Fig. 5: Patients-associated mutations of PDK1 confer to PDK1 membrane location and AKT kinase activation.
From: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions
a A schematic presentation of the patients-associated PDK1 mutations occurred around S6K1-mediated PDK1 phosphorylation or 14-3-3γ binding region. b IB analysis of WCL and IP products derived from 293 T cells transfected with the indicated constructs. c IB analysis of WCL and GST-pull-down derived from 293 T cells transfected with GST-14-3-3γ and the indicated constructs. d IB analysis of PIP3 pull-down products and WCL derived from 293 T cells transfected with Flag-PDK1(WT, R544K, R546P, S549N, P551A, P551L). e IB analysis of cell fractionations separated from 293 T cells transfected with the indicated constructs. f PDK1/Tubulin or AIF ratio in e were calculated, (mean ± SD, n = 3), *P < 0.05. g Representative immunofluorescence images of 293 T cells transfected with the indicated constructs, scale bar, 10 μm. h Mean PDK1 fluorescence intensity at plasma membrane relative cytosol was determined, data represent mean ± SD, P = 0.022, 0.036, 0.011, 0.035, 0.026, 0.030. Greater than 60 cells were analyzed from 3 independent experiments. i IB analysis of WCL and IP products derived from 293 T cells transfected with HA-AKT1 and the indicated construct. j AKT1/PDK1 ratio in i were calculated, (mean ± SD, n = 3), *P < 0.05. k IB analysis of WCL derived from 293T-PDK1 knockout cells transfected with the indicated constructs. l pT308-AKT/AKT1 ratio in k were calculated, (mean ± SD, n = 3), *P < 0.05, **P < 0.01. Similar results were obtained in n ≥ 3 independent experiments in b, c, d. Statistical significance was determined by two-tailed Student’s t-test in f, h, j, l. *P < 0.05, **P < 0.01. Source data are provided in Source Data files. EV, empty vector. WCL, whole cell lysate. IP, immunoprecipitation. WT, wild type. PD, pulldown. Cyto, cytoplasm. Memb, membrane.
