Figure 7. SGK3 phosphorylation and kinase activity is controlled by its ability to bind PtdIns(3)P.

(A) U2OS stably expressing wild-type or indicated mutant of full-length SGK3 with a C-terminal FLAG-tag were treated in the absence or presence of the indicated PDK1 inhibitor (1 μM GSK2334470) for 1 h. The cells were lysed and SGK3 immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies (lower panel) after being assayed for SGK3 kinase activity by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ-³²P]ATP in a 30 min reaction (upper panel). Kinase reactions are presented as means±S.D. for triplicate reaction. The similar result was obtained in at two separate experiments. ****P≤0.0001.