1 Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, Syracuse, NY 13210
2 McGill Cancer Centre, McGill University, 3655 Sir William-Osler, Montreal, Quebec, H3G1Y6 Canada
* Author for correspondence (e-mail: Turnerce{at}upstate.edu)
Accepted 14 September 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Rac, Cytoskeleton, Phosphatase, Focal adhesions, Tyrosine phosphorylation, Cell migration
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Paxillin is a 68 kDa multi-domain focal adhesion protein that functions as a molecular adaptor or scaffold to facilitate integrin signaling (Brown and Turner, 2004; Playford and Schaller, 2004
). It is essential for embryonic development and plays an important role in the regulation of cell attachment, spreading and motility (Brown and Turner, 2004
; Hagel et al., 2002
). Paxillin contains five leucine-rich LD motifs in its N-terminus that function as interaction sites for actin-binding proteins such as vinculin and actopaxin and for signaling proteins such as integrin-linked kinase (ILK), focal adhesion kinase (FAK), and ADP-ribosylation-factorGTPase-activating proteins (ARF-GAPs). The latter include the G protein coupled receptor kinase (GRK) interacting ARF GAP (GIT1) and the paxillin kinase linker (PKL)/GIT2 (Brown and Turner, 2004
). The N-terminus also contains several tyrosine residues that, when phosphorylated in response to adhesion or growth factor stimulation, provide docking sites for the Src homology 2 (SH2) domains of Crk and p120RasGAP (Petit et al., 2000
; Tsubouchi et al., 2002
). The C-terminus of paxillin comprises four LIM domains that are required for paxillin localization to focal adhesions and also serve as binding sites for the protein tyrosine phosphatase (PTP)-PEST and tubulin (Brown and Turner, 2002
; Brown and Turner, 2004
; Côté et al., 1999
; Shen et al., 1998
).
Tyrosine phosphatases such as PTP-PEST, along with the tyrosine kinases FAK and Src, are necessary for coordinating the dephosphorylation/phosphorylation of focal adhesion proteins to promote focal adhesion turnover and thereby stimulate cell migration (Mitra et al., 2005; von Wichert et al., 2003
; Webb et al., 2004
). Both the over-expression and the genetic ablation of PTP-PEST causes a profound inhibition of cell motility (Angers-Loustau et al., 1999
; Garton and Tonks, 1999
) consistent with a requirement for a precise balance of PTP-PEST function in regulating cell adhesion. PTP-PEST interacts with several focal adhesion proteins including p130cas, FAK/Pyk2, and paxillin (Brown and Turner, 2002
; Côté et al., 1999
; Garton et al., 1996
; Shen et al., 2000
; Shen et al., 1998
; Spencer et al., 1997
) and contributes to the dephosphorylation of these proteins, although only p130 Cas and Pyk2 appear to be direct substrates (Angers-Loustau et al., 1999
; Lyons et al., 2001
; Shen et al., 2000
).
The Rho family GTPases Cdc42, Rac and RhoA are activated by integrin engagement with the ECM, and control the formation of filopodia, lamellipodia and stress fibers, respectively (Burridge and Wennerberg, 2004). Coordination of Rho GTPase activity is required for efficient migration; regulating membrane protrusion at the leading edge, focal adhesion turnover, cytoskeletal contractility and cell retraction (Burridge and Wennerberg, 2004
; Schmitz et al., 2000
; Wittchen et al., 2005
). Both paxillin and PTP-PEST have been linked to the regulation of Rho family signaling. Paxillin contributes to the regulation of Rho GTPase function, in part via the protein networks that signal through the LD4 motif and tyrosine residues 31 and 118 (Petit et al., 2000
; West et al., 2001
). Assembly of a paxillin LD4-PKL-PIX-Pak-Nck complex at focal adhesions is crucial for normal Rac activation, cell spreading, polarization and directed migration (Brown et al., 2002
; Turner et al., 1999
; West et al., 2001
; Zhao et al., 2000
). Interestingly, PKL was shown to be tyrosine phosphorylated in response to cell adhesion (Bagrodia et al., 1999
) and recent studies in our lab have shown that PKL tyrosine phosphorylation facilitates its focal adhesion localization in response to Rac activation (Brown et al., 2005
). Tyrosine phosphorylation of paxillin residues 31 and 118 and subsequent interaction with the Crk/Cas/DOCK180 complex is also implicated in Rac activation and cell motility in certain cell types (Lamorte et al., 2003
; Petit et al., 2000
; Valles et al., 2004
). Finally, a role for PTP-PEST in the suppression of cell spreading and control of cell motility through inhibition of Rac was identified (Sastry et al., 2002
), although the pathway through which this regulation occurred has not been determined.
