Article |
Address correspondence to Christopher E. Turner, Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Tel.: (315) 464-8598. Fax: (315) 464-8535. E-mail: turnerce{at}upstate.edu
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Abstract |
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Key Words: paxillin; PKL; LD motif; Rho family GTPases; cell spreading and motility
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Introduction |
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Regulation of the actin cytoskeletal dynamics that occur after the activation of integrins via engagement with the ECM is effected primarily by the Rho family of small GTPases, i.e., Cdc42, Rac, and Rho (Hall, 1994, 1998; Hotchin and Hall, 1995; Clark et al., 1998; Price et al., 1998). Activation of Cdc42 and Rac elicit the formation of filopodia, and lamellipodia and peripheral membrane ruffles respectively, as well as focal contacts (Ridley et al., 1992; Nobes and Hall, 1995). Rho activation propagates the formation of actin stress fibers and focal contacts (Ridley and Hall, 1992). Multiple effector molecules have been implicated in Rho family signaling including the p21-activated kinase (PAK), the partner of Rac 1 (POR1), the Wiskott-Aldrich syndrome protein, the Rho kinase family, and PI-4-P5 kinase (Bishop and Hall, 2000).
PAK binds to and is activated by both Cdc42 and Rac (Manser et al., 1994; Martin et al., 1995; Knaus and Bokoch, 1998) and has been implicated in both Cdc42 and Rac-dependent changes in the actin cytoskeleton (Sells et al., 1997, 1999; Obermeier et al., 1998; Zhao et al., 1998; Kiosses et al., 1999). Interestingly, POR1, a scaffolding protein, has been shown to be involved in both Rac-mediated membrane ruffling, as well as ARF6-dependent cytoskeletal rearrangements (Van Aelst et al., 1996; D'Souza-Schorey et al., 1997). Furthermore, it is believed that the small GTPase ARF1 plays a role in regulating recruitment of paxillin to focal adhesions and in stimulating Rho activation leading to stress fiber formation (Norman et al., 1998). Thus, it appears that in certain cases the outcomes of Rho and ARF family activation are linked via the use of common downstream effectors.
Paxillin is a multidomain focal contact adapter protein involved in integrin and growth factor signaling (Turner, 2000). The NH2 terminus of paxillin contains five leucine-rich domains, termed LD motifs, which mediate proteinprotein interactions (Brown et al., 1996, 1998). The LD4 motif of paxillin binds a complex of proteins previously implicated in actin cytoskeletal regulation. These include the Cdc42/Rac guanine nucleotide exchange factor PIX, PAK, and the SH2-SH3 adapter protein Nck. This complex is linked to paxillin through the putative ARF-GTPaseactivating protein (ARF-GAP) paxillin kinase linker (PKL) (Turner et al., 1999), although PAK has been suggested to also bind directly to paxillin via the LD4 motif (Hashimoto et al., 2001). Consequently, paxillin, through these associations, may serve as a point of integration in the control of actin cytoskeleton dynamics by both the Rho and ARF family GTPases.
In this study, we have evaluated the importance of the paxillinPKL interaction in mediating cytoskeletal changes associated with cell adhesion and motility. We demonstrate that in CHO.K1 fibroblasts, perturbation of the interaction between paxillin and PKL interferes with the proper regulation of Rac activity and induces dramatic effects on cell spreading, morphology, and motility after plating on fibronectin.
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Results |
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To ensure these results were not due to a clonal phenomenon, full-length wild-type avian paxillin and wild-type avian paxillin engineered with a deletion of LD4 were transiently expressed in CHO.K1 cells. Analysis of these cells in spreading assays on fibronectin yielded results similar to those described above (data not shown). Since the paxillin WT and parental nontransfected cells behaved indistinguishably during spreading assays, paxillin WT cells were used for much of the remaining experimentation described herein.
To determine whether this morphological change was attributable solely to the absence of the paxillin LD4 motif, we introduced a full-length wild-type avian GFPpaxillin fusion (GFPpaxillin) into both paxillinLD4 and paxillin WT cells and examined the cell's phenotype during spreading on fibronectin. Although GFPpaxillin had no effect on the morphology of paxillin WT cells, its introduction into paxillin
LD4 cells reduced the generation of multiple broad lamellipodia to
20%, as compared with
60% in paxillin
LD4 cells not containing GFPpaxillin (Fig. 2, B
, a and c, and C). This effect was entirely due to the reintroduction of wild-type GFPpaxillin since expression of GFP alone did not disrupt the morphological changes observed in paxillin
LD4 cells (data not shown). The above observations suggest that the LD4 motif of paxillin is involved in the regulation of actin cytoskeletal changes that occur during cell spreading.
