Figure 8.

Tyrosine phosphorylation of ACK and inhibition of PAK1 kinase activity in response to fibronectin matrix removal by III1-C. (A) Tyrosine phosphorylation of ACK: KD fibroblasts were treated with 20 μM III1-C for 16 h. HUVECs were treated with 20 μM III1-C for 4 h or 15 min, or were stimulated by hyperosmotic shock (0.8 M NaCl for 10 min) or by decreasing the temperature (25°C for 30 min). Serum-free medium was used as a control (medium). ACK was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (PY20). (B) PAK1 in vitro kinase assay using MBP as the substrate. HUVECs were pretreated with 20 μM III1-C for 4 h in serum-free medium to remove fibronectin matrix (+), or were treated with the medium alone (−), and where indicated, were stimulated by anisomycin (10 μg/ml), bFGF (10 ng/ml), or EGF (10 ng/ml) for 5 min before lysis. This experiment was carried out four times with similar results, and representative results are shown. (C) Controls confirmed that an equivalent amount of PAK protein was immunoprecipitated from the III1-C treated and control cells.