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Previous Article | Next Article 
Molecular and Cellular Biology, May 2005, p. 3763-3773, Vol. 25, No. 9
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.9.3763-3773.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Hisataka Sabe,2
David R. Critchley,1 and
Jim C. Norman1*
Department of Biochemistry, University of Leicester, Leicester, United Kingdom,1 Department of Molecular Biology, Osaka Bioscience Institute, Suita, Osaka, Japan2
Received 24 May 2004/ Returned for modification 29 July 2004/ Accepted 10 January 2005
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ABSTRACT |
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INTRODUCTION |
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Using a proteomic approach, we have recently identified an association between paxillin and the mRNA-binding protein, PABP1. Moreover, the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling and is localized to sites of translation in the perinuclear endoplasmic reticulum and at the leading edge of migrating cells (29). PABP1 consists of an N-terminal portion that contains four tandem RNA-binding motifs (RRM) and a C-terminal region with homology to an ubiquitin E3 ligase, called HYD (Fig. 1A) (12). The RRM domains bind the mRNA poly(A) tail and also are known to interact with the eIF4G complex at the 5' mRNA cap. This PABP1-eIF4G interaction is important for the circularization of mRNA during translation, and PABP1 is also proposed to participate in mRNA polyadenylation and nuclear export (12). Interestingly, PABP1 contains two regions of sequence with similarity to proposed PBSs. One of these is in RRM1 (PABP1-PBS1; residues 17 to 30) and has some similarity to the PBS in actopaxin, and the other is in RRM4 (PABP1-PBS2; residues 345 to 358) and has similarity to the C-terminal PBS of p95PKL (Fig. 1B). The three-dimensional structure of RRM1 and RRM2 of PABP1 cocrystallized with poly(A)-RNA (6) reveals that PABP1-PBS1 corresponds to a surface-exposed loop connecting the first ß-sheet to the first 
-helix of PABP1. None of the residues in this loop are directly involved with the coordination of mRNA, making it an excellent candidate for a functional paxillin-binding site.
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MATERIALS AND METHODS |
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Expression plasmids.
The human sequence (residues 1 to 636) of PABP1, the indicated PABP1 mutants, and enhanced green fluorescent protein (EGFP)-tagged PABP1 (generated by PCR) were cloned into the pcDNA3 vector. EGFP-paxillin- and -ß were in the pEGFP-C1 vector as described previously (13). The mU6pro vector was used for the expression of RNA duplexes (21). A gene-specific sequence exclusive to murine paxillin was targeted (5'-CCCTGACGAAAGAGAAGCCTA-3'). All plasmids were purified by CsCl banding prior to transfection.
Cell culture and transfection. Swiss and NIH 3T3 mouse fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum as described previously (19) and transfected with Fugene 6 as specified by the manufacturer. The ratio of Fugene 6 to DNA was maintained at 3 µl of Fugene 6 to 1 µg of DNA. Where indicated, transfections were carried out using the Amaxa Nucleofector system. Briefly, cells were grown to 80% confluence, detached by trypsinization, washed in phosphate-buffered saline, and resuspended in Amaxa solution R together with 5 µg of DNA. Following electroporation (in the Nucleofector [program T-20]), the cells were replated and all experiments were carried out 24 h posttransfection. The efficiency of transfection in the Nucleofector was determined by immunofluorescence to be >90%.
Expression and purification of paxillin and PABP1 fusion proteins. The human sequence of PABP1 (residues 1 to 636) and the indicated PABP1 mutants were cloned into BamHI and SacI sites of the His-tagged bacterial expression vector pET15 (Qiagen). PET15-PABP1 was transformed into Escherichia coli strain BL-21, grown to a density of 0.3 (optical density at 600 nm) at 37°C, and then induced with 0.5 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for a further 2 h at 22°C. E. coli was lysed in a French press in a buffer containing 20 mM morpholinepropanesulfonic acid (MOPS) buffer (pH 7.4), 0.5 M NaCl, 20 mM imidazole, 2 mM benzamidine, 30 µg of leupeptin per ml, 15 µg of aprotinin per ml, and 1 mM 4-(2-aminoethyl)benzynesulfonyl fluoride (AEBSF). Then 1.0% Igepal CA-630 was added, the lysates were clarified by centrifugation, and His-PABP1 was recovered by incubation with 50% nitrilotriacetate-agarose beads for 1 h at 4°C. His-PABP1 was eluted with a buffer containing 100 mM EDTA at 4°C and dialyzed against phosphate-buffered saline (pH 7.4). The construct encoding GST-tagged human paxillin (residues 1 to 591) was expressed and purified as described previously (29).
