Figure 3. SGK3 activity is induced by growth factors, insulin and oncogenic Ras.

(A) HEK293 cells were serum-starved for 16 h prior to stimulation with or without the indicated concentrations of IGF1, EGF, insulin, FGF, TPA, H₂O₂ and serum for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM ³²PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies. (B) As in (A), except that HEK293 cells were transiently transfected when 60% confluent, with KRAS[G12C], KRAS[G12D], NRAS[G12D], ERBB2[V842I] and EGFR[L833R] (all HA epitope tagged). Forty eight hours later, WT and transfected cells were serum-starved for 16 h before cells lysed without further stimulation. (C) As in (B), except that HEK293 cells were transiently transfected with KRAS[G12C] and KRAS[G12D] and treated as indicated with VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) prior to stimulation with 50 ng/ml IGF1 for 10 min. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.