FIGURE 5. Increased VAV2 precipitation in PTP-PEST null cells.

A, affinity precipitation with a G15A mutant of Rac1 that mimics nucleotide-free Rac1 (NF Rac1) was used to indirectly assess GEF activity. CHOK1 cells were transfected with Myc-tagged wild type VAV2, then held in suspension or plated on FN for 60 min. GST-15ARac1 was used to pull down VAV2. The amount of VAV2 precipitated by NF-Rac1 when cells were plated on FN was increased relative to cells in suspension. WB, Western blot. S, suspension. B, NF-Rac1 precipitates an increased amount of VAV2 from PTP-PEST −/− cells compared with re-expressers in response to adhesion to FN. C, SOS-1 activity is not affected by PTP-PEST and serves as a negative control. However, SOS-1 can be activated by platelet-derived growth factor (PDGF) stimulation in 293 cells. D, expression of a VAV2 mutant that is altered in its DH domain, VAV2 Arg → Ser, restores a normal phenotype to PTP-PEST −/− cells. Note the arrows show protrusions in untransfected null cells, whereas arrowheads point to cells that expressing the VAV2 mutant with a morphology resembling control re-expressers. All cells were plated for 3 h on FN-coated coverslips in serum-free medium.