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.

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