Figure 6.

PKA activation at the leading edge is independent of PI3-kinase activity and involved in GFP-PH-Akt accumulation during cell migration. (A) CHO-α4 cell monolayers on CS1 expressing pmAKAR3 were wounded and then treated with 30 μM LY294002 for 15 min and imaged in the continued presence of the inhibitor. (B) Wounded monolayers of GFP-PH-Akt–expressing CHO-α4 cells on CS1 were fixed and imaged to visualize accumulation of PtdIns(3,4,5)P₃. (C and D) CHO-α4 cell monolayers adhered on CS1 were wounded, allowed to migrate for 15 min, and then treated with 30 μM LY294002, 30 μM H89, or left untreated for another 15 min before fixation and staining for C phosphorylated PKA substrates [p(S/T)substrate PKA] and (D) phosphorylated α4 (phospho-α4) by using the appropriate antibodies. Bottom, magnified and merged images of the regions bounded by yellow rectangles. (E) CHO-α4 cells were adhered to fibronectin-coated wells for the indicated times, lysed, fractionated by SDS-PAGE, and immunoblotted to detect Akt phosphorylation at serine 473 (pS473-Akt). The blot was reprobed with antibody recognizing total Akt protein. LY, LY294002. (F) To ensure the efficacy of H89 inhibition of PKA, cells were treated with 50 μM Fsk in the presence or absence of H89 and immunoblotted with phospho-PKA substrate antibody.