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The generation of broad lamellipodia-like structures in paxillinLD4 cells correlates with a prolonged elevation in Rac activity
Activation of the Rho family member Rac induces the formation of lamellipodia and membrane ruffles and has been implicated in cell spreading and motility (Ridley et al., 1992; Clark et al., 1998; Price et al., 1998; Rottner et al., 1999). In paxillinLD4 cells, a dramatic increase in broad lamellipodia-like structures was observed during cell spreading along with increases in cell protrusive activity and random motility. This suggested a potentiation of Rac activity. To determine whether or not Rac plays a role in these processes, paxillin
LD4 and paxillin WT cells were detached and allowed to spread on fibronectin. Cell lysates from these cells were incubated with GST fusion proteins containing the p21-binding domain of PAK, (GSTPBD), which binds only active or GTP-bound Rac, or Cdc42 (Sander et al., 1998). The amount of activated endogenous Rac bound was analyzed by Western blotting using anti-Rac antibodies. Paxillin WT cells spreading on fibronectin showed an increase in Rac activity at 5 min, peaking at 15 min, followed by a gradual return to baseline (Fig. 4, A and B)
. In contrast, paxillin
LD4 cells exhibited a steady and prolonged activation of Rac that was maintained out to 360 min (Fig 4, A and B). The Rac activation profile observed in paxillin WT cells is similar to what has been reported for cells stimulated with exogenous growth factors, and to the activation of PAK during cell spreading (Price et al., 1998; Sander et al., 1999). However, this is the first study in which the time course of Rac activity during cell spreading has been directly measured.
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Although the localization of PKL to focal contacts was disrupted by the deletion of paxillin LD4, expression of this mutant had no affect on the distribution of either FAK or vinculin, two other focal contact proteins shown to associate with paxillin via multiple LD motif interactions (Fig. 6 C) (Turner and Miller, 1994; Brown et al., 1996; Turner et al., 1999).
To further demonstrate that the phenotypic changes observed in paxillinLD4 cells were not due to perturbation of normal FAK function through the disruption of a paxillinLD4-FAK interaction, the change in FAK activity in these cells during spreading was assessed by measuring phosphorylation of the FAK autophosphorylation site Y397 (Schaller et al., 1994; Ruest et al., 2000). Parental CHO.K1, paxillin WT, or paxillin
LD4 cells respread on fibronectin for the times indicated demonstrated similar general changes in phosphotyrosine phosphorylation levels (Fig. 6 D, top). Moreover, immunoblotting of FAK immunoprecipitates from these same cell lysates with FAK Y397 phosphospecific antisera indicated equivalent FAK activation in all three cell lines (Fig. 6 D, middle).
Introduction of PKL mutants defective in paxillin-binding, but not wild-type PKL, induce paxillin WT cells to exhibit a morphology characteristic of paxillinLD4 cells
We have previously localized the binding site of PIX to the NH2 terminus of PKL, and that of paxillin to the COOH terminus (Turner et al., 1999). However, PKL contains two putative paxillin-binding subdomains (PBSs), based on their homology with the paxillin-binding sites of vinculin and FAK (Wood et al., 1994; Tachibana et al., 1995; Brown et al., 1996). To identify the PKL PBS that mediates paxillin LD4 binding, radiolabeled full-length PKL proteins, as well as PKL proteins containing either a deletion of PBS1 or PBS2 (Fig. 7
A), were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST-coupled fusion proteins, GSTPIX and GSTLD4. Although GSTPIX was found to bind the wild-type and both mutant PKL proteins, GSTLD4 bound to both full-length PKL and the PBS1 deletion mutant, but no binding was observed to the PBS2 deletion mutant (Fig. 7 B). These results identify the PBS2 region of PKL as the paxillin-binding site.
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To test whether PKL binding to the paxillin LD4 motif via the PBS2 domain was necessary for normal cell spreading both paxillinLD4 and paxillin WT cells were transiently transfected with GFPPKL or GFPPKL
PBS2 and evaluated for spreading on fibronectin. Although the introduction of GFPPKL
PBS2 into paxillin
LD4 cells had no effect on cell morphology, expression of this construct in paxillin WT cells caused the generation of a paxillin
LD4-like phenotype in
50% of cells (Fig. 8, B and C)
. Expression of GFPPKL had no effect on the morphology of either paxillin
LD4 or paxillin WT cells (Fig. 8, B and C). However, although it can be seen that paxillin and PKL colocalize in paxillin WT cells expressing GFPPKL (Fig. 9
A, b and f), in paxillin
LD4 cells expressing either GFPPKL or GFPPKL
PBS2, PKL is cytoplasmic (Fig. 9 A, a and c), whereas paxillin is found in focal contacts (Fig. 9 A, e and g). In paxillin WT cells, GFPPKL
PBS2 remained cytoplasmic (Fig. 9 A, d), whereas paxillin was found in focal contacts (Fig. 9 A, h). Thus, the introduction of wild-type PKL or PKL lacking the paxillin-binding site does not affect paxillin localization. Similar effects on morphology and paxillin and PKL subcellular localization were also obtained by the introduction of either GFPPKL or GFPPKL
PBS2 into parental CHO.K1 cells (data not shown).
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Discussion |
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Cell spreading on ECM involves coordinated regulation of the activity of various Rho family members. In particular, previous studies using NIH3T3 cells have shown that early spreading events are characterized by the extension of lamellipodia, which correlates with a transient increase in Cdc42/Rac-dependent PAK activity. Maximum activity is achieved between 10 and 20 min after plating, followed by a steady decline in kinase activity to baseline levels (Price et al., 1998). A transient reduction in Rho activity during the initial phase of cell spreading has also been reported (Ren et al., 1999). The decline in PAK, and presumably Cdc42/Rac activity, is generally accompanied by the development of a more elongated angular phenotype associated with the activation of Rho (Rottner et al., 1999; Sander et al., 1999; Arthur et al., 2000). Our results indicate that in parental CHO.K1 cells, or CHO.K1 cells overexpressing wild-type paxillin (paxillin WT), there is a similar sequence of morphologic changes and time course of Rac activation/inactivation, reaching a maximum at 15 min after plating. In striking contrast, overexpression in CHO.K1 cells of a paxillin mutant lacking the LD4 motif (paxillinLD4) resulted in a dramatic increase in membrane activity during cell spreading. This included the generation of multiple broad lamellipodia-like structures and enhanced membrane protrusive activity that persisted for several hours; i.e., the cells failed to transition to the more typical angular phenotype. Interestingly, the phenotypic changes induced by the paxillin
LD4 were accompanied by a prolonged elevation in Rac activity, suggesting that paxillin, through its LD4 adapter function, is critical for the tight regulation of Rac activity during the spreading process.
