15 research outputs found
RNAi-mediated ablation of KIN-E caused a dramatic change of cell morphology.
<p>(<b>A</b>). Western blotting to monitor the efficiency of RNAi against KIN-E. KIN-E was endogenously tagged with a triple HA epitope in cells containing the KIN-E RNAi construct. TbKIN-E-3HA was detected by anti-HA antibody. TbPSA6, the alpha6 subunit of the 26S proteasome, served as the loading control. (<b>B</b>). Effect of KIN-E RNAi on cell proliferation. (<b>C</b>). Percentages of 1N1K cells of trypomastigote and epimastigote-like morphology in control and KIN-E RNAi cells. Cell morphology was determined based on the position of the kinetoplast relative to the nucleus as shown in panel D below. A total of 100 1N1K cells were counted for each time point. Error bars indicated S.D. from three independent experiments. (<b>D</b>). Cell morphology of control and KIN-E RNAi cells examined under a light microscope. N, nucleus; K, kinetoplast DNA. Scale bar: 5 μm. (<b>E</b>). Cell morphology of control and KIN-E RNAi cells examined by scanning electron microscopy. Shown in panels a, c, and e are control cells, whereas panels b, d, and f are KIN-E RNAi cells. The arrow in panel b indicates the long, unattached flagellum. Arrows in panels d and f indicate the long, unattached new flagellum, and the arrowhead in panel f indicates the long, unattached old flagellum. Scale bars: 5 μm. (<b>F</b>). Effect of KIN-E RNAi on cell cycle progression examined by quantitation of cells with different numbers of nucleus (N) and kinetoplast DNA (K). A total of 120 cells were counted for each time point. Error bars indicated S.D. from three independent experiments. (<b>G</b>). Flow cytometry analysis of EP procyclin expression in control and KIN-E RNAi cells. Cells were immunostained with anti-EP procyclin and FITC-conjugated mouse IgG, and then analyzed by flow cytometry. (<b>H</b>). Western blotting to assess the level of EP procyclin in control and KIN-E RNAi cells. EP procyclin was detected with anti-EP procyclin mAb and FITC-conjugated anti-mouse IgG, whereas TbPSA6 was detected with anti-TbPSA6 pAb and IRDye 680LT anti-rabbit IgG. (<b>I</b>). Quantitation of the level of EP procyclin detected by western blotting in panel H. Error bars indicate S.D. calculated from three independent western blotting experiments. **, <i>p</i><0.01; ***, <i>p</i><0.001.</p
Analysis of the structural motifs in KIN-E, an orphan kinesin in <i>T</i>. <i>brucei</i>.
<p><b>(A).</b> Schematic illustration of the structural motifs in KIN-E. The putative nucleotide-binding motif and the putative microtubule-binding motif of the kinesin motor domain of KIN-E were aligned with that of other orphan kinesins reported previously. The conserved glycine, lysine and threonine/serine residues in the P-loop of the nucleotide-binding motif were highlighted in red. mCL, m-Calpain domain III-like domain. CC, coiled coil. (<b>B</b>). Alignment of the putative importin α-like domain in KIN-E with the importin α protein from <i>Saccharomyces cerevisiae</i> (PBD code: 1BK5). The α-helical structures were indicated at the top of the aligned sequences. (<b>C</b>). Homology modeling of the importin α-like domain in KIN-E, using the <i>S</i>. <i>cerevisiae</i> importin α protein (PBD code: 1BK5) as the template. Note that the importin α-like domain in KIN-E is only about half size of the <i>S</i>. <i>cerevisiae</i> importin α protein. (<b>D</b>). Alignment of the m-calpain domain III-like domains (mCL#1 and mCL#2) of KIN-E with the domain III of the human m-calpain protein (PBD code: 1KFU). The α-helix structures and the β-sheet structures were indicated at the top of the aligned sequences. (<b>E</b>). Homology modeling of the m-calpain domain III-like domains in KIN-E, using the human m-calpain domain III (PBD code: 1KFU) as the template.</p
KIN-E localizes to the flagellum and is enriched at the flagellar tip.
