Figure 4: Effects of the P-switch on the global activities of RhoA and Rac1.

(a) Depletion of TNS3 from MCF10A activated RhoA, but not Rac1. GTP-bound RhoA or Rac1 was pulled down (PD) with Rhotekin-RBD or PAK-PBD agarose beads from cell lysates and blotted with an anti-RhoA or anti-Rac1 antibody. WCL IB was included to show the total levels of TNS3, RhoA and Rac1. (b) Depletion of PTEN augmented the activity of Rac1, but not RhoA. The same set of experiments as in a were carried out in MCF10A cells transfected with a PTEN-specific siRNA or a scrambled oligo (siCtrl). (c) Overexpression of TNS3–ABD, but not PTEN nor a mutant of either protein, led to inactivation of RhoA. HEK293 cells expressing GFP fusion TNS3–ABD, PTEN or the indicated mutants were subjected to a Rhotekin-PD assay. (d) PTEN and its point mutants, but neither a PTEN truncation mutant missing the catalytic domain (▵CAT) nor TNS3–ABD and its point mutants, repressed Rac1 activity. A PAK-PBD PD assay was used to assess Rac1 activity in HEK293 cells co-expressing PI3K (that is, FLAG-p85 plus Myc-p110) and the indicated PTEN or TNS3–ABD proteins. (e) The specificity-switching mutant, TNS3–ABD–Y321T, but neither PTEN, PTEN–T319Y, nor TNS3–ABD, inactivated RhoA. DLC1-expressing HEK293 cells were serum starved and treated with EGF for 30 min before the Rhotekin-PD assay. (f) wt PTEN and the specificity-switching mutant, PTEN–T319Y, but neither TNS3–ABD nor the ABD–Y321T mutant, inhibited the gross activation of Rac1. Cells were treated the same way as in e. Data shown are representative of three independent experiments.