In view of the direct interaction between paxillin and PTP-PEST, we have examined the importance of this association in the regulation of integrin-mediated signaling events. Herein we show, through the reconstitution of paxillin/ mouse embryo fibroblast (MEF) cells and PTP-PEST/ MEF cells, that PTP-PEST-dependent regulation of cell spreading and migration depends on a direct interaction with paxillin and involves a complex relationship that requires binding of PTP-PEST to the paxillin C-terminal LIM domains and signaling through the tyrosine 31 and 118 phosphorylation sites and the LD4 motif of the paxillin N-terminus. Furthermore, we show that paxillin is necessary for PTP-PEST suppression of Rac activity during cell spreading. Finally, we identified the paxillin LD4 motif binding partner PKL as a new PTP-PEST substrate and show that a functional PKL-paxillin interaction is required for PTP-PEST to regulate cell spreading. These data provide mechanistic insight into how the paxillin-PTP-PEST interaction contributes to the regulation of cell migration.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and transfection
Normal mouse embryo fibroblasts (MEF) and paxillin/ MEF cells (Hagel et al., 2002) (a gift from Sheila Thomas, Harvard University, Boston, MA), and PTP-PEST/ cells (Angers-Loustau et al., 1999
; Côté et al., 1998
) have been previously described. MEFs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Atlas Biologicals), 50 U/ml penicillin, 50 µg/ml streptomycin and kanamycin (complete medium) at 37°C in a humidified chamber with 5% CO2. Chinese hamster ovary (CHO) K1 cells were cultured in modified Ham's F-12 medium supplemented with 10% (v/v) heat-inactivated FBS (Atlas Biologicals), 50 U/ml penicillin, 50 µg/ml streptomycin and kanamycin. Cells were transfected using Fugene6 (Roche) or Metafectene (Biontex) at a 3:1 ratio with 1 µg of total DNA according to the manufacturer's instructions. Transfection efficiencies were monitored by calculating the percentage of GFP-positive cells 24 hours post transfection and was approximately 60% for paxillin/ cells and greater than 80% for PTP-PEST/ cells. PTP-PEST re-expression was approximately fourfold endogenous levels. The expression level with the median impact on cell spreading (0.7 µg, supplementary material Fig. S1) was used for all experiments.
Cell spreading and area analysis
Cells were resuspended in 1 ml PBS-EDTA solution washed twice in serum-free DMEM containing 0.025% trypsin inhibitor and 0.1% BSA. Cells were maintained in suspension in the same media at 150,000 cells/ml for 1 hour at 37°C. Cells (approximately 75,000) were plated for the times indicated on 10 µg/ml fibronectin-coated coverslips. Serum was reintroduced for cells maintained overnight. Cells were fixed at indicated time points and processed for indirect immunofluorescence microscopy as described previously (West et al., 2001). For quantification of cell spreading, cells were scored as round (unspread) when they were attached to the substrate but had not extended stable lamellipodia. At least 100 GFP-positive cells from three independent experiments were counted for each condition. Spreading area was quantified for 50 cells in three independent experiments.
Protrusive analysis
Transfected cells were spread on fibronectin-coated 35-mm dishes as described above. 3 hours post replating, cells were washed and transferred to prewarmed serum-, sodium bicarbonate-, phenol-red-free DMEM supplemented with 25 mM Hepes pH 7.5 and 0.1% BSA. Plates, covered with mineral oil, were maintained at 37°C in a Harvard Apparatus PDMI 35-mm dish microincubation chamber mounted on a Nikon E600 microscope. GFP-expressing cells were identified and images were captured every 5 minutes for up to 2 hours using Compix Simple PCI software and a Spot RT CCD camera. Cell protrusion was quantified for 1 hour at 10-minute intervals as described previously (Kinley et al., 2003; West et al., 2001
) by comparing two images (10 minutes apart) thresholding images and subtracting overlapping regions. Protrusions were quantified as percentage of cell area and averaged over ten cells.
Modified Boyden chamber migration assays
PTP-PEST/ cells were transfected with GFP, PTP-PEST and paxillin constructs as indicated. Transfection efficiency, as measured by the percentage of GFP-positive cells 24 hours post transfection, was approximately equivalent for all cell populations and was routinely greater than 80%. Cells were harvested and modified Boyden chamber migration assays were performed as previously described (Riedy et al., 1999) using 8-µm-pore membranes pre-coated with 100 µg/ml gelatin (Sigma-Aldrich). Twenty-thousand cells were added to the top well and the chamber was incubated for 12 hours at 37°C in 5% CO2 before the filter was fixed and evaluated (Riedy et al., 1999
). Assays were performed in triplicate. The motility of PTP-PEST+/ cells was evaluated in parallel.