We have previously demonstrated that the paxillin LD4 motif is capable of binding directly to several proteins including the focal adhesion tyrosine kinase FAK, the actin-binding protein vinculin, and more recently PKL, a putative ARF-GAP (Turner and Miller, 1994; Brown et al., 1996; Turner et al., 1999). The paxillinLD4 mutant protein localized as efficiently as wild-type paxillin to focal complexes and focal contacts (Fig. 1 B), consistent with the use of the focal contact targeting sequence within the paxillin LIM domains (Brown et al., 1996). Importantly, overexpression of the paxillin
LD4 mutant failed to affect the localization to focal contacts of either FAK or vinculin (Fig. 6 C). In contrast, PKL localization to these structures was lost in the paxillin
LD4-expressing cells (Fig. 6 A). This difference is likely due to the fact that, although FAK and vinculin can bind to additional LD motifs within the NH2 terminus of paxillin, PKL binds exclusively to the LD4 motif (Turner and Miller, 1994; Brown et al., 1996; Turner et al., 1999).
In addition to increased cell spreading, enhanced protrusiveness, and multiple lamellipodia, paxillinLD4 mutant cells exhibit long tail-like retraction fibers suggestive of highly motile cells and/or a defect in rear release (Figs. 1 B and 3 A). We found that these cells display an increased random motile capacity (Fig. 3, AD). This stimulation of motility is consistent with data demonstrating that perturbation of the normal pathway of PAK function by expression of either kinase-dead or constitutively active PAK mutants increases basal cell motility (Sells et al., 1999). However, PAK kinase activity primarily functions to effect directional motility (Sells et al., 1999). Previously we demonstrated that microinjection of GSTLD4 into NIH 3T3 cells inhibited directed migration in a scrape wound assay (Turner et al., 1999). We confirm in this report that although deletion of paxillin LD4 increases random motility, directional motility as measured by scrape wound assay is profoundly attenuated (Fig. 3 E). The existence of long retraction fibers in paxillin
LD4 mutant cells is consistent with impaired tail release. Efficient release is necessary for cell migration (Small et al., 1996; Horwitz and Parsons, 1999) and has been proposed to require proper PAK activity (Kiosses et al., 1999).
However, in addition to mediating PKL binding, paxillin LD4, as well as LD2, supports FAK association (Turner and Miller, 1994; Brown et al., 1996; Turner et al., 1999). A role for FAK in cell motility is well characterized (Ilic et al., 1995; Cary et al., 1996; Hauck et al., 2000; Sieg et al., 2000, 1999; Klingbeil et al., 2001). Further, a FAKpaxillin interaction has been implicated directly and indirectly (as measured by paxillin tyrosine phosphorylation) in cell spreading, neurite outgrowth, and cell migration (Ivankovic-Dikic et al., 2000). In addition, expression of the PKL-related protein GIT1 promotes cell motility in a FAK- and paxillin-dependent manner (Zhao et al., 2000). It has been proposed that GIT1 increases cell motility by paxillin-dependent recruitment to focal contacts where it causes loss of paxillin to increase focal complex dynamics, and through PIX and FAK binding, activates Rac and antagonizes Rho, respectively (Zhao et al., 2000). Importantly, PKL, unlike GIT1, does not contain the Spa2 homology domain that mediates FAK binding (Turner et al., 1999; Zhao et al., 2000). In addition, neither a change in FAK localization or activity was apparent in paxillinLD4 cells (Fig. 6, C and D, respectively), nor was any defect in motility observed with paxillin
LD2 mutant cells (Fig. 3 D). These data combined with the demonstration that PKL
PBS2 recapitulates the paxillin
LD4 phenotype (Figs. 8 and 9) are consistent with a role for paxillinPKL in regulating PAK function. Moreover, these data indicate a direct PAKpaxillin interaction (Hashimoto et al., 2001) cannot compensate for the perturbation of the PKLpaxillin connection. Thus, interference with the normal physiologic paxillinPKLPIXPAK cascade causes the profound phenotypic alterations we report in this study.
Although the role for FAK in cell motility is well characterized, the precise role for paxillin is less clear. Paxillin, and its tyrosine phosphorylation, has been implicated in the regulation of cell migration (Liu et al., 1999; Riedy et al., 1999; Salgia et al., 1999; Turner et al., 1999; Ito et al., 2000; Nakamura et al., 2000; Petit et al., 2000; Yano et al., 2000). However, overexpression of wild-type paxillin has been shown to stimulate (Nakamura et al., 2000; Yano et al., 2000), inhibit (Salgia et al., 1999), and also fail to affect cell motility (Petit et al., 2000). These apparent discrepancies may be due to differences in cell systems and assays, but also likely point to the complex interactions that occur between paxillin and its binding partners that serve to regulate cell motility. That notwithstanding, this study demonstrates that the interaction between paxillin and PKL is critical in regulating Rac-dependent cell shape change and motility.
