Fig. 1. PP2 inhibits β1 integrin-induced, but not EGF-induced, Akt kinase activity. GD25β1A, GD25β1B (A), and SYF cells (B) were stimulated either by EGF or adhesion to invasin, and the induced Akt kinase activity toward the GST-GSK-3 fusion protein was determined in vitro in a non-radioactive Akt kinase assay (see Materials and methods). Cells were pre-treated with PP2 as indicated.

Fig. 2. PP2 inhibits β1 integrin-induced, but not EGF-induced, phosphorylation of Ser473 and Thr308 of Akt. (A) GD25β1A and GD25β1B cells, pre-treated with PP2 as indicated, were stimulated with EGF or allowed to adhere to the β1 integrin ligand invasin. The phosphorylation of Akt Thr308 and Ser473 was analyzed by western blotting. (B) FAK−/− and SYF cells were stimulated by β1 integrin-mediated adhesion, and the phosphorylation of Akt Thr308 was analyzed by western blotting.

Fig. 3. Tyrosine phosphorylation of Akt is induced by EGF receptor but not by β1 integrins, in a Src-family kinase-dependent manner. (A) GD25β1A and GD25β1B cells, pre-treated with PP2 as indicated, were stimulated with EGF and whole cell lysates were analyzed for pTyr326 by western blotting. (B) GD25β1A and GD25β1B cells were stimulated by adhesion via the β1 integrins. Akt was immunoprecipitated from the cells and the immune complexes were analyzed for pTyr326 by western blotting. (C) SYF cells were stimulated with EGF as indicated, and analyzed for pTyr326 and pThr308 (as control) by western blotting.

Fig. 4. EGF receptor is not involved in the integrin-induced phosphorylation of Akt. (A) Dose-dependent inhibition of EGF-induced activation of Akt phosphorylations by ZD1839 (Gefitinib) in GD25β1A cells. (B, C) Lack of inhibition by ZD1839 (Gefitinib) of Akt phosphorylations after β1 integrin-stimulation in GD25β1A (B), GD25β1B, and FAK−/− cells (C).

Fig. 5. The EGF-dependent phosphorylation of Akt is independent of adhesion. Suspended GD25β1A, GD25β1B, and FAK−/− cells were incubated in the absence or presence of EGF. The presence of Ser473-phosphorylated Akt was detected by western blotting.

Fig. 6. A hypothetical model for EGF receptor and integrin-mediated activation of Akt. The activation of Akt by EGF receptor involves the Src-family kinase-mediated tyrosine phosphorylation. β1 integrins do not induce Akt tyrosine phosphorylation, but are proposed to engage an upstream PP2-sensitive tyrosine kinase in Akt activation. This suggests that EGF receptor and β1 integrins regulate Akt via different signaling pathways, which can potentially lead to different Akt-dependent cellular responses.