Journal: eLife

Article Title: Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes

doi: 10.7554/eLife.70714

Figure Lengend Snippet: Kidneys were harvested from wild-type (WT) and Lepr db/db diabetic mice (db/db) that were treated with placebo (vehicle) or DMOG ( A–B, E, G ), and from non-diabetic control (Ctrl) or diabetic (Db) wild-type (WT) and Egln1 +/- mice ( C–D, F, H ). ( A and C ) HIF-1α (green), pimonidazole (red, hypoxia marker) and DAPI (blue, nuclear staining) signals were detected by fluorescent immunohistochemistry, and relative HIF-1α expression levels were quantified ( A , n = 4–5; C , n = 4–6). Scale bar: 100 μm. ( B and D ) Renal ROS levels were detected using the OxiSelect HNE adduct competitive ELISA kit ( B , n = 7–10; D , n = 5–8). ( E and F ) Mitochondrial respiratory function was evaluated using high resolution respirometry ( E , n = 4–9; F , n = 11–17). ( G and H ) PDK1 gene expression in kidneys ( G , n = 4–9; H , n = 4–6). ( I and J ) mIMCD-3 cells were transfected with plasmids encoding GFP or GFP-PDK1,and exposed to hypoxia and 30 mM glucose (H30) for 24 hr. ( I ) Expression of GFP and GFP-HIF-1α (green) and nuclear DAPI staining (blue) were detected using confocal microscopy. Scale bar: 50 μm. ( J ) Mitochondrial ROS levels are shown (n = 4). Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 using one-way ANOVA ( A, B, E, G ) and two-way ANOVA ( C, D, F, H ) followed by multi-comparison post hoc tests, and unpaired two-sided Student t-test ( J ). This figure has three figure supplements. Source data are shown in . Figure 4—source data 1. HIF-1α, ROS, and mitochondrial respiration levels in mouse kidneys and PDK1 gene expression and Mitosox intensity in mIMCD3 cells.

Article Snippet: Recombinant DNA reagent , pCMV3-FLAG-PDK1 , Sino Biological Inc , Cat#: HG12312-NF , Plasmid encoding FLAG-tagged human PDK1.

Techniques: Control, Marker, Staining, Immunohistochemistry, Expressing, Competitive ELISA, Transfection, Confocal Microscopy, Comparison

Journal: eLife

Article Title: Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes

doi: 10.7554/eLife.70714

Figure Lengend Snippet: Under non-diabetic conditions (left panel), HIF-1 is induced by hypoxia and activates PDK1 expression which inhibits excess mitochondrial ROS production through inhibition of mitochondrial respiration. However, under diabetic conditions (right panel), HIF-1 is inhibited by high glucose levels through a PHD-dependent mechanism despite hypoxia. This results in decreased expression of PDK1, leading to increased mitochondrial respiration and excessive mitochondrial ROS production which causes tissue damage.

Article Snippet: Recombinant DNA reagent , pCMV3-FLAG-PDK1 , Sino Biological Inc , Cat#: HG12312-NF , Plasmid encoding FLAG-tagged human PDK1.

Techniques: Expressing, Inhibition

Journal: eLife

Article Title: Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes

doi: 10.7554/eLife.70714

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCMV3-FLAG-PDK1 , Sino Biological Inc , Cat#: HG12312-NF , Plasmid encoding FLAG-tagged human PDK1.

Techniques: Transfection, Construct, Sequencing, Negative Control, Recombinant, Plasmid Preparation, Reporter Assay, Caspase-Glo Assay, Live Cell Imaging, Competitive ELISA, DC Protein Assay, Reverse Transcription, SYBR Green Assay, Bradford Protein Assay, In Situ, Protease Inhibitor, Software, Modification

Journal: Cell systems

Article Title: Four key steps control glycolytic flux in mammalian cells

doi: 10.1016/j.cels.2018.06.003

Figure Lengend Snippet:

Article Snippet: PDK1: PDK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002610","term_id":"1677556781","term_text":"NM_002610"}} NM_002610 ) Human cDNA Clone , Origene , Cat# SC321678.