How might the interaction between paxillin and PKL regulate this cytoskeletal reorganization and Rac activity? Paxillin is linked, via PKL, to a PIXPAKNck complex (Turner et al., 1999), each member of which is critically involved in the regulation of actin cytoskeleton dynamics. The precise temporal activation and spatial distribution of these proteins has been shown to be critical for proper Rac and Cdc42 signaling to the cytoskeleton (Bokoch et al., 1996; Galisteo et al., 1996; Lu et al., 1997; Manser et al., 1997, 1998; Bagrodia et al., 1998; Frost et al., 1998; Obermeier et al., 1998; Sells et al., 1999, 1997). A major means of regulating PAK function is through its restricted capacity to localize to focal complexes. Wild-type PAK normally maintains a diffuse cytoplasmic localization and transiently translocates to focal contacts stimulated by active Cdc42 or Rac, but not RhoA (Manser et al., 1997). The primary mechanism by which PAK localizes to focal contacts is not clear. However, overexpression of either PAK mutants (G191 and A192) that cannot bind PIX or PIX SH3 mutants that can no longer bind PAK prevent the recruitment of PAK to G12VCdc42 focal contacts (Manser et al., 1998). Our previous results suggest that PIX may be targeted to these sites through an interaction with PKL (Turner et al., 1999). In fact, we have found that PAK localization to focal contacts is entirely dependent on the precise interaction between paxillin and PKL (unpublished data).
PaxillinPKL-mediated localization of PIX and PAK to focal contacts may be essential for the proper function of PAK as an upstream regulator of Rac (Obermeier et al., 1998). It had been suggested that PAK and an as yet unidentified 90-kD protein (PKL?) may regulate PIX activity (Obermeier et al., 1998). Subsequent work has confirmed that both PAK membrane targeting and PAK binding to PIX increases PIX GEF activity towards Rac (Obermeier et al., 1998). Furthermore, overexpression of ßPIX produced active Rac-like changes in cell morphology presumably through increased GEF activity (Manser et al., 1998). It is not yet known if PKL binding to PIX influences GEF activity directly, although it is plausible that improper localization and assembly of a PKLPIXPAK complex in the context of paxillinLD4 leads to aberrant Rac activity.
Finally, the ability of Rac to control actin cytoskeletal dynamics has been linked to its activation via translocation to the membrane (Bokoch and Knaus, 1994; Michaely et al., 1999; Kraynov et al., 2000). This process of Rac membrane localization is believed to be under the control of the ARF family of proteins, in particular ARF6 (Radhakrishna et al., 1999; Zhang et al., 1999; Al-Awar et al., 2000; Boshans et al., 2000). Several groups have recently demonstrated that overexpression of ARF-GAP mutants of the ASAP/PAP/PKL families resulted in marked effects on cell morphology (Di Cesare et al., 2000; Kondo et al., 2000; Randazzo et al., 2000). In light of the ability of the PKL
PBS2 mutant to alter cell morphology, it is interesting to speculate that mislocalization of a PKLPIXPAK complex may indirectly result in the maintenance of ARF6 activity at the plasma membrane and the sustained recruitment and activation of Rac, conceivably through an inability to control the nucleotide state of ARF6 via PKLARFGAP activity.
In conclusion, our observations demonstrate that proper paxillin adapter function is critical to the regulation of the actin cytoskeletal changes that accompany integrin engagement with the ECM, and subsequent cell spreading and motility. This involvement is a result of paxillin's ability to associate with the PKLARFGAP via the LD4 motif, and entails the modulation of Rac activity. Thus, paxillin's ability to influence Rac activation through PKL binding conceivably places paxillin at a junction between Rho- and ARF-familymediated changes in the actin cytoskeleton. Future investigation will be aimed at elucidating how paxillin, PKL, and their associated proteins act in concert to regulate these processes.
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Materials and methods |
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CHO.K1 cells expressing full-length wild-type avian paxillin (paxillin WT), wild-type paxillin engineered with a deletion of LD4 (amino acids 263282) (paxillinLD4), or wild-type paxillin engineered with a deletion of LD2 (amino acids 141160) (paxillin
LD2) was generated as previously described (Brown et al., 1996; Riedy et al., 1999b). Clonal paxillin
LD4, paxillin
LD2, and paxillin WT cell lines were maintained in the presence of 100 µg/ml G418. Transient transfections of paxillin
LD4 and paxillin WT clonal cell lines, as well as parental CHO.K1 cells, were performed using lipofectamine (GIBCO BRL). Plasmids used in this study include GFP fused to full-length wild-type avian paxillin
, (GFPpaxillin), wild-type paxillin engineered with a deletion of LD4 (amino acids 263282), GFPpaxillin
LD4 full-length wild-type PKL (GFPPKL), PKL containing a deletion of the second paxillin-binding subdomain (PBS2) (amino acids 643679) (GFPPKL
PBS2), Myc-tagged N17 Rac, and N17 Cdc42 (gifts from Dr. Marc Symons, Picower Institute for Molecular Research, Manhasset, NY). Ectopic expression was confirmed by indirect immunofluorescent analysis as described elsewhere (Turner and Miller, 1994).