Rac-activity assay
Transfected cells were either held in suspension or plated onto fibronectin-coated (10 µg/ml) 100-mm plates. Cells (1x106 paxillin/ or 1.5x106 PTP-PEST/ cells) were lysed [50 mM Tris pH 7.6, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF)] and cellular debris was removed. Equivalent amounts (500 µg protein) of lysates were incubated with 10 µg of glutathione-S-transferasePAK-binding domain (GST-PBD) fusion protein (generously provided by Rick Cerione, Cornell University, Ithaca, NY) as described (West et al., 2001). Samples were separated on a 12.5% polyacrylamide gel and Myc-tagged Rac was detected with 9E10 antibody. Quantification of the relative amounts of active to total Rac was performed with NIH-image.
Immunoprecipitations
PTP-PEST/ cells were placed in suspension and replated on 10 µg/ml fibronectin-coated dishes for 60 minutes. Cells were washed in ice-cold phosphate-buffered saline followed by lysis in radioimmunoprecipitation buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA 1% TX-100, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 20 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 0.2 mM sodium vanadate). Lysates were precleared at 21,000 g for 15 minutes at 4°C and immunoprecipitated by incubating 250 µg of cell lysate with the anti-GFP (purified IgG, Molecular Probes) antibody for 3 hours at 4°C followed by protein A/G agarose beads (Santa Cruz) for 1 hour at 4°C. Immunoprecipitates were washed extensively with lysis buffer then processed for SDS-PAGE analysis and western blotting.
Substrate trapping
PTP-PEST substrate trapping experiments were performed as described previously (Blanchetot et al., 2005; Côté et al., 1998
). Briefly, PTP-PEST/ cells were lysed in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 5 mM iodoacetic acid and proteases inhibitors. Dithiothreitol (DTT, 10 mM final) was added for 15 minutes at 4°C and lysates were precleared by centrifugation at 21,000 g for 15 minutes. One mg of cell lysate was incubated with 5 µg of GST protein or GST fused to the catalytic domain of PTP-PEST (wild type or C231S mutant) for 2 hours at 4°C. Beads were washed several times in lysis buffer and processed for SDS-PAGE and western blotting.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To further examine the requirement for paxillin binding to PTP-PEST for its effects on cell spreading, these experiments were repeated in PTP-PEST/ cells. Re-expression of PTP-PEST, with or without over-expression of paxillin, inhibited spreading up to three hours (Fig. 3A,B). These results suggest that endogenous levels of paxillin are sufficient for PTP-PEST inhibition of spreading in these cells. However, to determine whether paxillin-binding to PTP-PEST was necessary for PTP-PEST to inhibit spreading in this cell type, the paxillin C523S mutant was introduced along with PTP-PEST. Cells co-expressing PTP-PEST and paxillin C523S spread at a rate similar to the GFP-transfected population (Fig. 3A,B). Western blot analysis confirmed similar expression of all constructs (Fig. 3C). The original analysis of PTP-PEST effects on cell spreading was performed in CHO.K1 cells (Sastry et al., 2002). To evaluate a requirement for paxillin, spreading experiments were repeated in this cell type. As in the paxillin/ and PTP-PEST/ cells, CHO.K1 cells expressing PTP-PEST alone or PTP-PEST together with wild-type paxillin exhibited a significant delay in spreading. However, spreading was not inhibited when PTP-PEST was co-expressed with paxillin C523S (data not shown).
|
|
To further examine the effects of the paxillin-PTP-PEST interaction on cell spreading, we calculated the spreading area of transfected populations of paxillin/ cells at 45 minutes and 120 minutes post replating on a fibronectin matrix. Spreading areas of all populations were compared with the area of GFP-transfected cells. At 45 minutes, the area of paxillin/ cells co-expressing PTP-PEST and paxillin was 43% of the GFP-expressing population, whereas cells transfected with PTP-PEST alone or with PTP-PEST and paxillin C523S spread to 92% and 93% of GFP-transfected cells, respectively (Fig. 4E). Notably, cells expressing PTP-PEST in combination with either the paxillin LD4 deletion mutant or the paxillin Y31/118F mutant spread to 68% and 69% of GFP-transfected cells, respectively (Fig. 4E). Furthermore, when PTP-PEST was expressed with a paxillin Y31/118FLD4 mutant, cells spread to 100% of GFP-transfected cells. Taken together, these results support a mechanism in which both the LD4 motif, and phosphorylation of the paxillin residues Y31 and Y118 are involved in PTP-PEST-dependent regulation of spreading.