<p>(<b>A</b>). KIN-E localizes to the distal tip of the flagellum. KIN-E was endogenously tagged with a triple HA epitope, and detected with FITC-conjugated anti-HA antibody. Cells were co-immunostained with anti-PFR2 antibody (L8C4), and counterstained with DAPI for nuclear and kinetoplast DNA. Scale bar: 5 μm. (<b>B</b>). KIN-E localizes to the flagella connector region. Cells co-expressing endogenously 3HA-tagged KIN-E and PTP-tagged FC1 were co-immunostained with FITC-conjugated anti-HA monoclonal antibody and anti-Protein A polyclonal antibody, and counterstained with DAPI for DNA. Scale bar: 5 μm. (<b>C</b>). Subcellular localization of ectopically expressed KIN-E and its various mutants in the 29–13 cell line. Wild-type KIN-E, the importin α-like domain deletion mutant (KIN-E-ΔIMPα), and the m-calpain domain III (mCL#1& mCL#2) deletion mutant (KIN-E-ΔmCL) were each tagged with a triple HA epitope at the C-terminus and ectopically expressed in the 29–13 cell line. Cells were incubated with 1.0 μg/ml tetracycline for 24 h and immunostained with FITC-conjugated anti-HA mAb. Arrows indicate the enrichment of KIN-E and KIN-E-ΔIMPα at the flagellar tip, and the arrowhead indicates KIN-E-ΔmCL at the posterior tip. Scale bar: 5 μm.</p
Morphological analysis of the epimastigote-like cells generated by KIN-E RNAi.
<p>(<b>A</b>). Morphology of 1N1K cells from control and KIN-E RNAi cells. 1N1K cells from the control and KIN-E RNAi-induced population were co-immunostained with anti-CC2D antibody to label the FAZ filament and with anti-PFR2 (L8C4) antibody to label the flagellum. Cells were counterstained with DAPI for nuclear (N) and kinetoplast (K) DNA. The cartoon depicted a 1N1K cell used for measuring the length of cell body, flagellum, unattached flagellum, FAZ filament, kinetoplast (K) to posterior cell tip, and kinetoplast to nucleus (N). Scale bar: 5 μm. (<b>B</b>). Morphometric measurement of uninduced control cells and KIN-E RNAi-induced (24 h) cells. 1N1K cells from control and KIN-E RNAi cells (+Tet, 24 h) were immunostained with anti-CC2D and anti-PFR2 antibodies, and the length of the cell body, the FAZ, the flagellum, the unattached flagellum, kinetoplast to nucleus, and kinetoplast to the posterior tip were measured and plotted (<i>n</i> = 101 for control cells and <i>n</i> = 104 for RNAi cells). ***, <i>p</i><0.001; ns, no statistical significance. (<b>C</b>). Position of the flagellar basal body in control and KIN-E RNAi cells. Cells (1N1K) were co-immunostained with YL 1/2 antibody to label the mature basal body (mBB) and with anti-TbSAS-6 antibody to label both the mBB and the pro-basal body (pBB), and then counterstained with DAPI for nuclear and kinetoplast DNA. Scale bar: 5 μm. (<b>D</b>). Positions of the bilobed structure and flagellar pocket in control and KIN-E RNAi cells. Cells (1N1K) were co-immunostained with 20H5 antibody to label the bilobed structure and with anti-CRAM antibody to label the flagellar pocket (FP), and then counterstained with DAPI for nuclear and kinetoplast DNA. Scale bar: 5 μm.</p
The structural motifs in KIN-E required for FLAM3 interaction and localization.
<p>(<b>A</b>). Co-immunoprecipitation to examine the requirement of KIN-E structural motifs for interaction with FLAM3. FLAM3 was endogenously tagged with an N-terminal PTP epitope in KIN-E-3’UTR RNAi cells expressing 3HA-tagged wild-type and mutant KIN-E proteins. PTP-FLAM3 was precipitated by IgG beads, and the immunoprecipitated proteins were immunoblotted with anti-HA antibody to detect 3HA-tagged KIN-E and its mutants and with anti-Protein A (α-ProtA) antibody to detect PTP-FLAM3. Cells expressing PTP-FLAM3 only and KIN-E-3HA only were included as controls. (<b>B</b>). <i>In vitro</i> interaction of the importin-α-like domain of KIN-E with FLAM3. Shown are the GST pull-down results using purified recombinant GST-fused importin-α-like domain (IMPα) and m-calpain domain III-like domain (mCL) as the baits. GST alone served as the negative control. FLAM3 was tagged with an N-terminal PTP epitope and detected by anti-Protein A (α-ProtA) antibody, whereas GST and GST fusion proteins were detected by Coomassie Brilliant Blue (CBB) staining. (<b>C</b>). The requirement of KIN-E structural motifs for FLAM3 localization. The same cell lines used in panel A were used for immunofluorescence microscopy. Non-induced (-Tet) and tetracycline-induced (+Tet, 24 h) cells were immunostained with anti-Protein A (α-ProtA) pAb to detect PTP-FLAM3 and FITC-conjugated anti-HA mAb to detect 3HA-tagged KIN-E, KIN-E-ΔIMPα, or KIN-E-ΔmCL. Scale bar: 5 μm. (<b>D</b>). Subcellular distribution of FLAM3 protein in KIN-E-3’UTR RNAi cells expressing wild-type and mutant KIN-E proteins. Un-induced and tetracycline-induced cells were lysed in PEME buffer containing 1% NP-40. Cell lysate was spun down to separate cytosolic soluble (S) fraction and cytoskeletal pellet (P) fraction for western blotting with anti-Protein A antibody to detect PTP-FLAM3 and with anti-HA antibody to detect 3HA-tagged KIN-E and its mutants in the two fractions. The same membrane was re-probed with anti-α-tubulin antibody and anti-PSA6 antibody to serve as cytoskeleton and cytosol markers, respectively.</p
KIN-E RNAi disrupted the elongation of the new FAZ and the migration of the kinetoplast/basal body towards cell posterior.