Immunofluorescence microscopy
For immunofluorescence of endogenous PKL, cells were plated on fibronectin-coated 13-mm glass coverslips in serum-free media for the times described in the spreading assays. Cells were then fixed for 8 min in PBS containing 3.7% formaldehyde, washed in 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.01% NaN3 (TBS), permeabilized in TBS containing 0.2% Triton X-100 (Sigma-Aldrich), and processed as previously described (Nikolopoulos and Turner, 2000). Photographs were taken using a ZEISS Axiophot photomicroscope equipped with epifluorescence illumination using either T-max 400 film (Eastman Kodak Co.), or a SPOTTM RT slider camera (Diagnostic Instruments). Images were scanned using Coolscan IITM (Eastman Kodak Co.) and processed using Adobe® PhotoshopTM v3.0.5 or SPOTTM RT software v3.0 (Diagnostic Instruments).
Antibodies used in this study include avian-specific antipaxillin polyclonal antibody (Pax1), which has been described previously (Turner and Miller, 1994). Other antibodies used were antipaxillin (clone 349), anti-PKL (clone 13, generated in collaboration with Transduction Labs using a GSTPKL [amino acids 140472] fusion protein as an antigen), anti-Rac (clone 102) anti-p130cas (clone 21), and anti-FAK (clone 77) monoclonal antibodies (Transduction Labs), antivinculin (Vin 11-5) monoclonal antibody (Sigma-Aldrich), anti-phosphoY118 paxillin and anti-phosphoY397 FAK polyclonal antibodies (Biosource International), and anti-GFP polyclonal antibody (a gift from Dr. Pam Silver, Dana Farber Institute, Boston, MA). The anti-Myc monoclonal antibody (9E10) developed by Dr. J. Michael Bishop (University of California at San Francisco, San Francisco, CA) was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA.
Spreading and motility assays
Cells were removed from tissue culture dishes by washing the cells once with PBS containing 1 mM EDTA, followed by incubation for 35 min at 37°C with 0.2 ml trypsin-EDTA (Sigma-Aldrich) in 1 ml PBS (1 mM) EDTA. Cells were harvested and then washed twice with complete Ham's F-12 media (see above) containing 10 µg/ml soybean trypsin inhibitor (Sigma-Aldrich), and once with serum-free Ham's F-12 containing 10 µg/ml soybean trypsin inhibitor, and 1.1 x 105 cells were placed on a rocker in suspension for 1 h at 37°C in serum-free media containing 1% BSA. Cells were replated on fibronectin-coated (10 µg/ml; Sigma-Aldrich) coverslips in serum-free media and then processed for indirect immunofluorescence. For transiently transfected cells, assays were performed 1518 h after transfection.
Modified Boyden chamber migration assays were performed as previously described (Riedy et al., 1999a). Wound Assays were performed as follows. In brief, 2.5 x 105 cells were plated on 35-mm tissue culture dishes for 12 h at 37°C in complete media. Scrape wounds were generated in the confluent cell monolayers using a micropipet tip. Cells were then washed extensively and refed complete media. Phasecontrast images of the cells were taken at 0 h (t = 0) and 24 h (t = 24 h) after wounding using a Nikon Eclipse TE-300 microscope equipped with a 40x objective lens and a SpotTM RT Monochrome camera (Diagnostic Instruments). The images were processed using SPOTTM RT Software v3.0 (Diagnostic Instruments).
Time-lapse videomicroscopy was performed as previously described (Huttenlocher et al., 1998). In brief, cells were plated in serum-free phenol redfree CCM1 medium on fibronectin-coated plates for 1 h at 37°C and then placed on a 37°C heated stage. Phasecontrast images were acquired with a Nikon TE-200 inverted microscope fitted with a charge-coupled device camera (DAGE MT1). The images were taken using a 10x objective at 5-min intervals for 56 hours and organized into time-lapse movies using the NIH Image software. For immunofluorescence studies, a Nikon TE-300 inverted microscope with a Hamamatsu OrcaII cooled charge-coupled device camera (Hamamatsu-City, Japan) was used. The microscope was also equipped with a Ludl Electronic Products motorized XYZ stage and heating insert. Time-lapse images were acquired with 10x objective using the ISee software (Inovision). At the end of filming, fields were observed by immunofluorescence of GFP using a 40x objective to identify transfected cells whereupon images were analyzed using NIH Image software. For migration speed, the cell centroid was tracked and the average speed for the cell determined by computing the average net displacement of the cell centroid divided by the time interval at each time point. For protrusiveness analysis, cells were outlined at two time points separated by 10 min; the two images were thresholded and then subtracted to estimate the new area. The area measurements were calibrated using a micrometer scale.
