Enzyme activity of ITPKA is required for EGF-mediated migration. A, whole cell extracts of H1299 kd cells stably re-expressing a kinase-dead form of GFP-ITPKA (D416N+), a GFP-ITPKA fusion protein (ITPKA+), or solely GFP (kd) were probed with an anti-ITPKA antibody, and expression of ITPKA in these cells was compared with endogenous expression of ITPKA in H1299 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, H1299 control, kd, D416N+, and ITPKA+ cells were serum-starved for 24 h and seeded in Boyden chambers containing Dulbecco's modified Eagle's medium, 100 nm EGF. After 16 h of incubation, migration was measured by the transwell assay. Migration was expressed as relative amounts with respect to H1299 control cells (c) (considered as 100%). Data represent mean values ± S.D. of four independent experiments. *, p < 0.05; ***, p < 0.0001.