Fig. 5. Adhesion of MDCK cells to fibronectin activates HA-SGK1 in a wortmanin-insensitive manner. (A) Time course of HA-SGK1 activation upon cell adhesion to fibronectin-coated plates. Transfected MDCK cells were allowed to bind to fibronectin-coated plates, and HA-SGK1 activity was measured at the indicated times as described in the Materials and Methods. The insert depicts the level of HA-SGK1 present in the assay. (B) Adhesion-dependent activation of AKT. Cell extracts prepared as in (A) and were analyzed for the presence of phosphorylated AKT (pAKT). (C) Adhesion-mediated activation of HA-SGK1 is not blocked by wortmanin. HA-SGK1 activation by adhesion to fibronectin coated plates was studied in the presence or absence of wortmanin. After 30 minutes of adhesion to fibronectin, SGK1 activity was measured as in (A). The insert depicts the level of HA-SGK1 present in the assay. (D) Adhesion-dependent activation of AKT is blocked by wortmanin. Cells extracts were prepared as in (C) and were analyzed for the presence of phosphorylated AKT (T473) (pAKT).