Fig. 4: 14-3-3 binds phosphorylated PDK1 and dissociates it from the cytoplasm membrane.
From: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions
a IB analysis of WCL and IP products derived from 293 T cells transfected with Flag-PDK1 and the indicated various HA-tagged 14-3-3 constructs. b, c DLD1 cell lysates were subjected to IP with control IgG, anti-PDK1 or 14-3-3γ antibodies for IB analysis. d A schematic presentation of the evolutionarily conserved putative AGC kinase phosphorylation consensus in PDK1 and 14-3-3 recognized consensus. e IB analysis of WCL and IP products derived from 293 T cells transfected with indicated constructs. f IB analysis of WCL and IP products derived from 293 T cells transfected with HA-14-3-3γ and indicated constructs. g IB analysis of PIP3 pull-down products and WCL derived from 293 T cells transfected with the indicated constructs. h IB analysis of WCL and GST-pulldown derived from 293 T cells transfected with the indicated constructs. i IB analysis of WCL derived from 293 T cells transfected with the indicated Flag-PDK1 constructs that were serum-starved for 12 h and then treated with insulin (100 nM) for the indicated time periods before collection for IB analysis. pT308-AKT/AKT1 ratio were calculated. (mean ± SD, n = 3, P = 0.0003). j IB analysis of the indicated fractionations derived from the gel filtration experiment with 293 T cells transfected with Flag-PDK1. k IB analysis of WCL and GST-pull-down derived from 293 T control or 14-3-3γ knockdown cells transfected with GST-PDK1 and Flag-PDK1. l IB analysis of WCL derived from 293 T control or 14-3-3γ knockdown cells. m IB analysis of WCL derived from 293 T control or 14-3-3γ knockdown cells that were serum-starved for 12 h and then treated with insulin (100 nM) for the indicated time periods before collection for IB analysis. pT308-AKT/AKT1 ratio were calculated. (mean ± SD, n = 3, P = 0.0001). n Proposed model for 14-3-3 involved in the PDK1/AKT pathway regulation. Red arrows indicate positive regulation, and blue arrows indicate negative regulation. Similar results were obtained in n ≥ 3 independent experiments in a, b, c, e, f, g, h. Statistical significance was determined by two-way ANOVA in i, m. **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. Scr, scramble.