Immunoprecipitations and pull-down assays. Cells were grown to 60% confluence, washed twice in ice-cold phosphate-buffered saline, and lysed in a buffer containing 200 mM NaCl, 75 mM Tris-HCl (pH 7.0), 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA, 7.5 mM EGTA, 1.5% (vol/vol) Triton X-100, 0.75% (vol/vol) Igepal CA-630, 50 µg of leupeptin per ml, 50 µg of aprotinin per ml, and 1 mM AEBSF (1.14 µl/cm2 [culture area]). The cells were scraped from the dish with a rubber policeman, and lysates were passed three times through a 27-gauge needle and centrifuged at 10,000 x g for 10 min at 4°C. For immunoprecipitations, magnetic beads conjugated to sheep anti-mouse IgG were blocked in phosphate-buffered saline containing 0.1% (wt/vol) BSA and then bound to mouse anti-paxillin, anti-HA or the control IgG1 antibody, MOPC. For pull-down assays, anti-mouse-conjugated magnetic beads were bound to mouse anti-rabbit IgG followed by rabbit anti-GST and finally GST or GST-paxillin (residues 1 to 591). Magnetic beads that were coated with antibodies or GST fusion proteins were incubated with lysates for 2 h at 4°C with constant rotation. Unbound proteins were removed by extensive washing in lysis buffer, and coimmunoprecipitating material was analyzed by Western blotting as described previously (20).
Immunofluorescence. For immunofluorescence analysis, cells were fixed in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline for 20 min at room temperature and permeabilized with 0.2% (vol/vol) Triton X-100 in phosphate-buffered saline for 5 min, and nonspecific binding sites were blocked for 1 h with phosphate-buffered saline containing 1% (wt/vol) BSA. The cells were incubated with the primary antibodies at room temperature for 1 h. Detection was with FITC- or Texas red-conjugated secondary antibodies. The actin cytoskeleton was visualized by counterstaining with FITC- or Texas red-conjugated phalloidin in phosphate-buffered saline for 10 min at room temperature. Coverslips were mounted in Profade antifade mountant (Molecular Probes) and viewed on a Leica confocal laser-scanning microscope, with EGFP fluorescence being collected into the fluorescein channel.
Cell-spreading assays. Tissue culture plates (24 wells) were coated overnight at 4°C with fibronectin (20 µg/ml) and then blocked with 2% (wt/vol) BSA. Cells were transfected with PABP1 or mutant PABP1s in conjunction with a ß-galactosidase expression construct, and 24 h later the cells were harvested by trypsinization and collected by centrifugation in the presence of 20 µg of soyabean trypsin inhibitor per ml. The cell suspensions were added immediately to fibronectin-coated wells in serum-free DMEM containing 10 ng of PDGF-BB per ml. The cells were allowed to attach for 60 min, and nonadherent cells were removed by six washes with phosphate-buffered saline. Attached cells were fixed for 1 min in 0.2% glutaraldehyde containing 5 mM EGTA, and ß-galactosidase-expressing cells were visualized by incubation with 5 mM potassium ferricyanide and 1 mg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) per ml overnight at 37°C. To obtain an index of cell spreading, the area of cells expressing ß-galactosidase was determined by delineation of the cell envelope, using NIH Image software (21).
