Fig. 1. Activation of SGK1 by HGF is wortmanin dependent. (A) Time course of SGK1 activation by HGF. MDCK cells were transiently transfected with HA-SGK1, stimulated with HGF and at the indicated times, cells were harvested and HA-SGK1 activity was measured as described in the Materials and Methods. The lower insert depicts the amount of HA-SGK1 present in the assay at the indicated times. (B) Time course activation of AKT by HGF. Cell extracts prepared as in (A) were separated in a SDS-PAGE and analyzed for the presence of phosphorylated AKT (T473)(pAKT) or total AKT as described in the Materials and Methods. (C) HGF activation of HA-SGK1 or AKT is blocked by wortmanin. MDCK cells were transfected with HA-SGK1, stimulated with HGF for 10 minutes in the presence or absence (DMSO) of wortmanin, and the kinase activity associated with HA-SGK1 was measured as in (A). The lower insert depicts the amount of phosphorylated AKT (T473)(pAKT), total AKT and HA-SGK1 present under the conditions described. Despite HGF-treated extract being under-loaded in the AKT gel, it contains detectable levels of pAKT.