FIGURE 2.

p38γ promotes the Ras oncogenesis via stimulating PTPH1/Ser-459 phosphorylation. A, coupling of p-PTPH1 expression with the soft-agar growth in IEC-6/K-Ras cells expressed with p38γ and its AGF mutant. IEC-6 cells were stably expressed with p38γ and p38γ/AGF by G418 selection, and resulting cells were then transduced with K-Ras, which were analyzed by PTPH1 IPs/WB (A, open arrowhead, p-p38γ) and by the soft-agar growth (supplemental Fig. S2A). B and C, PTPH1 dependent on Ser-459 for its oncogenic activity. Transformed IEC-6/K-Ras cells (16) were transfected with PTPH1 and PTPH1/S459A through G418 selection, and resistant cells were pooled and analyzed by the soft-agar growth (C) and p-p38 IP/WB (B). Results in C are from three separate experiments with each in triplicate as described in supplemental Fig. S2A and expressed as relative to vector controls (error bars, S.D.; *, p < 0.05; and **, p > 0.05 versus vector, Student's t test). Colonies are from one representative experiment shown in supplemental Fig. S2D. D–F, IEC-6/K-Ras/p38γ and IEC-6/K-Ras/PTPH1 cells incubated with either 50 μg/ml PFD or dimethyl sulfoxide for 24 h for IP/WB (D) or with 50 μg/ml PFD present in the top layer of soft-agar continuously for about 10–14 days for the soft-agar growth (18). E and F, mean of three separate experiments, with each in triplicate, * p < 0.05 versus dimethyl sulfoxide).