<p>(<b>A</b>). Morphology of 2N2K cells from control and KIN-E RNAi cells. 2N2K cells from control and KIN-E RNAi-induced population were immunostained with anti-CC2D and anti-PFR2 (L8C4) antibodies to label the FAZ and the flagellum, respectively. Cells were counterstained with DAPI to stain nuclear (N) and kinetoplast (K) DNA. NF, new flagellum; OF, old flagellum. Scale bar: 5 μm. (<b>B</b>). Morphometric measurement of uninduced control cells and KIN-E RNAi-induced (24 h) cells. 2N2K cells from control and KIN-E RNAi cells (+Tet, 24 h) were immunostained with anti-CC2D and anti-PFR2 antibodies. The length of the cell body, the new FAZ, the old FAZ, the new and old flagella, the unattached new and old flagella, posterior kinetoplast to cell posterior distance, and inter-kinetoplast distance were measured and plotted (<i>n</i> = 103 for control cells and <i>n</i> = 97 for RNAi cells). ***, <i>p</i><0.001; ns, no statistical significance. (<b>C</b>). Position of the flagellar basal body in control and KIN-E RNAi cells. Shown are 2N2K cells that were co-immunostained with YL 1/2 antibody to label the mature basal body (mBB) and with anti-TbSAS-6 antibody to lable the total basal body (BB) that is composed of mBB and pro-basal body (pBB). Cells were then counterstained with DAPI for nuclear and kinetoplast DNA. Scale bar: 5 μm. (<b>D</b>). Positions of the bilobe structure and the flagellar pocket in control and KIN-E RNAi cells. Shown are 2N2K cells that were co-immunostained with 20H5 antibody and anti-CRAM antibody to label the bilobe structure and the flagellar pocket (FP), respectively, and then counterstained with DAPI for nuclear and kinetoplast DNA. Scale bar: 5 μm.</p
The structural motifs required for KIN-E function.
<p>(<b>A</b>). Subcellular localization of ectopically expressed KIN-E and its various mutants in KIN-E 3’UTR RNAi cell line. Wild-type KIN-E, the importin α-like domain deletion mutant (KIN-E-ΔIMPα), and the m-calpain domain III (mCL#1& mCL#2) deletion mutant (KIN-E-ΔmCL) were each tagged with a triple HA epitope at the C-terminus and ectopically expressed in KIN-E-3’UTR RNAi cell line. Arrows indicate KIN-E and KIN-E-ΔIMPα at the flagellar tip, whereas the arrowhead indicates KIN-E-ΔmCL at the posterior tip. Scale bar: 5 μm. (<b>B</b>). Western blotting to monitor the knockdown of endogenous KIN-E, which was tagged with an N-terminal PTP epitope, and ectopically expressed wild-type and mutant KIN-E proteins, which were tagged with a C-terminal triple HA epitope. TbPSA6 served as the loading control. (<b>C</b>). Complementation of KIN-E-3’UTR RNAi by 3HA-tagged KIN-E and its mutants. Shown are the growth curves of KIN-E-3’UTR RNAi cell line and the KIN-E-3’UTR RNAi cell lines expressing wild-type or mutant KIN-E proteins at an ectopic locus. (<b>D</b>). Quantification of 1N1K cells of trypomastigote morphology and epimastigote-like morphology in KIN-E-3’UTR RNAi cell line and RNAi complementation cell lines. A total of 100 1N1K cells were counted for each time point, and three repeats were performed. Error bars indicate S.D.</p
DYRK2 Negatively Regulates Type I Interferon Induction by Promoting TBK1 Degradation via Ser527 Phosphorylation
<div><p>Viral infection activates the transcription factors NF-κB and IRF3, which contribute to the induction of type I interferons (IFNs) and cellular antiviral responses. Protein kinases play a critical role in various signaling pathways by phosphorylating their substrates. Here, we identified dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) as a negative regulator of virus-triggered type I IFN induction. DYRK2 inhibited the virus-triggered induction of type I IFNs and promoted the K48-linked ubiquitination and degradation of TANK-binding kinase 1 (TBK1) in a kinase-activity-dependent manner. We further found that DYRK2 phosphorylated Ser527 of TBK1, which is essential for the recruitment of NLRP4 and for the E3 ubiquitin ligase DTX4 to degrade TBK1. These findings suggest that DYRK2 negatively regulates virus-triggered signaling by targeting TBK1 for phosphorylation and priming it for degradation, and these data provide new insights into the molecular mechanisms that dictate the cellular antiviral response.</p></div
DYRK2 promoted TBK1 degradation via K48-linked ubiquitination.
