Figure 2

Protein kinase activities of ILK (A) and PKBα/Akt (B) and GSK-3β phosphorylation at the amino-acid residue Ser9 (C) were examined in A549 and CCD32 cells attached to polystyrene (P) or FN at 5 or 60 min after irradiation with 6 Gy (right panels). Basal kinase activities were strongly stimulated by FN in both tumour and normal cells (left panels). Radiation-dependent increases of ILK and PKBα/Akt activity and GSK-3β phosphorylation demonstrated to be pronounced on polystyrene and less prominent on FN. Additionally, ILK, PKBα/Akt and GSK-3β protein were detected to exclude changes in total protein amounts and β-actin served as loading control (data not shown). Each data point shown represents the fold induction (means±s.d.) of protein kinase activity or Ser9 phosphorylation of GSK-3β from the densitometric protein band analysis of three independent experiments in relation to untreated controls (mock). Inset, photographic demonstration of one exemplary protein kinase assay used for densitometric analysis. To examine the dependence of ILK, PKBα/Akt activity and GSK-3β phosphorylation on the PI3-K pathway, cells attached to FN or polystyrene were incubated with the PI3-K-specific inhibitors LY294002 (50 μ M) (D) or wortmannin (100 nM) (E) in serum-free medium and then irradiated with 6 Gy (IR). The data uncovered PI3-K-dependent decrease of basal and radiation-induced kinase activities (ILK and PKBα/Akt at 5 min; GSK-3β at 60 min) in cells grown on FN or polystyrene. However, irradiation on FN was able to stimulate ILK and GSK-3β independent of PI3-K indicating the involvement of yet unknown signalling pathways.