PTP-PEST inhibits membrane protrusion through a paxillin interaction
Both paxillin and PTP-PEST are involved in the regulation of membrane dynamics. PTP-PEST/ cells spread at an increased rate (Angers-Loustau et al., 1999) and the expression of the catalytically inactive PTP-PEST mutant C231S has been shown to cause prolonged protrusions in CHO.K1 cells (Sastry et al., 2002
). Paxillin/ cells also have irregular membrane dynamics (Hagel et al., 2002
) and introduction of paxillin small interference RNA (siRNA) into HeLa cells results in abnormal membrane activity with an increase in protrusion formation (Yano et al., 2004
). To determine whether the paxillin-PTP-PEST interaction is involved in the regulation of membrane dynamics after cells have spread, we quantified protrusive activity in paxillin/ cells at 180 minutes post replating on fibronectin using time-lapse video microscopy. Cell images were taken every 10 minutes for 60 minutes, consecutive images were overlayed and protrusions were quantified by excluding overlapping regions (see Materials and Methods for details). Protrusive activity of the cell was averaged over the 60-minute period and results are displayed in a box-and-whisker plot format (Fig. 5). The expression of PTP-PEST had very little effect on protrusions, whereas the expression of paxillin alone resulted in a significant reduction in protrusions. Interestingly, co-expression of paxillin and PTP-PEST caused a significant additional reduction in protrusive activity when compared to paxillin alone. To determine whether the direct interaction of paxillin and PTP-PEST was necessary for the regulation of protrusion, we expressed the paxillin C523S mutant with PTP-PEST. In this case, protrusive activity returned to levels observed in GFP-transfected paxillin/ cells (Fig. 5). (For representative movies of GFP-transfected, PTP-PEST plus wild-type paxillin transfected, and PTP-PEST plus C523S paxillin transfected paxillin/ cells see supplementary material Fig. S2.)
|
A paxillin-PTP-PEST interaction is required for PTP-PEST-regulated cell migration
Paxillin/ and PTP-PEST/ cells both exhibit defects in cell migration (Angers-Loustau et al., 1999; Hagel et al., 2002
). Since the phenotypic changes and signaling events accompanying cell spreading are considered a relevant model for the events occurring during extension of lamellipodia at the leading edge of migrating cells, we employed modified Boyden chamber assays to determine whether the paxillin-PTP-PEST interaction is important for the regulation of cell migration. Prior to these experiments, we performed a migration assay of PTP-PEST/ cells transfected with increasing amounts of PTP-PEST to determine the optimal rescue concentration (supplementary material Fig. S4). PTP-PEST/ cells exhibit reduced migration rates compared with PTP-PEST+/ cells (Fig. 6) (Angers-Loustau et al., 1999
). This defect was partially rescued by the overexpression of PTP-PEST (Fig. 6) to a level consistent with published reports (Angers-Loustau et al., 1999
) and was unaffected by the co-expression of paxillin with PTP-PEST. However, this rescue was completely abolished when PTP-PEST was co-expressed with the paxillin C523S mutant, which is defective for PTP-PEST binding (Fig. 6). Thus PTP-PEST-dependent cell migration requires a functional interaction with paxillin.
|
PTP-PEST influences Rac activity through paxillin
PTP-PEST and paxillin have both been coupled to the regulation of the Rho GTPase Rac (Brown and Turner, 2004; Sastry et al., 2002
; West et al., 2001
). Rac is activated during cell spreading and is required for lamellipodia extension (Price et al., 1998
). The role of the paxillin-PTP-PEST interaction in the regulation of Rac activity was tested using Pak-binding-domain (PBD) pull-down assays. PTP-PEST/ cells were transfected with PTP-PEST or with PTP-PEST and the paxillin C523S mutant defective for PTP-PEST binding, and compared with a GFP-transfected control population. Rac activity was measured in cells that had either been held in suspension or had been replated for 60 minutes on fibronectin. Consistent with previous studies (Sastry et al., 2002
), Rac activity was elevated when PTP-PEST/ cells were plated on fibronectin, and this increase in Rac activity was suppressed when PTP-PEST was reintroduced (Fig. 7A,C). Interestingly, disruption of the paxillin-PTP-PEST interaction by co-transfecting the paxillin C523S mutant with PTP-PEST resulted in elevated Rac activity in response to cell spreading, similar to GFP-transfected control cells (Fig. 7A,C). Parallel experiments were performed in paxillin/ cells to examine the role of paxillin in the PTP-PEST-dependent regulation of Rac in a paxillin/ background. Cells transfected with GFP exhibited increased Rac activity during cell spreading on fibronectin (Fig. 7B,D). There was still an induction of Rac activity when PTP-PEST was expressed alone or with the paxillin C523S mutant. However, the adhesion-induced increase in Rac activity was effectively blocked following the introduction of wild-type paxillin and PTP-PEST (Fig. 7B,D), consistent with the ability of this combination to inhibit cell spreading (Fig. 2). PTP-PEST also failed to inhibit Rac activity when expressed with the paxillin Y31/118F
LD4 mutant (data not shown).
