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First published online 29 November 2005
doi: 10.1242/jcs.02693

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Paxillin is essential for PTP-PEST-dependent regulation of cell spreading and motility: a role for paxillin kinase linker

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

View larger version (28K): [in a new window]   Fig. 1. Paxillin is required for PTP-PEST inhibition of cell spreading. (A) Paxillin+/+ and paxillin–/– cells were transfected with PTP-PEST and GFP or with GFP control vector alone. Cells were harvested and maintained in suspension for 60 minutes at 37°C and then replated on fibronectin-coated slips for 45 minutes or overnight. Cells were fixed at 45 minutes and stained with rhodamine phalloidin. The number of unspread transfected cells (GFP-positive) was evaluated (see Materials and Methods for details). Expression of PTP-PEST inhibited spreading of the paxillin+/+ but not paxillin–/– cells. Cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar, 25 µm. (B) Quantification of PTP-PEST-dependent inhibition of spreading was measured as the percentage of round GFP-positive cells. A minimum of 100 cells were counted per condition in three independent experiments. Results show mean±s.d. **P<0.001. (C) Western immunoblotting of total cell lysates demonstrates the absence of paxillin and upregulation of Hic-5 in paxillin–/– cells compared with the paxillin+/+ control cells.

View larger version (38K): [in a new window]   Fig. 2. A direct paxillin–PTP-PEST interaction is necessary for PTP-PEST to regulate cell spreading. (A) Paxillin–/– cells were transfected with PTP-PEST and paxillin, PTP-PEST and the paxillin C523S mutant (which is defective for PTP-PEST binding) or the PTP-PEST Pro II mutant (defective for paxillin binding) alone, in the absence or presence of paxillin. Cells were plated on fibronectin and assayed for spreading at 45 minutes post plating. Co-transfection with GFP allowed identification of transfected cells. Cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar, 25 µm. Perturbation of the paxillin-PTP-PEST interaction blocks PTP-PEST inhibition of cell spreading. (B) Quantification of round, GFP-positive cells observed in A. Results show the mean±s.d. A minimum of 100 cells were counted in three independent experiments. **P< 0.001. (C) Western immunoblot analysis confirms equivalent expression of paxillin and PTP-PEST constructs. (D) Localization of paxillin and paxillin C523S mutant to focal adhesions in paxillin–/– cells was visualized by indirect immunofluorescence microscopy. Cells were also stained with antibody against vinculin to visualize focal adhesions. Bar, 15 µm.

View larger version (25K): [in a new window]   Fig. 3. PTP-PEST reintroduced into PTP-PEST–/– cells inhibits spreading through paxillin. (A) Immunofluorescence staining of PTP-PEST–/– cells transfected with GFP, GFP and PTP-PEST, PTP-PEST and paxillin, or PTP-PEST and paxillin C523S and plated on fibronectin-coated coverslips for 60 minutes. Cells were co-stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar, 25 µm. (B) Inhibition of spreading was measured as the percentage of round GFP-positive cells observed in A. A minimum of 100 cells were counted per condition in three independent experiments. Results show the mean±s.d. **P<0.001, *P<0.05. (C) Western blots of transfected cells were probed for anti-paxillin and anti-PTP-PEST antibodies to demonstrate equal expression of constructs.

View larger version (37K): [in a new window]   Fig. 4. Paxillin N-terminus, LD4 motif and tyrosine phosphorylation sites Y31 and Y118 are required for PTP-PEST-dependent inhibition of spreading. Paxillin–/– cells were transiently transfected with GFP, paxillin alone or together with PTP-PEST, or the following paxillin constructs with and without PTP-PEST: (A) LIM1-4, (B) LD4, (C) LD1 or LD2 and (D) Y31/118F, and plated on fibronectin-coated coverslips. Cells were fixed after 45 minutes and processed for indirect immunofluorescence. Results represent the percentage of round GFP-positive cells. Results show the mean±s.d. A minimum of 100 cells were counted in three independent experiments. **P<0.001, * P<0.05 (E) GFP-transfected paxillin–/– cells that had also been transfected with PTP-PEST, paxillin and PTP-PEST, PTP-PEST and Pax C523S, PTP-PEST and Pax LD4, PTP-PEST and Pax Y31/118F, or PTP-PEST and Pax Y31/118F LD4 were plated on fibronectin-coated coverslips for 45 and 120 minutes. Cells were fixed and processed for indirect immunofluorescence as described above. Total cell area was obtained from these images using SpotTM RT software. Results represent the mean±s.d. of a minimum of 50 cells for each time point and three independent experiments. **P<0.001.

