FIGURE 8:

(A, B) Differential role of T678 and S885 in TNF-α–induced ERK activation. (A) LLC-PK₁ cells grown in 6-cm dishes were transfected with 100 nM NR or GEF-H1–specific siRNA and 24 h later with GFP-GEF-H1^T678A or GFP- GEF-H1^TS885A along with HA-ERK2. (B) Cells were transfected with GEF-H1 shRNA along with HA-ERK with or without GFP- GEF-H1^T678A or GFP- GEF-H1^TS885A. Details of the transfection are described in Materials and Methods. Cells were treated with 10 ng/ml TNF-α as indicated, and HA-ERK was immunoprecipitated and its phosphorylation detected using Western blotting as in Figure 2D. GEF-H1 and GFP were also detected in the cell lysates to assess down-regulation of endogenous GEF-H1 and expression of the GFP-tagged mutants. (C, D) Role of S885 in GEF-H1 activation toward RhoA. LLC-PK₁ cells were transfected with GFP-GEF-H1^WT, GFP-GEF-H1^S885A, or GFP-GEF-H1^T678D (labeled as TD) or GFP-GEF-H1^T678D/S885A (labeled as TD/SA) as indicated. Activated GFP-GEF-H1 was precipitated using RhoA(G17A) and detected by Western blotting with anti-GFP, as described earlier. In C, p38 in the cell lysates was also detected. Note that the transfected FLAG-tagged p38 is visualized as an additional, higher band (see arrows). Throughout the figure representative blots of three independent experiments are shown.