<p>(A) Overexpression of DYRK2 induced TBK1 degradation in a dose-dependent manner. The 293T cells (2×10<sup>5</sup>) were transfected with the Flag-TBK1, HA-β-actin and HA-DYRK2 plasmids (0.1, 0.2 or 0.4 μg) and were treated with dimethyl sulfoxide (DMSO) or MG132. The cells were lysed, and the lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibodies. (B) Overexpression of wild-type DYRK2 but not mutant DYRK2, promoted the ubiquitination of TBK1. The 293 cells (1×10<sup>7</sup>) were transfected with the indicated plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-TBK1 antibody. The immunoprecipitates were analyzed by immunoblotting with an anti-Myc antibody (upper). Protein expression was analyzed by immunoblotting with the indicated antibodies (lower). (C) Effects of DYRK2 RNAi on the SeV-induced ubiquitination of endogenous TBK1. The 293 cells (5×10<sup>7</sup>) were transfected with control or DYRK2 RNAi (#2) plasmids. Twenty hours after transfection, the cells were infected or not infected with SeV for 10 h. The cell lysates were immunoprecipitated with an anti-TBK1 antibody. The immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody (top). The expressions of related proteins were examined by immunoblotting with the indicated antibodies (bottom). (D) DYRK2 promoted K48-linked but not K63-linked ubiquitination of TBK1. The 293 cells (2×10<sup>6</sup>) were transfected with HA-tagged Lys-48-only or Lys-63-only ubiquitin plasmids and the other indicated plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-TBK1 antibody and then analyzed by immunoblotting with an anti-HA antibody (upper panel). The expressions of related proteins were examined by immunoblotting with the indicated antibodies (lower panel).</p
Effects of RNAi-mediated knockdown of DYRK2 on SeV-induced signaling and IRF3 activation.
<p>(A) Effects of DYRK2 RNAi on the expression of transfected and endogenous DYRK2. In the upper panel, 293 cells (2×10<sup>5</sup>) were transfected with expression plasmids for Flag-DYRK2 and HA-NEK6 (0.1 μg each) and the indicated RNAi plasmids (1 μg). Twenty-four hours after transfection, the cell lysates were analyzed by immunoblotting with the indicated antibodies. In the lower panel, the 293 cells (2×10<sup>5</sup>) were transfected with the control or indicated DYRK2 RNAi plasmids (1 μg each) for 36 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Effects of DYRK2 RNAi on the SeV-induced activations of the ISRE, NF-κB and the IFN-β promoter. The 293 cells (1×10<sup>5</sup>) were transfected with the ISRE, NF-κB or IFN-β promoter reporters (0.05 μg) and the indicated RNAi plasmids (0.5 μg each) for 36 h and then infected or not infected with SeV for 10 h before the luciferase assays were performed. The graphical data are presented as the means ± the SDs (n = 3). (C) Knockdown of DYRK2 promoted SeV-induced IRF3 dimerization. The 293 cells (2×10<sup>5</sup>) were transfected with control or DYRK2 RNAi (#2) plasmids (1 μg). Thirty-six hours after transfection, the cells were infected with or without SeV for 0, 4, 8, or 12 h. The cell lysates were separated by native (top) or SDS (bottom) PAGE, and the blots were analyzed using the indicated antibodies. (D) Effects of DYRK2 RNAi on the SeV-induced endogenous gene transcriptions of IFNB1 and RANTES. The 293 cells (2×10<sup>5</sup>) were transfected with the indicated RNAi plasmids (1 μg each) for 36 h and then infected or not infected with SeV for 10 h before reverse transcription PCR was performed. (E) Effects of DYRK2 RNAi on SeV-, VSV- and HSV-1-induced transcriptions of IFNB1 and RANTES in THP-1 cells. RNAi-transduced stable THP-1 cells (2×10<sup>5</sup>) were infected or not infected with SeV/VSV/HSV-1 for 8 h before qPCR was performed. (F) Effects of DYRK2 RNAi on SeV- and HSV-1-induced secretion of IFN-β. RNAi-transduced stable THP-1 cells (1×10<sup>5</sup>) were infected with SeV or HSV-1 for 12 h. The culture medium was collected for quantitation of the indicated cytokines by ELISA. (G) Effects of DYRK2 RNAi on virus replication. RNAi-transduced stable THP-1 cells (1×10<sup>5</sup>) were mock-transfected or transfected with poly(I:C) (1μg) for 16 h and then infected with VSV or HSV-1 (MOI = 0.1). The supernatants were harvested 24 h after infection for standard plaque assays.</p