View larger version (13K): [in a new window]   Fig. 5. Paxillin is required for PTP-PEST to inhibit cell protrusiveness. Paxillin–/– cells transfected with GFP and the PTP-PEST and paxillin constructs as indicated were plated on 10 µg/ml fibronectin-coated coverslips for 180 minutes; thereafter, time-lapse images were taken every 10 minutes. Changes in cell protrusion area of a minimum of 10 transfected cells were quantified at 10-minute intervals for 1 hour as described in Materials and Methods. **P<0.001, *P<0.05.

View larger version (11K): [in a new window]   Fig. 6. A productive paxillin–PTP-PEST interaction is required for PTP-PEST-regulated cell migration. Modified Boyden chamber assays were performed as described in Materials and Methods to determine whether the paxillin–PTP-PEST interaction was important for the regulation of cell migration. PTP-PEST–/– cells were transiently transfected with GFP alone or with PTP-PEST, PTP-PEST and paxillin, or PTP-PEST and paxillin C523S. PTP-PEST–/– cells exhibit reduced migration rates compared with PTP-PEST+/– cells. This defect was partially rescued by overexpression of PTP-PEST. However, this rescue was completely abolished when PTP-PEST was co-expressed with the paxillin C523S mutant. Results show the mean±s.d. and represent three independent experiments. **P<0.001.

View larger version (36K): [in a new window]   Fig. 7. PTP-PEST regulates Rac1 activation through an interaction with paxillin. (A) PTP-PEST–/– cells or (B) paxillin–/– cells were co-transfected with Myc-tagged wild-type Rac1 together with GFP, PTP-PEST, PTP-PEST and paxillin, or PTP-PEST and paxillin C523S, and either held in suspension or plated on fibronectin-coated culture dishes for 60 minutes. The level of activated Rac was determined using a GST-PAK-PBD pull-down assay as described in Materials and Methods. Total and active Rac levels were visualized by western blotting and quantitative densitometry was performed. (C,D) Results show the mean±s.d. of three independent experiments. PTP-PEST inhibited adhesion-induced Rac activation only in the presence of paxillin. This effect was reversed by introduction of the paxillin C523S mutant defective in PTP-PEST binding. **P<0.001.

View larger version (27K): [in a new window]   Fig. 8. Rac1 rescues paxillin-dependent PTP-PEST suppression of cell spreading. (A) Paxillin–/– cells were cotransfected with constitutively active (V12 Rac) or dominant negative (N17 Rac) Rac together with GFP, and with PTP-PEST and paxillin, PTP-PEST and paxillin LD4, PTP-PEST and paxillin Y31/118F or PTP-PEST and paxillin Y31/118FLD4. Cells were plated on fibronectin-coated coverslips for 45 minutes, then fixed and processed for indirect immunofluorescence. Cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar, 25 µm. (B) Quantification of cell spreading was accomplished by counting round versus spread GFP-positive cells. A minimum of 100 cells were counted per condition in three independent experiments. Results show mean±s.d. **P<0.001, *P<0.05.

View larger version (40K): [in a new window]   Fig. 9. PKL is a substrate of PTP-PEST (A) PTP-PEST–/– cell lysates were incubated with GST-catalytic-domain fusions of either wild-type PTP-PEST or the C231S catalytically inactive `trapping' mutant. Pull-down assays were blotted with antibodies against phosphotyrosine (4G10), PKL or -actinin. `Trapped' proteins of 130, 95 and 70 kDa were observed. The PKL immunoblot shows that a significant amount of PKL is trapped, with only a modest amount being pulled down by the wild-type fusion protein. DLS, detergent soluble lysate. (B) GFP, GFP PKL or GFP-PKL triple YF mutant were expressed in PTP-PEST–/– cells in the presence or absence of PTP-PEST. Cells were plated on fibronectin-coated dishes for 60 minutes and cell lysates were immunoprecipitated with the anti-GFP antibody. The precipitates were immunoblotted with anti-phosphotyrosine (4G10) and anti-GFP antibodies. The phosphorylation of PKL was reduced in the presence of PTP-PEST. (C) PTP-PEST suppresses PKL phosphorylation in the presence of constitutively active Rac. PTP-PEST–/– cells expressing GFP or GFP PKL in the presence or absence of PTP-PEST and constitutively active Rac (V12 Rac) were plated for 60 minutes on fibronectin-coated dishes, cell lysates were then immunoblotted with anti-phosphotyrosine (4G10) and anti-GFP antibodies.

View larger version (17K): [in a new window]   Fig. 10. PKL-paxillin interaction is necessary for PTP-PEST to regulate cell spreading. PTP-PEST–/– cells were transiently transfected with GFP with or without PTP-PEST, together with paxillin LD4, PKL or the PKL PBS2 mutant, plated on fibronectin-coated coverslips for 60 minutes and processed for immunofluorescence. Results represent the percentage of round GFP-positive cells. Results show mean±s.d. A minimum of 100 cells were counted in three independent experiments. **P<0.001, *P<0.05.

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