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doi: 10.1242/10.1242/jcs.00105

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PTP-PEST controls motility through regulation of Rac1

Department of Cell and Developmental Biology and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (29K): [in a new window]   Fig. 1. PTP-PEST is activated by integrins and localizes to the tips of membrane protrusions. (A) CHOK1 fibroblasts were held in suspension (S) or plated on fibronectin-coated (FN, 10 µg/ml) or poly-L-lysine-coated (PL, 10 µg/ml) plates for 1 hour in serum-free medium. PTP-PEST was immunoprecipitated from TX-100 lysates. PTPase activity was determined by incubating the immunoprecipitates with a 32P-labeled peptide substrate (32P polyglu-tyr) and measuring the amount of 32P released. PTP-PEST activity is 2-3-fold higher in cells plated on FN compared with cells in suspension (S) or plated on a non-specific substrate (PL). The results represent three independent experiments. (Lower panel) Immunoprecipates of PTP-PEST from CHOK1 cells in suspension, plated on FN or PL, show that equal amounts of PTP-PEST were precipitated for the activity assay. (B) Swiss 3T3 fibroblasts were plated on FN-coated coverslips for 20 minutes and then immunostained for PTP-PEST (left) and actin (right). PTP-PEST transiently localizes at the tips of protrusions.

View larger version (19K): [in a new window]   Fig. 2. PTP-PEST effect on motility depends on its catalytic activity. (A) Transient expression of GFP, PTP-PEST or PTP-PESTC231S in CHOK1 cells. Western blots for the KT3 epitope tag (KT3) or for GFP show expression levels of transfected proteins. (B) CHOK1 cells transfected with wild-type PTP-PEST, PTP-PESTC231S, or GFP were analyzed for their ability to migrate towards a FN substrate using a haptotactic migration assay. The lower membrane of a transwell chamber was coated with 10 µg/ml FN. While expression of a mock gene, GFP, did not affect CHOK1 migration, expression of wild-type PTP-PEST significantly impaired their migration. In contrast, expression of a catalytically inactive mutant, PTP-PESTC231S, did not inhibit CHOK1 migration, indicating that the catalytic function is critical for motility. The number of cells per field for five fields were scored in three separate experiments. Migration of transfected cells is expressed as a percentage of CHOK1 controls.

View larger version (66K): [in a new window]   Fig. 3. PTP-PEST regulates the formation of membrane protrusions. CHOK1 cells were transiently transfected with expression plasmids encoding wild-type PTP-PEST, a catalytically inactive mutant PTP-PESTC231S, or a mock vector encoding GFP. 24 hours after transfection, cells were trypsinized and replated onto FN-coated coverslips in serum-free medium for indicated times. PTP-PEST or PTP-PESTC231S-transfected cells were fixed and co-stained for PTP-PEST with the KT3 mAb and for actin with Texas-Red-phalloidin. GFP-transfected cells were co-stained for actin only. MOCK-transfected cells spread on a FN substrate extending prominent lamellipodia and form membrane ruffles. WT PTP-PEST-transfected cells are inhibited in spreading. These cells are able to attach but have a rounded morphology. They do not extend protrusion or form ruffles. Catalytically inactive PTP-PEST(C231S) enhances membrane ruffling at early times of adhesion (30-60 min). At later times after plating (2 hours) these cells extend numerous protrusions in all directions.

View larger version (82K): [in a new window]   Fig. 4. PTP-PEST impairs membrane ruffling and motility in response to PDGF. (A) Parental Rat-1 fibroblasts or Rat-1 cells stably overexpressing PTP-PEST were serum starved (left panels) and then stimulated with PDGF-BB (20 ng/ml, 15 minutes, right panels) to assess membrane ruffling. Whereas control cells reorganize actin into ruffles, those that express PTP-PEST show less prominent membrane ruffling. (B) Parental Rat-1 fibroblasts or those stably expressing PTP-PEST were added to transwell filters in which the bottom membrane was coated with FN in the presence or absence of PDGF-BB in the lower chamber. While control cells are induced to migrate in the presence of PDGF, PTP-PEST-expressing cells exhibit reduced motility in response to PDGF.

View larger version (49K): [in a new window]   Fig. 5. PTP-PEST overexpression suppresses adhesion- and growth factor-dependent activation of Rac1. Rac1 activity was measured using an affinity precipitation assay in which a GST fusion protein of the Rac1-binding domain of the Rac1 effector PAK3 (GST-PBD), which specifically binds GTP-Rac1 (active), is used to `pulldown' active Rac1 from cell lysates. The amount of GTP-Rac1 isolated by GST-PBD immobilized on glutathione beads was determined by a Rac1 immunoblot. (A) A timecourse of Rac1 activation in response to CHOK1 attachment and spreading on a FN substrate shows that Rac1 activity is stimulated 10-20 minutes after attachment, remains active for 30-40 minutes and then begins to decrease by 60 minutes. Rac1 activity in the pulldown is normalized against total Rac1 in the lysate. Overexpression of PTP-PEST, but not GFP (MOCK), decreases the level of Rac1 activity in response to cell attachment to FN for 45 minutes. (B) Parental or PTP-PEST-expressing Rat-1 fibroblasts were serum starved and then stimulated with PDGF-BB (50 ng/ml) for the times indicated. PDGF robustly activates Rac1 in parental cells. PTP-PEST-expressing cells have a lower baseline of Rac1 activity and respond poorly to PDGF.

View larger version (33K): [in a new window]   Fig. 6. PTP-PEST is required to regulate Rac1 activity. PTP-PEST null fibroblasts (PTP-PEST -/-) or null fibroblasts that re-express PTP-PEST (PTP-PEST RE) were maintained in suspension or plated on FN in serum-free medium for 30, 60 or 120 minutes. Rac1 activity was determined using the GST-PBD pulldown assay. In cells that re-express PTP-PEST (left), Rac1 is transiently activated by adhesion to FN within 30 minutes and then returns to baseline. In PTP-PEST -/- cells (right), Rac1 is activated by adhesion after 30 minutes to a greater degree than in control cells. This elevated activity is sustained throughout the time course.

View larger version (45K): [in a new window]   Fig. 7. Activated Rac1 overcomes the phenotypic effects of PTP-PEST overexpression. Wild-type PTP-PEST and GFP-tagged Q61LRac1 were co-expressed in CHOK1 cells and assayed for cell spreading and migration. (A) For cell spreading, co-transfected cells were plated on FN-coated coverslips for 45 minutes in serum-free medium. Cells were fixed and then triple-stained for PTP-PEST overexpression (left), GFP (middle) and actin (right). Compared with PTP-PEST overexpression alone (see Fig. 2C), co-expression of activated Rac1 resulted in cells that were able to spread. (B) Co-expression of activated Rac1 also restored the ability of PTP-PEST-transfected cells to migrate towards FN in a haptotaxis transwell assay.

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