Migration assays. For wound-healing assays, NIH 3T3 fibroblasts were transfected with PABP1 or mutant PABP1s by using the Amaxa Nucleofector, plated on 3-cm tissue culture plates, and allowed to grow to confluence over 24 h. Crosses were scraped into the confluent monolayer by using a plastic pipette tip. The cells were washed with phosphate-buffered saline and allowed to migrate in DMEM supplemented with 1% (vol/vol) fetal calf serum and 10 ng of PDGF-BB per ml at 37°C. Photographs of the wounds were taken at 0, 6, 14, and 28 h postwounding. Wound closure was determined by quantification of these images using NIH image software. For transmigration assays, cells were transfected with PABP1 or mutant PABP1s using the Amaxa Nucleofector and seeded into the top well of a Boyden chamber by using Transwell filter inserts that had been coated with 10 µg of fibronectin per ml on the lower surface. Migration was allowed to proceed for 3 h at 37°C with 100 ng of LPA added to the lower chamber. The number of cells that had migrated to the lower chamber was determined by staining with 1% toludine blue and expressed as a proportion of the total quantity of cells added to the assay. For time-lapse fluorescence video microscopy, cells transfected with EGFP-tagged PABP1s were plated onto coverslips coated with 3 µg of fibronectin per ml and incubated for 1 h at 37°C. The cells were then transferred to a heated microscope stage, and their migration was observed by fluorescence time-lapse video microscopy in the presence of 10 ng of PDGF-BB per ml. Images were collected at 1-min intervals for 12 min.
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RESULTS |
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Paxillin association is required for efficient nuclear export of PABP1. We used fluorescence microscopy to study the distribution of mutant PABP1s expressed in NIH 3T3 fibroblasts. Wild-type HA-tagged PABP1 presented a predominantly cytoplasmic distribution (Fig. 4A), which was indistinguishable from that of endogenous PABP1 (not shown), indicating appropriate localization of this epitope-tagged protein. Quantification of three-dimensional reconstructions of confocal image stacks indicated that less than 5% of cellular PABP1 was present in the nucleus (Fig. 4E). PABP1-PBS1RN, a mutant that associates with cellular paxillin, had an intracellular distribution similar to that of the wild-type protein (Fig. 4B) and colocalized closely with paxillin in the perinuclear region (Fig. 5A). By contrast, mutants of PABP1 that do not associate with paxillin have an altered intracellular distribution. Approximately 40% of cellular PABP1-PBS2RN and PABP1-PBS1/2RN accumulated in the nucleus (Fig. 4C to E) and had reduced colocalization with paxillin in the cytoplasm (Fig. 5B). In addition, it is notable that the size (Fig. 5C) and paxillin content (Fig. 5D) of focal adhesions was increased by the expression of PBS2 mutants of PABP1.
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(Fig. 6E) and GFP-paxillin-ß (Fig. 6F) but not by GFP (Fig. 6G) or an empty vector control (Fig. 6D). Quantification of confocal images indicated that the degree of nuclear accumulation of PABP1 that occurred following paxillin RNAi was similar to that found for the PBS2 mutants of PABP1 (Fig. 6H). Addition of leptomycin B (which blocks nuclear export of PABP1 and paxillin [29]) caused paxillin, PABP1, and mutants of PABP1 to accumulate in the nucleus (data not shown). These data indicate that although the nuclear import of PABP1 does not require paxillin association, PABP1 must recruit paxillin in order to be efficiently exported to the cytoplasm.
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DISCUSSION |
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Nature of the paxillin-PABP1 interaction. The sequences proposed to interact with paxillin LD motifs (termed PBSs) have been identified by mutational and deletion analysis of a number of paxillin ligands, including vinculin, FAK, actopaxin, and p95PKL (23). Therefore, identification of potential paxillin-binding sites in PABP1 was initially based on sequence alignments with these proteins, and short sequences in RRM1 and RRM4 of PABP1 were found that matched the PBSs of actopaxin and p95PKL, respectively. Interestingly, these sequences are conserved in species from Xenopus to human that express paxillin or paxillin homologues but are not present in PABP1 of yeast, an organism that lacks paxillin. Subsequent mutagenesis confirmed that these PBSs are key determinants of the interaction between PABP1 and paxillin. Nevertheless, recent nuclear magnetic resonance spectroscopy (NMR) and crystallographic studies of the interaction between paxillin and FAK (1, 10) warn against a simple model for the interaction between paxillin LD motifs and their various binding partners. The FAK-paxillin interaction involves two hydrophobic patches on opposite faces of the FAK four-helix bundle, and the residues previously proposed to comprise the PBSs are not directly involved in contacting the paxillin LD domains. Similarly, our preliminary NMR studies indicate that although a single paxillin LD domain binds PABP1, a tandem array of two paxillin LD motifs binds far more tightly (data not shown). Further studies are required to provide a more detailed account of the intermolecular contact between paxillin and PABP1.