|
Rac has previously been shown to function downstream of paxillin and PTP-PEST (Brown and Turner, 2004; Sastry et al., 2002
). To determine whether Rac is a downstream effector of the paxillin-PTP-PEST interaction, we co-transfected constitutively-active Rac (G12V Rac) or dominant-negative Rac (T17N Rac) with PTP-PEST, and also in combination with wild-type or mutant paxillin, and analysed cell spreading. In paxillin/ cells that re-express wild-type paxillin and PTP-PEST, the introduction of G12V Rac promoted spreading despite a functional paxillin-PTP-PEST interaction (Fig. 8). By contrast, T17N Rac suppressed spreading in all cell populations not inhibited by PTP-PEST alone (Fig. 8). Experiments performed in PTP-PEST/ cells showed similar results (data not shown). Together, these data suggest that Rac is a downstream effector of the paxillin-PTP-PEST interaction and that this association must be maintained for PTP-PEST to regulate Rac activity during cell spreading.
|
|
To determine whether PKL-binding can account for the role of the paxillin LD4 motif in PTP-PEST-dependent regulation of cell spreading (Fig. 4), we introduced wild-type PKL and a PKL mutant that had previously shown to be defective in paxillin binding (PKL PBS2) (West et al., 2001
) into PTP-PEST/ cells in the presence and absence of PTP-PEST (Fig. 10). PTP-PEST-dependent inhibition of spreading was maintained in the presence of wild-type PKL. By striking contrast, expression of the PKL
PBS2 mutant with PTP-PEST resulted in normal spreading-kinetics, suggesting that a functional paxillin-PKL interaction is required for PTP-PEST to inhibit cell spreading.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PTP-PEST/ cells exhibit enlarged focal adhesions and elevated tyrosine phosphorylation of the focal adhesion proteins paxillin, p130 Cas and FAK, leading to the speculation that PTP-PEST stimulates cell migration by promoting dephosphorylation of these proteins, which, in turn, contributes to increased focal adhesion turnover (Angers-Loustau et al., 1999; Garton et al., 1996
; Shen et al., 2000
). Interestingly, focal adhesion turnover and cell migration are also reduced in paxillin/ cells (Hagel et al., 2002
; Webb et al., 2004
), indicating that these cells might be deficient in PTP-PEST signaling. The failure of paxillin/ cells, when compared to paxillin+/+ cells, to exhibit impaired cell spreading following overexpression of PTP-PEST (Fig. 1) confirmed this speculation. This result was somewhat surprising because the paxillin/ cells have elevated levels of the paxillin family member Hic-5 (Fig. 1). Clearly, although Hic-5 shares a similar domain-structure to paxillin (Brown and Turner, 2004
) and has also been shown to share many paxillin-binding partners including PTP-PEST (Nishiya et al., 1999
), Hic-5 is unable to compensate for the loss of paxillin in the regulation of spreading, consistent with the embryonic lethality of paxillin/ mice (Hagel et al., 2002
). The lack of well-conserved tyrosine residues in Hic-5 that correspond to Y31 and Y118 of paxillin, and the different spatial organization of the LD motifs within the N-terminus might account for this difference (Brown and Turner, 2004
).
PTP-PEST binds to paxillin through the LIM3-4 domains (Côté et al., 1999). In a previous study, we showed that overexpression of a paxillin mutant lacking LIM4 resulted in reduced cell migration of CHO.K1 cells. Additionally, introduction of the LIM3-4 domain only, which is primarily cytosolic in its distribution, caused increased cell spreading (Brown and Turner, 2002
). It is now evident that both of these phenotypes can be accounted for by a perturbation in paxillin-mediated recruitment of PTP-PEST to focal adhesions. However, in paxillin/ cells the expression of the LIM1-4 domains of paxillin, which contain both focal adhesion targeting and PTP-PEST binding sites (Brown et al., 1996
; Côté et al., 1999
), was not sufficient for PTP-PEST to inhibit cell spreading (Fig. 4A), demonstrating that the role of the interaction with paxillin is not exclusively to target PTP-PEST to the focal adhesions. Instead, the LD4 motif and the tyrosine phosphorylation sites Y31 and Y118 within the paxillin N-terminus, are also required for PTP-PEST function (Figs 4 and 5).