Cell lysate preparation, immunoprecipitation, and Western immunoblotting
Cell lysates were prepared using assay-specific lysis buffers: (a) standard lysis buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1.0% Triton X-100, and 0.1% sodium deoxycholic acid with 1 mM PMSF and 10 µg/ml leupeptin [Sigma-Aldrich]); (b) coimmunoprecipitation lysis buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% NP-40 [Sigma-Aldrich] and 10% glycerol [Sigma-Aldrich]); and (c) denaturing lysis buffer for FAK assay (1% SDS, 1% Triton-X 100, 0.1% sodium deoxycholic acid, 20 mM Hepes, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 10% glycerol, 1 mM PMSF, 10 µg/ml leupeptin, and phosphatase inhibitors [25 mM NaF, 25 mM ß-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM p-nitrophenylphosphate; Sigma-Aldrich]). Cell lysates were cleared of insoluble material by centrifugation at 14,000 g at 4°C for 10 min. Protein concentrations were determined using the DcTM protein assay (Bio-Rad Laboratories). Immunoprecipitation was performed by incubating the appropriate primary antibody and protein A/G PLUS-agarose (Santa Cruz Biotechnology) with cell lysate (for standard or denaturing immunoprecipitation, 200300 µg of protein was used; for coimmunoprecipitation, 8001,000 µg of protein was used) for 12 h at 4°C, rotating. Protein from detergent-soluble cell lysates (for cell lysates, 2030 µg of protein was mixed 1:1 with 2x SDS-PAGE loading buffer) and immunoprecipitates were separated by SDS-PAGE using 10% polyacrylamide gels and transferred to Immobilon-NC (Millipore). Western immunoblotting analysis using the appropriate primary antibodies, followed by incubation with either antimouse or rabbit secondary antibodies conjugated to horseradish peroxidase (Bio-Rad Laboratories) was performed to detect the proteins of interest. Chemiluminescence was used to visualize the specific proteins (Amersham Pharmacia Biotech) by exposure to blue-sensitive autoradiographic film (Marsh Bio Products, Inc.).
GST fusion protein binding assays
GST fusion proteins of ßPIX (a gift from Dr. R. Cerione, Cornell University, Ithaca, NY) and paxillin LD4 motif were expressed in Escherichia coli (DH5) and purified on glutathioneagarose beads as previously described (Turner and Miller, 1994; Riedy et al., 1999b; Turner et al., 1999). Radiolabeled full-length PKL and PKL deletion mutants of either PBS1 (amino acids 119155) or PBS2 (amino acids 643679) were generated by coupled transcription/translation reaction in a cell-free reticulocyte lysate system (TNT and Promega) as previously described (Nikolopoulos and Turner, 2000; Turner et al., 1999).
Rac activation assays were a modified version of that designed by Ren et al. (1999) using a GST fusion protein containing the p21-binding domain of PAK (GSTPBD) (a gift from Dr. R. Cerione). Immunodetection of total and active Rac was performed using Rac-specific antisera (Transduction Labs). Densitometry analysis was performed on a Macintosh computer using the public domain NIH Image program (developed at the National Institutes of Health and available at http://rsb.info.nih.gov/nih-image). The amount of PBD-bound Rac was normalized to the total amount of Rac in the lysates for comparison of Rac activity in different samples. Depending on the different cell types used (either paxillinLD4 or paxillin WT cells), the PBD-bound Rac accounts for
0.43.2% of total Rac.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM 47607 (C.E. Turner) and GM23244 (A.F. Horwitz), and a Grant-in-Aid from the American Heart Association (C.E. Turner).
Submitted: 12 January 2001
Revised: 20 April 2001
Accepted: 24 May 2001
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References |
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---|
Al-Awar, O., H. Radhakrishna, N.N. Powell, and J.G. Donaldson. 2000. Separation of membrane trafficking and actin remodeling functions of ARF6 with an effector domain mutant. Mol. Cell. Biol. 20:59986007.
Arthur, W.T., L.A. Petch, and K. Burridge. 2000. Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism. Curr. Biol. 10:719722.[Medline]
Bagrodia, S., S.J. Taylor, K.A. Jordon, L. Van Aelst, and R.A. Cerione. 1998. A novel regulator of p21-activated kinases. J. Biol. Chem. 273:2363323636.
Bishop, A.L., and A. Hall. 2000. Rho GTPases and their effector proteins. Biochem. J. 348:241255.[Medline]
Bokoch, G.M., and U.G. Knaus. 1994. Ras-related GTP-binding proteins and leukocyte signal transduction. Curr. Opin. Hematol. 1:5360.[Medline]
Bokoch, G.M., Y. Wang, B.P. Bohl, M.A. Sells, L.A. Quilliam, and U.G. Knaus. 1996. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271:2574625749.
Boshans, R.L., S. Szanto, L. van Aelst, and C. D'Souza-Schorey. 2000. ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA. Mol. Cell. Biol. 20:36853694.
Brown, M.C., J.A. Perrotta, and C.E. Turner. 1996. Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J. Cell Biol. 135:11091123.[Abstract]
Brown, M.C., M.S. Curtis, and C.E. Turner. 1998. Paxillin LD motifs may define a new family of protein recognition domains. Nat. Struct. Biol. 5:677678.[Medline]
Burridge, K., and M. Chrzanowska-Wodnicka. 1996. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12:463518.[Medline]
Cary, L.A., J.F. Chang, and J.L. Guan. 1996. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109:17871794.
Clark, E.A., W.G. King, J.S. Brugge, M. Symons, and R.O. Hynes. 1998. Integrin-mediated signals regulated by members of the rho family of GTPases. J. Cell Biol. 142:573586.
D'Souza-Schorey, C., R.L. Boshans, M. McDonough, P.D. Stahl, and L. Van Aelst. 1997. A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements. EMBO J. 16:54455454.
