Figure 9. Class I and class III PI3K inhibitors rapidly inactivate SGK3 and evidence that SGK2 is not regulated by Vps34.

(A and B) U2OS stably expressing wild-type full-length SGK3 with a C-terminal FLAG-tag were treated in the absence or presence of the 1 μM VPS34-IN1 (A) or 0.5 μM GDC-0941 (B) for the indicated times. The cells were lysed and SGK3 immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies (top 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 (bottom panel). Kinase reactions are presented as means±S.D. for triplicate reaction. (C) As above except U2OS stably expressing wild-type full-length SGK2 with a C-terminal FLAG-tag were treated in the absence or presence of the indicated inhibitors for 1 h. Similar results was obtained in at least two separate experiments for all data shown in this Figure. ***P≤0.001, **P≤0.01 and *P≤0.05. ns, not significant.