The LD4 motif of paxillin has several binding partners, including the ARF GAP PKL/GIT2 and actopaxin, through which PTP-PEST might regulate adhesion-induced Rac signaling, and thus spreading, protrusion and migration (Brown and Turner, 2004; Clarke et al., 2004
; Turner et al., 1999
). Interestingly, PKL is tyrosine phosphorylated during cell spreading (Bagrodia et al., 1999
; Brown et al., 2005
) and recently we have shown that this phosphorylation, which is stimulated in response to Rac activation, is necessary for efficient recruitment of PKL to focal adhesions (Brown et al., 2005
). Herein, using a substrate-trapping mutant, we show that PKL is a substrate of PTP-PEST (Fig. 9) and might indeed correspond to the 95-kDa protein that was reported to exhibit elevated tyrosine phosphorylation in PTP-PEST/ cells (Côté et al., 1998
). Importantly, by using the PKL
PBS2 mutant that is defective for paxillin binding and focal adhesion targeting (West et al., 2001
), we show that the paxillin-PKL interaction is necessary for PTP-PEST to inhibit spreading (Fig. 10). Since overexpression of either a paxillin LD4-deletion mutant or PKL
PBS2 results in abnormal cell spreading and sustained activation of Rac (Brown et al., 2002
; West et al., 2001
), we speculate that PTP-PEST, by dephosphorylating PKL present in focal adhesions, provides a mechanism to terminate localized Rac activity. Whether this occurs through dissociation of, as yet unidentified, SH2-domain-containing binding partners for phosphorylated PKL, activation of PKL ARF GAP activity which has been linked to Rac inhibition in the related GIT1 protein (Nishiya et al., 2005
) or through modulation of the guanine nucleotide exchange factor (GEF) activity of the PKL-associated PAK-interacting exchange factor (PIX) (Manser et al., 1998
; Turner et al., 1999
) will require further research.
How do tyrosine residues 31 and 118 of paxillin facilitate PTP-PEST function? Phosphorylation of these sites is induced during cell spreading (Brown and Turner, 2004; Petit et al., 2000
). Phosphorylated paxillin binds to CrkII, which, in turn, can bind to the atypical Rac GEF DOCK180/ELMO complex along with p130 Cas, a PTP-PEST substrate (Gumienny et al., 2001
; Klemke et al., 1998
), to stimulate spreading and cell migration by activating Rac (Feller, 2001
; Valles et al., 2004
). In the context of cell spreading, PTP-PEST that binds to the paxillin C-terminus in focal adhesions might therefore suppress Rac activation by dephosphorylating p130 Cas and thus inhibit DOCK180 Rac GEF activity. Indeed, p130 Cas was dephosphorylated more efficiently in PTP-PEST/ cells when PTP-PEST was reintroduced together with wild-type paxillin as opposed to the paxillin C523S mutant, which is defective for PTP-PEST binding (data not shown). Alternatively, PTP-PEST might also interfere with this signaling axis at the level of the paxillin-Crk interaction, because overexpression of PTP-PEST results in a decrease in the phosphorylation levels of paxillin (Shen et al., 2000
). However, if this is the case, interference is probably indirect, because the use of a substrate-trapping mutant of PTP-PEST indicated that paxillin itself is not a direct substrate of PTP-PEST (Côté et al., 1999
).
Finally, although this study focused on dissecting the contribution of paxillin in the control of cell spreading, membrane protrusion and Rac activation by PTP-PEST, we have also shown that binding to paxillin is essential for reintroduced PTP-PEST to rescue cell migration in PTP-PEST/ cells. It is generally accepted that, the signaling events that control lamellipodia extension in a spreading cell represent the molecular events that occur during the membrane extension and adhesion phases of cell migration. The translation of these events into productive cell movement undoubtedly requires successive rounds of activation and inactivation of relevant signaling pathways, including phosphorylation and dephosphorylation, and cycling of Rho family GTPase activities. Our current study suggests that, PTP-PEST contributes to this process through binding to the focal adhesion protein paxillin and modulates signaling to Rac by obtaining access to other proteins, such as PKL, that bind through multiple domains within the N-terminus of paxillin. Future studies will be directed towards understanding the complex interrelationship of these various signaling moieties.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angers-Loustau, A., Cote, J. F., Charest, A., Dowbenko, D., Spencer, S., Lasky, L. A. and Tremblay, M. L. (1999). Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts. J. Cell Biol. 144, 1019-1031.
Bagrodia, S., Bailey, D., Lenard, Z., Hart, M., Guan, J. L., Premont, R. T., Taylor, S. J. and Cerione, R. A. (1999). A tyrosine-phosphorylated protein that binds to an important regulatory region on the cool family of p21-activated kinase-binding proteins. J. Biol. Chem. 274, 22393-22400.
