Fig. 2. Activation of HA-SGK1 by Rac1V12 is wortmanin independent. (A) MDCK cells were co-transfected with HA-SGK1 and either Rac1V12 (lane 1), RhoL63 (lane 2), Rap1V12 (lane3) or HA-SGK alone (lane 4), and the kinase activity associated with HA-SGK1 was assayed as described in the Materials and Methods. The results are presented as the fold increase in activation over the activity obtained in the absence of the small GTP-binding proteins. The lower insert depicts the amount of HA-SGK1 present in the assay. (B) Dose-dependent activation of HA-SGK1 by Rac1V12. MDCK cells were cotransfected with HA-SGK1, and the indicated amounts of Rac1V12 and the kinase activity was assayed as described in the Materials and Methods. The lower insert depicts the amount of HA-SGK1 present in the assay. (C) The effects of wortmanin on RacV12 activation of HA-SGK1. HA-SGK1 was cotransfected with Rac1V12, and cell extracts were prepared from wortmanin or vehicle-treated cells as described in the legend to Fig. 1C. The lower insert depicts the amount of HA-SGK1 present in the assay. (D) The HGF-mediated activation of HA-SGK1 is not blocked by RacN17. MDCK cells were cotransfected with HA-SGK and an empty vector or a vector containing myc-tagged RacN17. The amount of SGK in the kinase assay is depicted in the HA-SGK blot. Verification of myc-RacN17 expression is shown in the lower blot. The kinase activity was carried out as described in the legend to Fig. 2A.

Return to article