Di Cesare, A., S. Paris, C. Albertinazzi, S. Dariozzi, J. Andersen, M. Mann, R. Longhi, and I. de Curtis. 2000. p95-APP1 links membrane transport to Rac-mediated reorganization of actin. Nat. Cell Biol. 2:521530.[Medline]
Frost, J.A., A. Khokhlatchev, S. Stippec, M.A. White, and M.H. Cobb. 1998. Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation. J. Biol. Chem. 273:2819128198.
Galisteo, M.L., J. Chernoff, Y.C. Su, E.Y. Skolnik, and J. Schlessinger. 1996. The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1. J. Biol. Chem. 271:2099721000.
Hall, A. 1994. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell Biol. 10:3154.
Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science. 279:509514.
Hashimoto, S., A. Tsubouchi, Y. Mazaki, and H. Sabe. 2001. Interaction of paxillin with p21-activated kinase (PAK). Association of paxillin with the kinase-inactive and the Cdc42-activated forms of PAK3. J. Biol. Chem. 276:60376045.
Hauck, C.R., D.A. Hsia, and D.D. Schlaepfer. 2000. Focal adhesion kinase facilitates platelet-derived growth factor-BB-stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells. J. Biol. Chem. 275:4109241099.
Horwitz, A.R., and J.T. Parsons. 1999. Cell migrationmovin' on. Science. 286:11021103.
Hotchin, N.A., and A. Hall. 1995. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J. Cell Biol. 131:18571865.[Abstract]
Huttenlocher, A., M. Lakonishok, M. Kinder, S. Wu, T. Truong, K.A. Knudsen, and A.F. Horwitz. 1998. Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J. Cell Biol. 141:515526.
Ilic, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, and T. Yamamoto. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 377:539544.[Medline]
Ito, A., T.R. Kataoka, M. Watanabe, K. Nishiyama, Y. Mazaki, H. Sabe, Y. Kitamura, and H. Nojima. 2000. A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J. 19:562571.
Ivankovic-Dikic, I., E. Gronroos, A. Blaukat, B.U. Barth, and I. Dikic. 2000. Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins. Nat. Cell Biol. 2:574581.[Medline]
Kiosses, W.B., R.H. Daniels, C. Otey, G.M. Bokoch, and M.A. Schwartz. 1999. A role for p21-activated kinase in endothelial cell migration. J. Cell Biol. 147:831844.
Klingbeil, C.K., C.R. Hauck, D.A. Hsia, K.C. Jones, S.R. Reider, and D.D. Schlaepfer. 2001. Targeting Pyk2 to ß1-integrincontaining focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells. J. Cell Biol. 152:97110.
Knaus, U.G., and G.M. Bokoch. 1998. The p21Rac/Cdc42-activated kinases (PAKs). Int. J. Biochem. Cell Biol. 30:857862.[Medline]
Kondo, A., S. Hashimoto, H. Yano, K. Nagayama, Y. Mazaki, and H. Sabe. 2000. A new paxillin-binding protein, PAG3/Pap/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration. Mol. Biol. Cell. 11:13151327.
Kraynov, V.S., C. Chamberlain, G.M. Bokoch, M.A. Schwartz, S. Slabaugh, and K.M. Hahn. 2000. Localized rac activation dynamics visualized in living cells. Science. 290:333337.
Liu, S., S.M. Thomas, D.G. Woodside, D.M. Rose, W.B. Kiosses, M. Pfaff, and M.H. Ginsberg. 1999. Binding of paxillin to 4 integrins modifies integrin-dependent biological responses. Nature. 402:676681.[Medline]
Lu, W., S. Katz, R. Gupta, and B.J. Mayer. 1997. Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck. Curr. Biol. 7:8594.[Medline]
Manser, E., T. Leung, H. Salihuddin, Z.S. Zhao, and L. Lim. 1994. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 367:4046.[Medline]
Manser, E., H.Y. Huang, T.H. Loo, X.Q. Chen, J.M. Dong, T. Leung, and L. Lim. 1997. Expression of constitutively active -PAK reveals effects of the kinase on actin and focal complexes. Mol. Cell. Biol. 17:11291143.[Abstract]
Manser, E., T.H. Loo, C.G. Koh, Z.S. Zhao, X.Q. Chen, L. Tan, I. Tan, T. Leung, and L. Lim. 1998. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell. 1:183192.[Medline]
Martin, G.A., G. Bollag, F. McCormick, and A. Abo. 1995. A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20. EMBO J. 14:4385.[Medline]
Michaely, P.A., C. Mineo, Y.S. Ying, and R.G. Anderson. 1999. Polarized distribution of endogenous Rac1 and RhoA at the cell surface. J. Biol. Chem. 274:2143021436.
Mitchison, T.J., and L.P. Cramer. 1996. Actin-based cell motility and cell locomotion. Cell. 84:371379.[Medline]
Nakamura, K., H. Yano, H. Uchida, S. Hashimoto, E. Schaefer, and H. Sabe. 2000. Tyrosine phosphorylation of paxillin is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells. J. Biol. Chem. 275:2715527164.
Nikolopoulos, S.N., and C.E. Turner. 2000. Actopaxin: a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion. J. Cell Biol. 151:14351448
Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 81:5362.[Medline]
Norman, J.C., D. Jones, S.T. Barry, M.R. Holt, S. Cockcroft, and D.R. Critchley. 1998. ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts. J. Cell Biol. 143:19811995.
Obermeier, A., S. Ahmed, E. Manser, S.C. Yen, C. Hall, and L. Lim. 1998. PAK promotes morphological changes by acting upstream of Rac. EMBO J. 17:43284339.