Techniques: Recombinant, Modification, Transfection, Clone Assay, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Lactate Dehydrogenase Assay, Bicinchoninic Acid Protein Assay, PK Assay, Plasmid Preparation, Sequencing, Expressing, Variant Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-141 and MicroRNA-146b-5p Inhibit the Prometastatic Mesenchymal Characteristics through the RNA-binding Protein AUF1 Targeting the Transcription Factor ZEB1 and the Protein Kinase AKT *

doi: 10.1074/jbc.M114.593004

Figure Lengend Snippet: AUF1 binds and stabilizes the PDK1 mRNA. A, whole cell lysates were prepared from the indicated cells and used for immunoblotting analysis utilizing antibodies against the indicated proteins. B, total RNA was prepared from the indicated cells and utilized to amplify the indicated transcripts by qRT-PCR using specific primers. C, U2OS and EH1 cells expressing the indicated constructs were treated with actinomycin D and then reincubated for the indicated periods of time. Total RNA was extracted, and the remaining amount of the PDK1 mRNA was assessed using qRT-PCR. The dashed lines indicate the PDK1 mRNA half-life. Error bars, S.E. values of three different experiments. D, biotinylated PDK1 3′-UTR bearing either wild type or mutated sequence of the AUF1 binding site was incubated with cytoplasmic cellular lysate from the indicated cells, and the association of AUF1 with these RNAs was detected by immunoblotting using anti-AUF1 antibody. E, U2OS cells expressing AUF1 siRNA or a control plasmid were stably transfected with the luciferase reporter vector bearing either the wild-type PDK1 3′-UTR or a mutated sequence for the binding site of AUF1 (residues 556–562). The reporter activity was assessed at 48 h post-transfection. Data (mean ± S.E., n = 4) were presented as a percentage change in reporter activity as compared with the negative control cells (*) or with the wild-type 3′-UTR (**). * and **, p < 0.00003.

Article Snippet: U2OS cells were plated at 1 × 10 5 cells/well on 6-well plates and transfected with 3 μg of the luciferase/ Renilla reporter vector containing either human AUF1 3′-UTR (871 bp), mutated sequence of the miR-141 or miR-146b-5p seed sequence, human ZEB1 3′-UTR (75 bp), mutated sequence of the AUF1 binding site in the corresponding sequence, human PDK1 3′-UTR, or the mutated sequence of the AUF1 binding site as well as a control sequence with no AU-rich conserved elements (GeneCopoeia).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Construct, Sequencing, Binding Assay, Incubation, Control, Plasmid Preparation, Stable Transfection, Transfection, Luciferase, Activity Assay, Negative Control

Journal: Cell Reports

Article Title: Pak1 Kinase Maintains Apical Membrane Identity in Epithelia

doi: 10.1016/j.celrep.2018.01.060

Figure Lengend Snippet: An RNAi Screen for Cdc42 Effectors Contributing to Epithelial Polarization Identifies Pak1 (A) RNAi knockdown of Wasp-Arp2/3 complex, Pak3, Pak4, or MRCK/Gek does not have polarity phenotype, whereas Pak1 knockdown causes a partial epithelial polarity disruption. (B) The cdc42 3 mutant phenotype is similar to but stronger than that of Pak1 loss of function. Note that RNAi knockdown of Pak1 or induction of pak1 14 null mutant clones throughout the epithelium causes a mild disruption of aPKC. (C) Pak1-GFP is recruited to the plasma membrane by active Cdc42. Coexpression of Cdc42 V12 with Pak1-GFP results in translocation of Pak1-GFP from cytoplasmic punctae to the plasma membrane. (D) Epithelial polarity is not affected in follicle cells expressing RNAi against β-integrins (mys) or in the triple Rac mutant (rac1, rac2, mtl).

Article Snippet: For in vitro kinase assays, high-pressure liquid chromatography (HPLC)-purified peptide substrates were incubated with either 150 pg recombinant human Pak1 kinase domain (AbNOVA) or 0.1 μM recombinant human aPKCiota kinase domain (a gift from N. McDonald) for 30 min at 30°C in kinase reaction buffer (250 mM HEPES [(4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid] pH 7.5, 50 mM MgCl 2 , 5 mM EGTA, 0.05% Brij35) containing 10 μM cold ATP and 3 μCi Y-P32 ATP (PerkinElmer).

Techniques: Knockdown, Disruption, Mutagenesis, Clone Assay, Clinical Proteomics, Membrane, Translocation Assay, Expressing

Journal: Cell Reports

Article Title: Pak1 Kinase Maintains Apical Membrane Identity in Epithelia

doi: 10.1016/j.celrep.2018.01.060

Figure Lengend Snippet: aPKC and Pak1 Act Redundantly to Maintain Apical Domain Identity (A–C) aPKC (A), Crb-GFP (B), and Baz/Par3 (C) localize apically in control follicle cells treated with DMSO, but are partially lost upon treatment with either aPKC kinase inhibitors or Pak1 kinase inhibitors, and are completely lost upon treatment with both inhibitors. (D) aPKC localizes apically in control follicle cells, is partially lost in aPKC psu265 mutant follicle cells or upon RNAi knockdown of Pak1, and is completely lost upon RNAi knockdown of Pak1 in aPKC psu265 mutant follicle cells. (E) RNAi knockdown of Pak1 in aPKC psu265 mutant follicle cells (GFP + cells) causes the loss of aPKC from the apical domain and the ectopic spreading of Dlg. (F) RNAi knockdown of Pak1 in myr-Lgl-GFP expressing follicle cells (an inhibitor of aPKC kinase) causes the loss of aPKC from the apical domain.

