Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) All three members of human TIM family interact with NUR77 as shown by coimmunoprecipitation assay. N=3 independent experiments (B) Top panel: Domain architecture of TIM-1. Middle Panel: Coimmunoprecipitation experiment demonstrating that only the entire extracellular domain (EED) and IgV domain (IgVD) of TIM-1 interact with NUR77 and not the mucin (MD) or the cytoplasmic domain (CD). Bottom panel: long exposure of the input blot showing the CD protein band. N=2 independent experiments. (C) The transactivation function of NUR77 is repressed by all three human TIM proteins in a dose dependent manner (Top panel). Full length TIM-1 protein is required for repression of NUR77 transcriptional activity (Bottom panel). Data from at least three independent experiments is presented. (D) Binding of NUR77 to its response elements (NBRE) is abrogated by the full length TIM proteins in an electrophoretic mobility shift assay using p32 labeled NBRE oligo. Competition with unlabelled probe (cold competition) reveals the specificity of NUR77 binding. A representative autoradiograph is shown (left) and the relative amount of NUR77 protein binding to NBRE probe was quantified from three independent experiments (right). ***, P<0.001, **, P<0.01, *,P<0.05.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Co-Immunoprecipitation Assay, Activity Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Autoradiography, Protein Binding

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) Quantitative RT-PCR measurement of NUR77 transcript induced by PMA and ionomycin treatment in control and TIM-1 shRNA stable knockdown clones of 769-P cells (N=3 experiments). (B) Western analysis of the protein samples from (A) indicating the decrease in NUR77 abundance in the presence of TIM-1. Con, control. N= 3 independent experiments. (C) Overexpression of TIM-1 in HK-2 cells resulted in decreased NUR77 protein abundance induced by desferrioxamine (Des) (compare lanes: 3 and 4), which is perturbed by chloroquine treatment (CQ) (Lanes: 7 and 8). N=3 independent experiments. (D) Silencing of TIM-1 expression in HK-2 cells resulted in increased cell death in an in vitro epithelial cell injury model induced by a combination of ATP and glucose depletion and calcium overload. N= 3 independent experiments. IRI, Ischemia reperfusion injury (E) Confocal microscopy analysis of the localization pattern of endogenous hTIM-1 (green) in 769-P cells (Top panel) and GFP-tagged hTIM-1 stably expressed in Jurkat cells (Bottom panel). Scale bar, 10 µm. N=2 experiments. (F) Colocalization of ectopically expressed TIM-1 (green) in Cos-7 cells with markers of early endosomes (EEA1), Golgi complex (Giantin), and lysosomes (Lamp2a). Scale bar, 5 µm. Colocalization coefficients: hTIM-1/EEA1:0.34±0.06; hTIM-1/Giantin: 0.74±0.04; hTIM-1/Lamp2a: 0.55±0.08; n=5 cells each from two independent experiments (G). Colocalization of TIM-1 (blue) and ectopically expressed NUR77 (green) with lysosomes (Lysotracker red) in 769-P cells. Colocalization coefficients: hTim-1/NUR77: 0.59±0.12; hTIM-1/Lysotracker: 0.47±0.09; NUR77/Lysotracker: 0.51±0.08; and HK-2 cells; Colocalization coefficients: hTim-1/NUR77: 0.52+−.06; hTIM-1/Lysotracker: 0.56+0.08; NUR77/Lysotracker: 0.62+0.07; n=5 cells each from two independent experiments. Scale bar, 10 µm.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Quantitative RT-PCR, shRNA, Clone Assay, Western Blot, Over Expression, Expressing, In Vitro, Confocal Microscopy, Stable Transfection

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) Left panel: Mutations in the MILIBS of TIM-1 abrogated TIM-1-mediated degradation of NUR77 protein (top). The double mutant of cytoplasmic tyrosine residues Y299 and Y335 partially restored the protein abundance of NUR77, but did not affect that of GFP (bottom). Right panel: Quantitation of NUR77 and GFP abundance in the presence of wild type and mutant TIM-1 based on three independent experiments. (B) Confocal microscopy analysis of the localization pattern of wild type, MILIBS, cytoplasmic tyrosine residue mutants of TIM-1, revealing increased cell surface localization of the MILIBS TIM-1 mutants. Scale bar, 5 µm (Left). Quantification of the localization of wild-type TIM-1 and the MILIBS mutant. N=10 cells each from three independent experiments (Right). (C) Inability of MILIBS mutants to mediate NUR77 degradation does not arise from lack of interaction with NUR77: Wild type and MILIBS mutant TIM-1 interact with NUR77 with equal efficiency demonstrated by GST pull down assay (Middle panel). IgV and mucin domains served as positive and negative controls of the interaction between TIM-1 and NUR77. Shown is a representative of three independent experiments.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Mutagenesis, Quantitation Assay, Confocal Microscopy, Pull Down Assay