Petit, V., B. Boyer, D. Lentz, C.E. Turner, J.P. Thiery, and A.M. Valles. 2000. Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells. J. Cell Biol. 148:957970.
Price, L.S., J. Leng, M.A. Schwartz, and G.M. Bokoch. 1998. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell. 9:18631871.
Radhakrishna, H., O. Al-Awar, Z. Khachikian, and J.G. Donaldson. 1999. ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements. J. Cell Sci. 112:855866.
Randazzo, P.A., J. Andrade, K. Miura, M.T. Brown, Y.Q. Long, S. Stauffer, P. Roller, and J.A. Cooper. 2000. The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton. Proc. Natl. Acad. Sci. USA. 97:40114016.
Ren, X.D., W.B. Kiosses, and M.A. Schwartz. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18:578585.
Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 70:389399.[Medline]
Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 70:401410.[Medline]
Riedy, M.C., M.C. Brown, C.J. Molloy, and C.E. Turner. 1999a. Activin A and TGF-ß stimulate phosphorylation of focal adhesion proteins and cytoskeletal reorganization in rat aortic smooth muscle cells. Exp. Cell Res. 251:194202.[Medline]
Riedy, M.C., M.C. Brown, and C.E. Turner. 1999b. Analysis of paxillin as a multidomain scaffolding protein. Methods in Signal Transduction-Signaling Through Cell Adhesion Molecules. Vol. 1. J.-L. Guan, editor. CRC Press LLC, Boca Raton, FL. 6380.
Rottner, K., A. Hall, and J.V. Small. 1999. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9:640648.[Medline]
Ruest, P.J., S. Roy, E. Shi, R.L. Mernaugh, and S.K. Hanks. 2000. Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site. Cell Growth Differ. 11:4148.
Salgia, R., J.L. Li, D.S. Ewaniuk, Y.B. Wang, M. Sattler, W.C. Chen, W. Richards, E. Pisick, E. Shapiro, B.J. Rollins, et al. 1999. Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility. Oncogene. 18:6777.[Medline]
Sander, E.E., S. van Delft, J.P. ten Klooster, T. Reid, R.A. van der Kammen, F. Michiels, and J.G. Collard. 1998. Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cellcell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J. Cell Biol. 143:13851398.
Sander, E.E., J.P. ten Klooster, S. van Delft, R.A. van der Kammen, and J.G. Collard. 1999. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J. Cell Biol. 147:10091022.
Schaller, M.D., J.D. Hildebrand, J.D. Shannon, J.W. Fox, R.R. Vines, and J.T. Parsons. 1994. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol. Cell. Biol. 14:16801688.[Abstract]
Schwartz, M.A., M.D. Schaller, and M.H. Ginsberg. 1995. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11:549599.[Medline]
Sells, M.A., U.G. Knaus, S. Bagrodia, D.M. Ambrose, G.M. Bokoch, and J. Chernoff. 1997. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7:202210.[Medline]
Sells, M.A., J.T. Boyd, and J. Chernoff. 1999. p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts. J. Cell Biol. 145:837849.
Sieg, D.J., C.R. Hauck, and D.D. Schlaepfer. 1999. Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J. Cell Sci. 112:26772691.
Sieg, D.J., C.R. Hauck, D. Ilic, C.K. Klingbeil, E. Schaefer, C.H. Damsky, and D.D. Schlaepfer. 2000. FAK integrates growth-factor and integrin signals to promote cell migration. Nat. Cell Biol. 2:249256.[Medline]
Small, J.V., K. Anderson, and K. Rottner. 1996. Actin and the coordination of protrusion, attachment and retraction in cell crawling. Biosci. Rep. 16:351368.[Medline]
Tachibana, K., T. Sato, N. D'Avirro, and C. Morimoto. 1995. Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK. J. Exp. Med. 182:10891099.[Abstract]
Turner, C.E. 2000. Paxillin and focal adhesion signalling. Nature Cell Biology. 2:E231E236.[Medline]
Turner, C.E., and J.T. Miller. 1994. Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region. J. Cell Sci. 107:15831591.
Turner, C.E., M.C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: a role in cytoskeletal remodeling. J. Cell Biol. 145:851863.
Van Aelst, L., T. Joneson, and D. Bar-Sagi. 1996. Identification of a novel Rac1-interacting protein involved in membrane ruffling. EMBO J. 15:37783786.[Abstract]
Wood, C.K., C.E. Turner, P. Jackson, and D.R. Critchley. 1994. Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin. J. Cell Sci. 107:709717.
Yano, H., H. Uchida, T. Iwasaki, M. Mukai, H. Akedo, K. Nakamura, S. Hashimoto, and H. Sabe. 2000. Paxillin and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation. Proc. Natl. Acad. Sci. USA. 97:90769081.
Zhang, Q., J. Calafat, H. Janssen, and S. Greenberg. 1999. ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting. Mol. Cell. Biol. 19:81588168.
Zhao, Z.S., E. Manser, X.Q. Chen, C. Chong, T. Leung, and L. Lim. 1998. A conserved negative regulatory region in PAK: inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1. Mol. Cell. Biol. 18:21532163.
Zhao, Z.S., E. Manser, T.H. Loo, and L. Lim. 2000. Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly. Mol. Cell. Biol. 20:63546363.