Figure 3.

Requirement of FAK Tyr397 phosphorylation for FAK-mediated cell proliferation and migration in gastric carcinoma cells. A: A fivefold excess of exogenous FAK or mutant FAK relative to endogenous FAK was expressed in AGS cells via an adenovirus-based gene expression system harboring a GFP reporter, as described in the Materials and Methods section. The amount of pY397 was dramatically elevated in cells in which wild-type FAK was overexpressed, whereas it was absent in cells overexpressing either the Y397F mutant or FRNK. B: AGS cells overexpressing wild-type FAK, the Y397F mutant, or FRNK were subjected to BrdU incorporation assays as described in the Materials and Methods section. Results show the mean ± SD of the relative percentage of BrdU-positive cells from at least three independent experiments. C: Cell migration assays were performed with AGS cells overexpressing wild-type FAK, the Y397F mutant, or FRNK. The capability of cells to migrate toward fibronectin (10 and 20 μg/ml) or EGF (25 ng/ml) was measured using a modified Boyden chamber as described in the Materials and Methods section. Compared to cells expressing wild-type FAK or GFP alone, cell migration was impaired in Y397F- and FRNK-overexpressing cells. Cell migration was quantified by counting the number of cells that migrated toward the chemoattractants in the bottom wells. Mean cell counts from at least nine fields of polycarbonate membranes (under an Olympus IX71 20X objective microscopic lens) and three experiments are shown. Error bars represent ± SD. *P ≤ 0.05, as compared with GFP controls; **P < 0.01 as compared with wild-type FAK.