Figure 3.

CIIA promotes EGF-induced SOS1–Rac1 signaling. (A) HeLa cells transfected with GFP or SOS1 siRNA for 12 h were deprived of serum for 16 h and then incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were pulled down (PD) with GST-CRIB and assayed for Rac1 activity. (B) 293T cells were transfected for 48 h with vectors for the indicated proteins, after which cell lysates were subjected to a pull-down assay with GST-CRIB as in A. (C) The GEF activity of SOS1-DHPH on Rac1 was determined in vitro in the absence or presence of CIIA by fluorescence spectroscopy with the use of the GEF exchange assay kit as described in Materials and methods. (top) Relative fluorescence intensity data from one representative experiment. (bottom) Data are the means ± SD of three independent experiments. Results are expressed as the mant-GTP incorporation into GST-Rac1 after 480 s relative to time 0 after adding His₆-SOS1-DHPH. *, P < 0.05. (D) MDCK/CIIA-Flag and MDCK/control cells were deprived of serum for 16 h and incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were subjected to Rac1 activity assay as in A. (E) HeLa cells were transfected for 24 h with the indicated combinations of GFP, CIIA, or E3B1 siRNA and a vector encoding CIIA*-Myc. Then, the cells were deprived of serum for 16 h and incubated for 5 min in the presence or absence of 100 ng/ml EGF. Cell lysates were assayed for Rac1 activity as in A. (F) HeLa-siGFP or HeLa-siCIIA cells were deprived of serum for 16 h and then incubated for 5 min with or without 100 ng/ml EGF. Cell lysates were examined by immunoblotting (IB) with antibodies to phospho(Thr⁴²³)-PAK1, PAK1, or CIIA.