Blanchetot, C., Chagnon, M., Dube, N., Halle, M. and Tremblay, M. L. (2005). Substrate-trapping techniques in the identification of cellular PTP targets. Methods 35, 44-53.[CrossRef][Medline]
Brown, M. C. and Turner, C. E. (2002). Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility. Int. J. Biochem. Cell Biol. 34, 855-863.[CrossRef][Medline]
Brown, M. C. and Turner, C. E. (2004). Paxillin: adapting to change. Physiol. Rev. 84, 1315-1339.
Brown, M. C., Perrotta, J. A. and Turner, C. E. (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, 1109-1123.[Abstract]
Brown, M. C., West, K. A. and Turner, C. E. (2002). Paxillin-dependent paxillin kinase linker and p21-activated kinase localization to focal adhesions involves a multistep activation pathway. Mol. Biol. Cell 13, 1550-1565.
Brown, M. C., Cary, L. A., Jamieson, J. S., Cooper, J. A. and Turner, C. E. (2005). Src and FAK kinases cooperate to phosphorylate PKL, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness. Mol. Biol. Cell 9, 4316-4328.[CrossRef]
Burridge, K. and Wennerberg, K. (2004). Rho and Rac take center stage. Cell 116, 167-179.[CrossRef][Medline]
Clarke, D. M., Brown, M. C., LaLonde, D. P. and Turner, C. E. (2004). Phosphorylation of actopaxin regulates cell spreading and migration. J. Cell Biol. 166, 901-912.
Côté, J. F., Charest, A., Wagner, J. and Tremblay, M. L. (1998). Combination of gene targeting and substrate trapping to identify substrates of protein tyrosine phosphatases using PTP-PEST as a model. Biochemistry 37, 13128-13137.[CrossRef][Medline]
Côté, J. F., Turner, C. E. and Tremblay, M. L. (1999). Intact LIM 3 and LIM 4 domains of paxillin are required for the association to a novel polyproline region (Pro 2) of protein-tyrosine phosphatase-PEST. J. Biol. Chem. 274, 20550-20560.
DeMali, K. A., Wennerberg, K. and Burridge, K. (2003). Integrin signaling to the actin cytoskeleton. Curr. Opin. Cell Biol. 15, 572-582.[CrossRef][Medline]
Feller, S. M. (2001). Crk family adaptors-signalling complex formation and biological roles. Oncogene 20, 6348-6371.[CrossRef][Medline]
Garton, A. J. and Tonks, N. K. (1999). Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST. J. Biol. Chem. 274, 3811-3818.
Garton, A. J., Flint, A. J. and Tonks, N. K. (1996). Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST. Mol. Cell. Biol. 16, 6408-6418.[Abstract]
Gumienny, T. L., Brugnera, E., Tosello-Trampont, A. C., Kinchen, J. M., Haney, L. B., Nishiwaki, K., Walk, S. F., Nemergut, M. E., Macara, I. G., Francis, R. et al. (2001). CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 107, 27-41.[CrossRef][Medline]
Hagel, M., George, E. L., Kim, A., Tamimi, R., Opitz, S. L., Turner, C. E., Imamoto, A. and Thomas, S. M. (2002). The adaptor protein paxillin is essential for normal development in the mouse and is a critical transducer of fibronectin signaling. Mol. Cell. Biol. 22, 901-915.
Juliano, R. L. (2002). Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu. Rev. Pharmacol. Toxicol. 42, 283-323.[CrossRef][Medline]
Kinley, A. W., Weed, S. A., Weaver, A. M., Karginov, A. V., Bissonette, E., Cooper, J. A. and Parsons, J. T. (2003). Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion. Curr. Biol. 13, 384-393.[CrossRef][Medline]
Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K. and Cheresh, D. A. (1998). CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J. Cell Biol. 140, 961-972.
Kurokawa, K., Nakamura, T., Aoki, K. and Matsuda, M. (2005). Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells. Biochem. Soc. Trans. 33, 631-634.[CrossRef][Medline]
Lamorte, L., Rodrigues, S., Sangwan, V., Turner, C. E. and Park, M. (2003). Crk associates with a multimolecular Paxillin/GIT2/beta-PIX complex and promotes Rac-dependent relocalization of Paxillin to focal contacts. Mol. Biol. Cell 14, 2818-2831.
Lim, L., Manser, E., Leung, T. and Hall, C. (1996). Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur. J. Biochem. 242, 171-185.[CrossRef][Medline]
Lyons, P. D., Dunty, J. M., Schaefer, E. M. and Schaller, M. D. (2001). Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation. J. Biol. Chem. 276, 24422-24431.
Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L., Tan, I., Leung, T. and Lim, L. (1998). PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1, 183-192.[CrossRef][Medline]
Mitra, S. K., Hanson, D. A. and Schlaepfer, D. D. (2005). Focal adhesion kinase: in command and control of cell motility. Nat. Rev. Mol. Cell. Biol. 6, 56-68.[CrossRef][Medline]
Nikolopoulos, S. N. and Turner, C. E. (2000). Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion. J. Cell Biol. 151, 1435-1448.
