Effect of Spry2 on Rac1 activation in HRas-transformed cells.
A, WCL from the HRas-transformed cell strain expressing a high
endogenous level of Spry2 (PH3MT-SC (SC)) or a reduced level of Spry2
(PH3MT-2A3 (2A3)) and the parental non-transformed human fibroblast
cell strain expressing a low endogenous level of Spry2 (empty vector)
(MSU1.1-VC (VC)) or expressing a high level of Spry2 (MSU1.1-S62
(S62)) were pulled down (PD) with PAK-CRIB-conjugated beads.
The amount of Rac1 bound to the beads, as well as the Rac1 present in the WCL
was determined. The amount of active Cdc42 was determined only in the PH3MT
cells. B, WCL from PH3MT-SC and PH3MT-2A3 cell strains were
immunoblotted or immunoprecipitated (IP) with an antibody specific to
HRas and then immunoblotted to detect Tiam1 and HRas with the indicated
antibodies. C, HRas-transformed fibroblasts (PH3MT) were stably
transfected with an empty vector or a vector encoding a Myc-tagged, dominant
negative form of Rac1 (Rac1N17). WCL from these stable clones were
analyzed by Western blotting for Rac1 and β-actin expression. The same
cell strains were treated and analyzed as in
Fig. 1A. The average
of two independent experiments is shown. D, HRas-transformed cells
with down-regulated Spry2 (PH3MT-2A3) but stably expressing
GFP-Rac1V12 (2A3-R1) or GFP alone (2A3-VC) were analyzed by Western
blotting for pAkt; Akt; p53; HDM2; GFP; Spry2; or β-actin. E,
the PH3MT-2A3 VC and PH3MT-2A3 R1 cell strains were UV-irradiated and analyzed
as in Fig. 1A. The
average of three independent experiments is shown. F, the indicated
cell strains (5,000 cells/dish) were grown in agarose in a culture medium
containing 2.5% serum for 3 weeks, as described in Ref.
31. Representative pictures of
the colonies that formed in agarose are shown. R1, PH3MT-2A3-R1.