Article Snippet: For in vitro kinase assays, high-pressure liquid chromatography (HPLC)-purified peptide substrates were incubated with either 150 pg recombinant human Pak1 kinase domain (AbNOVA) or 0.1 μM recombinant human aPKCiota kinase domain (a gift from N. McDonald) for 30 min at 30°C in kinase reaction buffer (250 mM HEPES [(4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid] pH 7.5, 50 mM MgCl 2 , 5 mM EGTA, 0.05% Brij35) containing 10 μM cold ATP and 3 μCi Y-P32 ATP (PerkinElmer).

Techniques: Control, Mutagenesis, Knockdown, Expressing

Journal: Cell Reports

Article Title: Pak1 Kinase Maintains Apical Membrane Identity in Epithelia

doi: 10.1016/j.celrep.2018.01.060

Figure Lengend Snippet: aPKC and Pak1 Both Contribute to Phosphorylation of Known aPKC Targets Such as Baz and Lgl (A) Consensus motifs for aPKC and Pak1 are similar, and both kinase domains (KDs; top) can phosphorylate known aPKC targets (Crb, Par6, Baz/Par3, Lgl) in vitro (bottom). (B) Phospho-Baz (p-Baz) staining of UAS.Baz-GFP expressing follicular epithelia in the presence of aPKC inhibitor, Pak1 inhibitor, or both. Note that loss of p-Baz requires inhibition of both kinases. (C) Lgl localization in UAS.Lgl-GFP expressing follicular epithelia in the presence of aPKC inhibitor, Pak1 inhibitor, or both. Note that apical localization of Lgl requires inhibition of both kinases.

Article Snippet: For in vitro kinase assays, high-pressure liquid chromatography (HPLC)-purified peptide substrates were incubated with either 150 pg recombinant human Pak1 kinase domain (AbNOVA) or 0.1 μM recombinant human aPKCiota kinase domain (a gift from N. McDonald) for 30 min at 30°C in kinase reaction buffer (250 mM HEPES [(4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid] pH 7.5, 50 mM MgCl 2 , 5 mM EGTA, 0.05% Brij35) containing 10 μM cold ATP and 3 μCi Y-P32 ATP (PerkinElmer).

Techniques: Phospho-proteomics, In Vitro, Staining, Expressing, Inhibition

Journal: Cell Reports

Article Title: Pak1 Kinase Maintains Apical Membrane Identity in Epithelia

doi: 10.1016/j.celrep.2018.01.060

Figure Lengend Snippet: Requirement for aPKC and Pak1 Kinases in Mammalian Colonic Epithelial Cell Polarity (A) Schematic diagram of aPKC and Pak1 activation by Cdc42. Note that two aPKC zeta/iota homologs and Pak1 localize apically in human colonic epithelia. (B) Mouse intestinal organoids grown in Matrigel and treated with both aPKC and Pak1 inhibitors exhibit loss of epithelial polarity more strongly in the double treatments than in the single treatments. (C) aPKC and Pak 1 inhibitor treatment in 3D Caco-2 cultures results in disorganized Caco-2 epithelial cysts. (D) Cdc42 siRNA in 2D plated Caco-2 cells results in a strong disruption of the apical marker ZO-1. (E) Double siRNA of aPKC and Pak1 results in a much stronger effect on ZO-1 than single knockdowns in Caco-2 cells, thus mimicking Cdc42 depletion. (F) Quantification of the effect of various siRNA treatments on the organization of ZO-1 and immunoblots showing the efficiency of the Pak1 and aPKC knockdowns in Caco-2 cells. (G) aPKC and Pak 1 inhibitor phenocopies siRNA treatment in 2D Caco-2 cultures.

Article Snippet: For in vitro kinase assays, high-pressure liquid chromatography (HPLC)-purified peptide substrates were incubated with either 150 pg recombinant human Pak1 kinase domain (AbNOVA) or 0.1 μM recombinant human aPKCiota kinase domain (a gift from N. McDonald) for 30 min at 30°C in kinase reaction buffer (250 mM HEPES [(4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid] pH 7.5, 50 mM MgCl 2 , 5 mM EGTA, 0.05% Brij35) containing 10 μM cold ATP and 3 μCi Y-P32 ATP (PerkinElmer).

Techniques: Activation Assay, Disruption, Marker, Western Blot

Journal: PLoS ONE

Article Title: Identification of Direct Target Engagement Biomarkers for Kinase-Targeted Therapeutics

doi: 10.1371/journal.pone.0026459

Figure Lengend Snippet: A ) Regulation of enzyme activity by reversible protein phosphorylation. Because the phosphorylation state of kinases often correlates with enzymatic activity, monitoring site-specific phosphorylation events are attractive biomarkers for measuring drug-target inhibition. B ) Previously reported phosphorylation sites on PDK1.