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) A form of TIM-1 lacking the signal peptide deleted TIM-1 is not glycosylated and unable to mediate degradation of NUR77. N= 2 independent experiments. (B) Perturbation of endocytic pathway using a dominant negative Dynamin construct (Dyn2K44A) did not affect TIM-1 mediated degradation of NUR77. N= 3 independent experiments (Left panel) and quantitation of protein abundance based on three independent experiments (Right panel). (C) Dominant negative constructs of Eps15 (DIII and D3delta2) substantially abrogated TIM-1 mediated degradation of NUR77. N=3 independent experiments (left panel). NUR77 protein abundance was quantified based on three independent experiments (right panel). (D) TIM-1 is constitutively endocytosed as revealed by the increased cell surface localization of transiently transfected TIM-1 in 293T cells, upon blockade of endocytosis using dominant negative constructs of dynamin-2 and Eps15 as assayed by flow cytometry. Perturbation of clathrin vesicle formation further enhanced the increased cell surface localization of the MILIBS mutant. Shaded histogram represents the control antibody staining and the open histogram represents hTIM-1 staining. Two peaks of TIM-1 staining reveal the dynamic cycling of TIM-1. N= 2 independent experiments.

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Dominant Negative Mutation, Construct, Quantitation Assay, Transfection, Flow Cytometry, Mutagenesis, Staining

Journal: Science signaling

Article Title: TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77 *

doi: 10.1126/scisignal.2003200

Figure Lengend Snippet: (A) Confocal immunofluorescence analysis of TIM-1 (green) colocalization with Clathrin (Top panel) and Caveolin-1 (Bottom panel) where yellow indicates colocalization. Scale bar, 10 µm. Colocalization coefficients: hTIM-1/Clathrin: 0.68±0.05; hTIM-1/Caveoli: 0.12±0.04; n=5 cells each from two independent experiments. (B). Retrograde translocation of TIM-1(green) from cell surface to lysosomes and endoplasmic reticulum through the trans-Golgi network in a clathrin-mediated pathway demonstrated by endocytosis assay; subcellular organelle markers are in red. Shown is a representative image. Scale bar, 10 µm. Colocalization coefficients: hTIM-1/EEA1: 0.57±0.05; hTIM-1/p230: 0.35±0.08; hTIM-1/Lamp2a (30 min): 0.46±0.09; hTIM-1/Clathrin: 0.38±0.06; hTIM-1/Lamp2a (60 min): 0.55±0.03; hTIM-1/Calnexin: 0.31±0.03; n=5 cells each from two independent experiments. (C to E) Model depicting the clathrin-mediated constitutive trafficking of TIM-1 and the phenomenon of TIM-1 mediated NUR77 degradation. TIM-1 and other TIM family members (red) are targeted to cell surface (C), prior to constitutive endocytosis through clathrin-coated vesicles (D). Following fusion with the trans-golgi network, TIM-1 undergoes retrograde translocation to endoplasmic reticulum (ER) and lysosomes (purple circles). TIM-1 might come in contact with NUR77 possibly in ER or vesicles leading to recruitment of this protein complex to PS-rich endosomes and lysosomes depending on the TIM-1-PS interaction, facilitating sorting of NUR77 to lysosomes and ultimately resulting in the degradation of NUR77 by lysosomal enzymes. TIM-1 is presumably retained in the vesicles (E).

Article Snippet: Western blotting and quantification of proteins Protein concentrations were quantified using Bio-rad DC kit and equal amounts of protein samples were separated on either 8% or 4–12% polyacrylamide gel and blotted onto PVDF membrane and probed with different antibodies as indicated (Anti-human NUR77 (Pharmingen), Anti-mouse NUR77 (BD), Anti-human TIM-1 (R&D), Anti-HA (Upstate/Millipore corp), Anti-β actin (Abcam), Anti-GFP (Abcam).

Techniques: Immunofluorescence, Translocation Assay, Endocytosis Assay