Fig. 3: S6K1-mediated phosphorylation of PDK1 inhibits PDK1 membrane localization and PIP3 binding.
From: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions
a The indicated Flag-PDK1 constructs with/without HA-S6K1-R3A were transfected into 293 T cells and anti-Flag immunoprecipitation was recovered as the kinase source to phosphorylate insect cells purified His-AKT1 in vitro. pT308-AKT/AKT1 ratio were calculated. (mean ± SD, n = 3). b IB analysis of WCL and GST-pulldown derived from 293 T cells transfected with GST-PDK1, Flag-AKT1 and increasing dose of HA-S6K1-R3A. pS549-PDK1/PDK1 ration were calculated. (mean ± SD, n = 3, P = 0.0001, 0.0002, 7.82E-10). c IB analysis of WCL and IP products derived from 293 T cells transfected with Flag-PDK1(WT, S549A, S549D) and HA-AKT1. Indicated protein were quantified. (mean ± SD, n = 3, P = 0.005, 0.004). d IB analysis of PIP3 pull-down products and WCL derived from 293 T cells transfected with the indicated constructs. Where indicated, empty beads (EV) serve as a negative control. e IB analysis of PIP3 pull-down products and WCL derived from HeLa cells transfected with Flag-PDK1 that were serum-starved for 24 h and then collected after insulin (100 nM) stimulation for 30 min, where indicated, the kinase inhibitors were added. f IB analysis of cell fractionations separated from 293 T cells transfected with indicated constructs. PDK1/Tubulin or AIF ratio were calculated. (mean ± SD, n = 3, P = 2.98E-06, 0.002). g Representative immunofluorescence images of 293 T cells transfected with indicated constructs, scale bar, 10 μm. Mean PDK1 fluorescence intensity at plasma membrane relative cytosol was determined, data represent mean ± SD, P = 0.008, 0.03. Greater than 60 cells were analyzed from 3 independent experiments. Similar results were obtained in n ≥ 3 independent experiments in d, e. Statistical significance was determined by two-tailed Student’s t-test in b, c, f. g N.S > 0.05, *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.
