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

Journal: The EMBO Journal

Article Title: Cellular stress promotes NOD1/2‐dependent inflammation via the endogenous metabolite sphingosine‐1‐phosphate

doi: 10.15252/embj.2020106272

Figure Lengend Snippet: A The de novo and the hydrolysis pathway of sphingolipid metabolism and inhibitors used in this study. B Lipidomic analysis of the abundance of various lipid classes in cells upon indicated stimulations. Human fibroblasts were treated with cytochalasin D (5 µM), vinblastine (10 µM), cycloheximide (25 µM), CCCP (5 µM), etoposide (5 µM), or tunicamycin (5 µg/ml) for 2 h. Cell lysates were collected for lipidomic profiling. Heatmap shows the mean of 2 independent experiments. CE: cholesteryl ester, Cer: ceramide, CL: cardiolipin, DAG, HexCer: hexosylceramide, LPA: lyso‐phosphatidate, LPC: lyso‐phosphatidylcholine, LPE: lyso‐phosphatidylethanolamine, LPE O‐: ether‐linked LPE, LPG: lyso‐phosphatidylglycerol, LPI: lyso‐phosphatidylinositol, PA: phosphatidate, PC: phosphatidylcholine, PC O‐: ether‐linked PC, PE: phosphatidylethanolamine, PE O‐: ether‐linked PE, PG: phosphatidylglycerol, PI: phosphatidylinositol, PS: phosphatidylserine, SM: sphingomyelin, and TAG: triacylglycerol. C–G qRT–PCR analysis of IL6 expression (C, E) and ELISA analysis of IL6 production (D, F, G) in THP‐1 cell (C, D), BMDMs (E, F), or human CD14 + monocytes (G) pretreated with various inhibitors against sphingolipid metabolism upon tunicamycin (5 µg/ml) stimulation. H Western blot analysis of MAPK activation in B6N WT, Sphk1 KO, and Sphk2 KO BMDMs upon tunicamycin stimulation. B6N WT, Sphk1 KO, and Sphk2 KO BMDMs were treated with tunicamycin (5 µg/ml) and at different time points, cell lysates were collected for SDS–PAGE. I Quantification of normalized protein levels of p‐p38, p‐JNK, and p‐ERK. The band intensity of each protein was measured with ImageJ and normalized to β‐actin. Then relative levels were calculated against corresponding controls (WT, Sphk1 KO, Sphk2 KO without treatment). Means ± SD of three independent experiments. J Western blot analysis of SPHK1 and SPHK2 in HeLa control (scrambled shRNA) and SPHK1/2 knockdown cells. Data information: (C‐G) Means ± SEM of three independent experiments (C‐F) or 4 different donors (G). Each dot represents one independent experiment (C‐F) or one donor (G). P values (C‐G) were calculated using one‐way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: S1P‐coated beads (Echelon Biosciences, S‐6110‐2) and sphingolipid‐coated bead pack (Echelon Biosciences, P‐B00SS), C2 ceramide, C16 ceramide, and sphingosine, were dissolved in ethanol.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, SDS Page, shRNA

Journal: The EMBO Journal

Article Title: Cellular stress promotes NOD1/2‐dependent inflammation via the endogenous metabolite sphingosine‐1‐phosphate

doi: 10.15252/embj.2020106272

Figure Lengend Snippet: A Fluidigm analysis of expression of genes involved in sphingolipid metabolism in human dermal fibroblasts (HDFs) upon indicated stimulations for 4 h. Heatmap shows means of 4 independent experiments. The color scale represents the relative gene expression compared to control from lower (blue) to higher levels (red). B Lipidomic profiling of sphingolipid metabolites in HDFs upon indicated stimulations for 2 h. Fold changes of each metabolite were calculated against corresponding controls. Heatmap shows means of three independent experiments. The color scale represents the relative levels of various lipids from lower (blue) to higher abundance (red). C ELISA analysis of cytosolic S1P production in HDFs upon indicated stimulation for 4 h. D qRT–PCR analysis of IL6 expression in HDFs upon indicated stimulations for 4 h in the absence or presence of the inhibitor of sphingosine kinases‐SKI‐II (SPHKi, 50 µM). E–G ELISA analysis of IL6 in supernatants of HDFs (E), HeLa NOD1 cells (F), or HeLa NOD2 cells (G) upon indicated stimulations for 20 h in the absence or presence of the inhibitor of sphingosine kinases. H qRT–PCR analysis of Cxcl2 expression in WT, Sphk1 KO, or Sphk2 KO BMDMs upon indicated stimulations for 4 h. I Multiplex analysis of CXCL2 production in supernatants of WT, Sphk1, and Sphk2 KO BMDMs upon indicated stimulations for 20 h. J ELISA analysis of IL8 production upon Shigella infection. HeLa scrambled control cells and SPHK1/2 double KD cells were infected with Shigella flexneri M90T for 3 and 6 h or stimulated with TNFα for 6 h. Data information: (C, E‐G, I, J) Means ± SEM of three independent experiments. Each dot represents one independent experiment. (D, H) Means ± SD of three technical replicates from one representative experiment out of three independent experiments. P values were calculated using one‐way or two‐way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: S1P‐coated beads (Echelon Biosciences, S‐6110‐2) and sphingolipid‐coated bead pack (Echelon Biosciences, P‐B00SS), C2 ceramide, C16 ceramide, and sphingosine, were dissolved in ethanol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Multiplex Assay, Infection