Article Snippet: Acquired MS data were uploaded to the Elucidator data analysis system (Rosetta Biosoftware, version 3.5) as previously described , for feature extraction, quantitative analysis and searched against a human PDK1 database obtained from National Center for Biotechnology Information (NCBI) http://pubchem.ncbi.nlm.nih.gov/ using SEQUEST.

Techniques: Activity Assay, Phospho-proteomics, Inhibition

Journal: PLoS ONE

Article Title: Identification of Direct Target Engagement Biomarkers for Kinase-Targeted Therapeutics

doi: 10.1371/journal.pone.0026459

Figure Lengend Snippet: A ) The T-loop phosphorylation site on PDK1 Ser241 is not modulated by PDK1 inhibitor treatment in PC3 cells whereas downstream substrates, such as p-AKT Thr308 and p-RSK Ser221 , are affected. B ) In PC3 cells the inhibition of p-RSK Ser221 correlates with the enzymatic potency of PDK1 inhibitors (N = 147). C ) LNCap cells don't exhibit a correlation between inhibition of p-AKT Thr308 , a bona fide PDK1 substrate (N = 279), and the D ) enzymatic potency of PDK1 inhibitors (N = 82). E ) Western blot of p-AKT Thr308 and p-RSK Ser221 in a variety of cell lines. F) PDK1 substrates are subject to feedback regulation and pathway cross-talk.

Article Snippet: Acquired MS data were uploaded to the Elucidator data analysis system (Rosetta Biosoftware, version 3.5) as previously described , for feature extraction, quantitative analysis and searched against a human PDK1 database obtained from National Center for Biotechnology Information (NCBI) http://pubchem.ncbi.nlm.nih.gov/ using SEQUEST.

Techniques: Phospho-proteomics, Inhibition, Western Blot

Journal: PLoS ONE

Article Title: Identification of Direct Target Engagement Biomarkers for Kinase-Targeted Therapeutics

doi: 10.1371/journal.pone.0026459

Figure Lengend Snippet: T293 cells grown in either light ( 12 C-Arg, 12 C-Lys) or heavy ( 13 C-Arg, 13 C-Lys) medium were lysed and combined at a 1∶1 ratio based on total protein quantity. PDK1 was immunoprecipitated and analyzed by mass spectrometry. MS raw data were quantified by the Elucidator software 3.5 and confirmed by manual validation as described in the .

Article Snippet: Acquired MS data were uploaded to the Elucidator data analysis system (Rosetta Biosoftware, version 3.5) as previously described , for feature extraction, quantitative analysis and searched against a human PDK1 database obtained from National Center for Biotechnology Information (NCBI) http://pubchem.ncbi.nlm.nih.gov/ using SEQUEST.

Techniques: Immunoprecipitation, Mass Spectrometry, Software, Biomarker Discovery

Journal: PLoS ONE

Article Title: Identification of Direct Target Engagement Biomarkers for Kinase-Targeted Therapeutics

doi: 10.1371/journal.pone.0026459

Figure Lengend Snippet: A ) SDS-PAGE and isolation of the PDK1 protein band for LC-MS/MS analysis. B ) PDK1 tool compounds representing three structurally distinct chemical classes. PDK1 enzymatic activity is shown (EC 50 ). C ) Mapping of phosphorylation sites of immunoprecipitated PDK1 from 293T cells identifies 12 Ser/Thr phosphorylation sites with ∼95% sequence coverage. D ) Relative quantification by SILAC-IAP-MS showed a greater than 4 fold reduction of two phosphorylation sites (pS410, and pT513) in samples treated with PDK1 inhibitors compared to samples treated with DMSO. In contrast, no change in phosphorylation was detected in the self-to-self control sample where 293T cells grown in heavy and light media were both treated with DMSO prior to mixing. E ) MS/MS spectra for pS241 (ANpSFVGTAQYVSPELLTEK), pS410 (DTGLPQRSGpSNIEQYIH), and pT513 (NFKpTFFVHTPNR) containing peptides.

Article Snippet: Acquired MS data were uploaded to the Elucidator data analysis system (Rosetta Biosoftware, version 3.5) as previously described , for feature extraction, quantitative analysis and searched against a human PDK1 database obtained from National Center for Biotechnology Information (NCBI) http://pubchem.ncbi.nlm.nih.gov/ using SEQUEST.

Techniques: SDS Page, Isolation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Phospho-proteomics, Immunoprecipitation, Sequencing, Quantitative Proteomics, Multiplex sample analysis, Control, Tandem Mass Spectroscopy