected. The deletion (p95-C3) or mutation (p95-K39) of the ARF-GAP domain also induce accumulation to Rab11-positive structures, showing that the alteration of the ARF-GAP domain of p95-APP1 is responsible for the formation of the abnormal recycling compartment. In vitro ARF-GAP activity of the highly homologous GIT1 on ARF6 has been recently demonstrated (Vitale et al., 2000). The strong homology between avian p95-APP1 and mammalian GIT1, and the colocalization of the N-terminal portion of p95-APP1 with N27-ARF6 in the endocytic compartment suggest that this protein is a candidate ARF6 regulator in vivo. The fact that we have not been able to detect any ARF-GAP activity of p95-APP1 on ARF6 in vitro indicates that the ARF-GAP activity of p95-APP1 may be finely regulated in the cell. The conserved arginine corresponding to arginine 39 of p95-APP1 dramatically reduces the ARF-GAP activity of a number of ARF-GAPs in vitro (Mandiyan et al., 1999; Randazzo et al., 2000; Szafer et al., 2000; Jackson et al., 2000). We would like to speculate that like ARF1 in the Golgi (Roth, 1999), ARF6 regulates vesicle formation during recycling between endosomes and the plasma membrane. According to our model (Fig. 11), the effects of the ARF-GAP mutants on the Rab11-recycling compartment is due to the inability of the mutated p95-APP1 to induce GTP hydrolysis, necessary for ARF-mediated vesicle budding from recycling endosomes. The overexpressed ARF-GAP mutants, by interacting via PIX with the endosomes, would compete with the endogenous p95-APP1, and act as dominant negative forms, inhibiting p95-APP1 function in vesicle formation. The abnormal recycling compartment would be generated by the accumulation of internalized membranes, not compensated by membrane recycling in cells overexpressing the ARF-GAP mutants.
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The absence of the PIX-binding region from the N-terminal p95-N4 construct leads to accumulation of this protein in a distinct population of endocytic vesicles. In contrast to the large Rab11-positive vesicles, the small p95-N4-positive vesicles are only weakly overlapping with the population of vesicles stained with anti-Rab11 antibodies. This localization of p95-N4 is mediated by the first ankyrin repeat (Di Cesare et al., 2000), and is independent of the SHD-mediated localization of p95 to the large vesicles. We have previously shown that p95-N4 and N27-ARF6 colocalize in these vesicles. Further work will be required to understand whether the two identified structures correspond to two altered, functionally distinct endosomal subcompartments. This hypothesis is supported by the finding that PIX and the transferrin receptor are excluded from the small p95-N4-positive vesicles, while both are present into the large Rab11-positive vesicles. The transferrin receptor cycles between the plasma membrane and the recycling endosomes, and can be induced to accumulate intracellularly by ligand internalization (Marsh et al., 1995). In non-transfected CEFs, a clear surface distribution of the transferrin receptor is observed. The concentration of the receptor in the large Rab11-positive vesicles in cells transfected with p95-C2 or p95-C3 indicates that recycling to the cell surface is altered in these cells, leading to accumulation into an abnormally enlarged recycling compartment.
The accumulation of p95-N4 in the endosomal compartment may be explained by the inability of the truncated N-terminal protein to be relocated to the plasma membrane, and implicates the C-terminal portion of p95-APP1 in targeting to the plasma membrane. One possible mechanism for targeting the p95-complex to the plasma membrane is Rac activation. In fact, overexpression of a constitutively active form of Rac induces accumulation of p95-C3 and p95-C2 to the plasma membrane together with Rac, while activated Rac does not affect the distribution of p95-N4. Therefore, PIX binding is necessary for Rac-mediated localization of the p95 complex to the cell membrane. Since PIX binds to PAK1, the localization of the p95-complex at the plasma membrane may be mediated by the binding of PAK1 to Rac (Fig. 11), although we cannot exclude the direct interaction between PIX and Rac. One could envisage that the amount of the p95-complex recruited at the plasma membrane would be dependent on the amount of active Rac at the cell surface. Under steady state conditions, the amount of endogenous GTP-Rac would not be sufficient to recruit efficiently the overexpressed p95-C2-complex to the cell surface. The p95-C2-complex would then accumulate at Rab11 endosomes via PIX. However, overexpressed V12Rac would be able to recruit a significant amount of the complex to the plasma membrane by interacting with PAK, by competing with the PIX-mediated binding of the complex to Rab11 endosomes. This idea is supported by the finding of p95-C2 at sites of endogenous Rac-mediated actin organization at the cell periphery. The results of the expression of p95-N5 and p95-C5 proteins have revealed the importance of the C-terminal paxillin binding domain on protrusive activity and paxillin relocalization, and argue in favour of a contribution of paxillin to the localization of the p95 complex at the cell periphery. In fact, the lack of the paxillin binding site leaves paxillin predominantly in focal adhesions in cells transfected with p95-N5. The p95-N5 with an intact ARF-GAP is affecting the morphology of the Rab11 compartment, although the p95-N5 protein localizes to vesicles which only partially overlap with the Rab11 compartment. Our findings imply a complex regulation of the subcellular distribution and trafficking of the p95 complex between endosomes and plasma membrane, which implicate in the process not only a functional ARF-GAP domain, but also the PIX/PAK complex and the focal adhesion protein paxillin. The lack of the paxillin binding site could affect the distribution of the protein, resulting in its partial association to a vesicular compartment that may correspond to a distinct intermediate during the recycling process. Future work on the complex network of events involved will help to further elucidate the proposed model. Interestingly, a number of ARF-GAPs including GIT2, a member of the same family of p95-APP1 (Mazaki et al., 2001), and PAPa, a member of the centaurin family (Kondo et al., 2000), have also been implicated in the regulation of paxillin distribution in the cell.
Movement of vesicles to areas of the plasma membrane involved in protrusive activity may represent a mechanism to help the forward movement of the front of migrating cells (Bretscher, 1996). The data presented in this study provide further support to a role of p95-APP1 in coordinating membrane traffic from recycling endosomes to sites of Rac-mediated actin reoganization. By using its multi-domain structure, p95-APP1 may bring together functions related to the regulation of recycling vesicular traffic, with components necessary to a productive protrusive activity driven by actin polymerization.
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ACKNOWLEDGMENTS |
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