Journal: Nature Communications

Article Title: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions

doi: 10.1038/s41467-022-28910-8

Figure Lengend Snippet: 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 PIP 3 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 PIP 3 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.

Article Snippet: PIP 3 beads (P-B00Ss) and label-free PIP 3 (P-3908) were purchased from Echelon Biosciences.

Techniques: Construct, Transfection, Immunoprecipitation, Purification, In Vitro, Derivative Assay, Negative Control, Immunofluorescence, Fluorescence, Two Tailed Test, Plasmid Preparation

Journal: Nature Communications

Article Title: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions

doi: 10.1038/s41467-022-28910-8

Figure Lengend Snippet: 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 PIP 3 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.

Article Snippet: PIP 3 beads (P-B00Ss) and label-free PIP 3 (P-3908) were purchased from Echelon Biosciences.

Techniques: Derivative Assay, Transfection, Construct, Filtration, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions

doi: 10.1038/s41467-022-28910-8

Figure Lengend Snippet: 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 PIP 3 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.

Article Snippet: PIP 3 beads (P-B00Ss) and label-free PIP 3 (P-3908) were purchased from Echelon Biosciences.

Techniques: Binding Assay, Derivative Assay, Transfection, Construct, Immunofluorescence, Fluorescence, Knock-Out, Two Tailed Test, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions

doi: 10.1038/s41467-022-28910-8

Figure Lengend Snippet: a The canonical activation of PI3K-AKT-mTOR pathway toward growth factors, such as Insulin or EGF treatment. b The negative feedback regulations of AKT kinase by S6K1-mediated phosphorylation of SIN1, PDK1 as well as IRS1. Among which, phosphorylation of IRS1 decreases PI3K-mediated PIP 3 generation; phosphorylation of SIN1 dissociates mTORC2 from membrane location; phosphorylation of PDK1 recruits 14-3-3 and dissociates from membrane location. These pathways together tightly negatively control growth factors-induced constant activation of AKT kinase. c Patient-associated PDK1 mutations could block S6K-mediated PDK1 phosphorylation (Type I mutations) or 14-3-3-mediated PDK1 membrane dissociation (Type II mutations), leading to AKT constitutive activation and oncogenic roles, such as accelerating cell proliferation and anti-apoptosis. Red and black arrows indicate positive and negative regulation, respectively. Solid and dotted lines indicate direct and indirect (multistep) regulation, respectively.

Article Snippet: PIP 3 beads (P-B00Ss) and label-free PIP 3 (P-3908) were purchased from Echelon Biosciences.

Techniques: Activation Assay, Blocking Assay