Nishiya, N., Iwabuchi, Y., Shibanuma, M., Cote, J. F., Tremblay, M. L. and Nose, K. (1999). Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain. J. Biol. Chem. 274, 9847-9853.
Nishiya, N., Kiosses, W. B., Han, J. and Ginsberg, M. H. (2005). An alpha4 integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migrating cells. Nat. Cell Biol. 7, 343-352.[CrossRef][Medline]
Petit, V., Boyer, B., Lentz, D., Turner, C. E., Thiery, J. P. and Valles, A. M. (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, 957-970.
Playford, M. P. and Schaller, M. D. (2004). The interplay between Src and integrins in normal and tumor biology. Oncogene 23, 7928-7946.[CrossRef][Medline]
Price, L. S., Leng, J., Schwartz, M. A. and Bokoch, G. M. (1998). Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell 9, 1863-1871.
Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T. and Horwitz, A. R. (2003). Cell migration: integrating signals from front to back. Science 302, 1704-1709.
Riedy, M. C., Brown, M. C., Molloy, C. J. and Turner, C. E. (1999). Activin A and TGF-beta stimulate phosphorylation of focal adhesion proteins and cytoskeletal reorganization in rat aortic smooth muscle cells. Exp. Cell Res. 251, 194-202.[CrossRef][Medline]
Sastry, S. K., Lyons, P. D., Schaller, M. D. and Burridge, K. (2002). PTP-PEST controls motility through regulation of Rac1. J. Cell Sci. 115, 4305-4316.
Schmitz, A. A., Govek, E. E., Bottner, B. and Van Aelst, L. (2000). Rho GTPases: signaling, migration, and invasion. Exp. Cell Res. 261, 1-12.[CrossRef][Medline]
Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A. and Schaller, M. D. (1998). Direct association of protein-tyrosine phosphatase PTP-PEST with paxillin. J. Biol. Chem. 273, 6474-6481.
Shen, Y., Lyons, P., Cooley, M., Davidson, D., Veillette, A., Salgia, R., Griffin, J. D. and Schaller, M. D. (2000). The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo. J. Biol. Chem. 275, 1405-1413.
Spencer, S., Dowbenko, D., Cheng, J., Li, W., Brush, J., Utzig, S., Simanis, V. and Lasky, L. A. (1997). PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase. J. Cell Biol. 138, 845-860.
Tsubouchi, A., Sakakura, J., Yagi, R., Mazaki, Y., Schaefer, E., Yano, H. and Sabe, H. (2002). Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration. J. Cell Biol. 159, 673-683.
Tumbarello, D. A., Brown, M. C., Hetey, S. E. and Turner, C. E. (2005). Regulation of paxillin family members during epithelial-mesenchymal transformation: a putative role for paxillin . J. Cell Sci. 20, 4849-4863.[CrossRef]
Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C., Nikolopoulos, S. N., McDonald, A. R., Bagrodia, S., Thomas, S. and Leventhal, P. S. (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, 851-863.
Valles, A. M., Beuvin, M. and Boyer, B. (2004). Activation of Rac1 by paxillin-Crk-DOCK180 signaling complex is antagonized by Rap1 in migrating NBT-II cells. J. Biol. Chem. 279, 44490-44496.
von Wichert, G., Haimovich, B., Feng, G. S. and Sheetz, M. P. (2003). Force-dependent integrin-cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2. EMBO J. 22, 5023-5035.
Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J. T. and Horwitz, A. F. (2004). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6, 154-161.[CrossRef][Medline]
West, K. A., Zhang, H., Brown, M. C., Nikolopoulos, S. N., Riedy, M. C., Horwitz, A. F. and Turner, C. E. (2001). The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL). J. Cell Biol. 154, 161-176.
Wittchen, E. S., van Buul, J. D., Burridge, K. and Worthylake, R. A. (2005). Trading spaces: Rap, Rac, and Rho as architects of transendothelial migration. Curr. Opin. Hematol. 12, 14-21.[CrossRef][Medline]
Wozniak, M. A., Modzelewska, K., Kwong, L. and Keely, P. J. (2004). Focal adhesion regulation of cell behavior. Biochim. Biophys. Acta 1692, 103-119.[Medline]
Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S. K., Matsuda, M. and Sabe, H. (2004). Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. J. Cell Biol. 166, 283-295.
Zhao, Z. S., Manser, E., Loo, T. H. and Lim, L. (2000). Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly. Mol. Cell Biol. 20, 6354-6363.
|