Interestingly, the nature of the paxillin-PABP1 interaction in vitro differs somewhat from that within the cell. When the interaction between purified proteins was studied, mutation of both PBS1 and PBS2 in PABP1 was required to abolish paxillin binding. However, only PBS2 was required for the interaction of PABP1 with paxillin in NIH 3T3 cells. This raises the possibility that within the cell, the PBS1 in PABP1 is occupied by an alternative LD domain-containing protein. LD domains have been identified in proteins other than those of the paxillin family, and binding of some of these to PBS sequences in target proteins is known to be mutually exclusive. For instance, binding of the LD domain protein E6-AP ubiquitin ligase to E6 protein is blocked by paxillin (25), and it will be important to establish whether the PBS1 in PABP1 recruits an LD domain-containing protein other than paxillin. Candidate proteins include the ubiquitin E3 ligase hyperplastic disk protein (HYD), which has homology to the C-terminal region of PABP1 (12). Paxillin ubiquitinylation has been proposed to displace it from focal adhesions (7), and our evidence suggests that association of paxillin with PABP1 promotes paxillin ubiquitinylation and is also required for efficient focal adhesion turnover.
Paxillin and PABP1 nucleocytoplasmic shuttling.
PABP1 mutants that do not associate with cellular paxillin accumulate in the nucleus. Since we can demonstrate no requirement for paxillin-PABP1 association in the nucleocytoplasmic shuttling of paxillin or the nuclear import of PABP1, we conclude that paxillin associates with PABP1 in the nucleus and facilitates its export to the cytoplasm. The fact that leptomycin B traps paxillin-PABP1 within the nucleus (29) indicates that it uses the CRM1-exportin pathway to exit the nucleus (28). Proteins with Rev-like leucine-rich nuclear export signal motifs generally utilize the CRM1 pathway, but no such sequence is present in PABP1, indicating that it is likely to leave the nucleus by virtue of nuclear export information contained within one of its binding partners. There are two sequences in paxillin that conform to the consensus (L***L**L**
) NES found in a number of proteins including c-Abl, PKI-, PKI-ß, Trip6, and LPP (26). One of these is located in paxillin LD2, and the other is near LD5. Our preliminary data indicate that the putative NES in LD2 is not on its own sufficient to mediate paxillin export, and further studies are needed to establish whether paxillin contains a functional NES.
PABP1 is thought to bind mRNAs in the nucleus, thereby determining the length of the poly(A) tail. It is also reported to facilitate the export of mature mRNAs and their associated proteins (mRNPs) to the cytoplasm (12). The fact that paxillin is involved in the nucleocytoplasmic shuttling of PABP1 therefore suggests that paxillin plays a role in exporting PABP1 and, by inference, mRNAs. We have also shown that polypyrimidine tract binding protein-associated splicing factor is associated with the paxillin-PABP1 complex (29), although whether such a complex plays a role in the splicing of specific transcripts awaits further investigation.
Paxillin and PABP1 localization in the cytoplasm. Once PABP1 has been exported from the nucleus, it must be localized to the appropriate compartment, and it is interesting that much of the cellular pool of paxillin colocalizes with PABP1 in the endoplasmic reticulum (29). Moreover, PABP1 (but not PABP2) colocalizes with paxillin at the leading edge of cells, and in situ hybridization studies suggest that focal adhesions contain mRNAs and also ribosomes (4). Thus, paxillin may also be involved in localizing PABP1 to sites involved in protein biosynthesis.
PABP1 mediates the circularization of mRNA during translation, and the PABP1 RRM1/2 domains associate with eIF4G of the translation complex (12). It is notable that eIF4G and eIF4E are absent from paxillin immunoprecipitates, indicating that paxillin is not part of the translation complex and suggesting that binding of paxillin and eIF4G to PABP1 is mutually exclusive. Indeed, since mRNAs are thought to be translationally repressed during transport (16), the various protein-protein interactions within mRNA transport complexes are likely to prevent association with the translational machinery, and paxillin may fulfil this role. Paxillin present in PABP1 immunoprecipitates is hypophosphorylated in comparison to total cellular paxillin (data not shown), suggesting that it is the nonphosphorylated form of paxillin that transports PABP1 out of the nucleus. Paxillin is extensively phosphorylated on both serine/threonine and tyrosine residues, and this is known to be one of the mechanisms by which the activity of the protein is regulated (24). It is therefore possible that phosphorylation of paxillin in the endoplasmic reticulum or at the lamellipodium triggers the release of PABP1, thereby activating translation.
