Overexpression of activated mutants of H-Ras, N-Ras, or M-Ras results in activation of Rac-1 via a mechanism, which is not inhibited by Ly294002.

Baf/3 cells were coelectroporated with 3 μg pEGFP-C1 empty vector, 3 μg wild-type, myc-tagged Rac-1, and 15 μg of (A and C) control plasmid (vector), a constitutively active mutant of PI-3K (p110*), or constitutively active mutants of one of 2 p21 Ras isoforms (G12 H-Ras and Q61 N-Ras), (B) control plasmid (vector), wild-type M-Ras (WT M-Ras), or a constitutively active mutant of M-Ras (Q71 M-Ras). At 16 hours after electroporation, cells were resuspended in serum-free medium for 2 hours. Ly294002 (25 μM dissolved in DMSO) was present for these 2 hours for N-RAS (A). Ly294002 (25 μM dissolved in ethanol) was present for the last 30 minutes for H-Ras (A), M-Ras (B), and H-Ras/p110* (C). After this, cells were lysed. Activated Rac-1 myc (Rac-1 myc GTP) was precipitated from lysates using GST-PAK bound to GT beads and detected by immunoblotting with a Rac-1 specific monoclonal antibody. The activation status of Akt and Erk was also determined from the same lysates using phospho-specific antibodies (p-Akt, p-Erk). Equivalency of loading was confirmed as described earlier (Akt, Erk).