Is paxillin involved in mRNA localization within cells? Cells expressing PABP1-PBS2 mutants have well-defined effects on cell physiology, indicating that it is unlikely that binding of paxillin to PABP1 is required for global protein synthesis. Thus, although cell migration and focal adhesion remodeling are reduced, two-dimensional gel analysis reveals that the overall pattern of cellular protein expression is unchanged following disruption of the paxillin-PABP1 interaction (data not shown), suggesting that the paxillin-PABP1 interaction must play a more specific role within the cell. In addition to binding poly(A) tracts, PABP1 can selectively bind to a subset of mRNAs. It can bind regions in the 5' UTR of its own mRNA thereby repressing its own translation (30), and it has also been shown to bind to sequences in certain 3' UTRs, e.g., the dendritic localizer sequence of the vasopressin mRNA, and to be involved in the subcellular localization of this mRNA (15). It is therefore tempting to speculate that paxillin is involved in the targeting of a subset of PABP1-mRNA complexes within the cell. Given that the PABP1 RRM4 domain has a preference for sequences other than poly(A) (3), it is perhaps significant that it is this domain that associates with cellular paxillin. It will be interesting to determine whether paxillin is able to influence the mRNA species recruited to PABP1 and whether these are enriched in transcripts for proteins that are involved in cell adhesion and migration.
The concept that certain mRNAs are selectively localized within the cell is well established and is known to be crucial to development in Drosophila and Xenopus. Moreover, cytoskeletal elements are thought to be involved in this process (2). Interestingly, proteins of the VICKZ family of RNA-binding proteins appear to coordinate the transport and localization of mRNAs with cell migration (8, 17). For example, the protein ZBP1 is thought to shuttle ß-actin mRNA to the leading edge of migrating fibroblasts (18), and IMP1, which binds the insulin-like growth factor II mRNA, is also localized to the leading edge in migrating cells (17). Delocalization of the ß-actin mRNA either by microinjection of antisense oligonucleotides corresponding to the 3' UTR of ß-actin mRNA (22) or by overexpressing the regions of ZBP (KH domains 1 to 4) that bind to this mRNA (8) affects fibroblast migration. As a consequence, directional cell movement is compromised and the migration speed is reduced somewhat.
However, ablation of the paxillin-PABP1 interaction inhibits cell migration in a way that is more reminiscent of FAK–/– mouse embryo fibroblasts, which exhibit profoundly decreased rates of spreading and migration and more abundant and larger focal adhesions (11). These migratory defects are accounted for by a 10-fold reduction in the rate of focal adhesion disassembly (27), and we show here that cells expressing PABP1-PBS2RN have larger focal adhesions and appear to be incapable of remodeling these during cell spreading. Thus, our results support a role for the paxillin-PABP1 complex in a pathway that is required for focal adhesion remodeling. One possibility is that the paxillin-PABP1 complex targets mRNAs encoding proteins that regulate focal adhesion dynamics during cell spreading and migration. Indeed, a recent proteomic study has found PABP1 to be one of a range of mRNA-binding proteins that associate with focal adhesion components during the early stages of cell spreading (5). These workers propose these interactions to occur within a novel type of adhesive structure, termed a spreading initiation center, and report that they are rich in a number of transcripts, including rRNAs, suggesting that actively translating ribosomes are recruited to the initial points of cell contact with the extracellular matrix (5). It is possible that the paxillin-PABP1 complex is found within such a structure and that it contributes to the localization of mRNA during cell spreading and migration, but we do not rule out alternative roles for the paxillin-PABP1 interaction in focal adhesion turnover. The fact that this interaction appears to be correlated with paxillin ubiquitinylation is of interest in this regard. Whether this reveals a novel function for PABP1 or whether this, too, is linked to the well-established role of PABP1 in binding mRNAs remains to be established.
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ACKNOWLEDGMENTS |
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We thank Kul Sikand and Sam Wattam their invaluable assistance with the fluorescence microscopy.
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FOOTNOTES |
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Present address: Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
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