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. 2006 Feb 13;172(4):497-504.
doi: 10.1083/jcb.200505079. Epub 2006 Feb 6.

A Novel GTPase, CRAG, Mediates Promyelocytic Leukemia Protein-Associated Nuclear Body Formation and Degradation of Expanded Polyglutamine Protein

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Free PMC article

A Novel GTPase, CRAG, Mediates Promyelocytic Leukemia Protein-Associated Nuclear Body Formation and Degradation of Expanded Polyglutamine Protein

Qingyu Qin et al. J Cell Biol. .
Free PMC article

Abstract

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Figures

Figure 1.
Figure 1.
NI body formation of CRAG by response to UV irradiation. (a) Amino acid sequence of CRAG. CRAG contains a Q-rich domain (dots), a Ras homology domain (blue), and an NLS sequence (red). (b) Comparison of CRAG with the related GTPase proteins centaurin-γ3 and phosphatidylinositol 3-kinase enhancer, short isoform. A, ankyrin repeat; P, proline-rich domain; PH, pleckstrin homology domain. (c) Northern blot analysis of CRAG in mouse tissues. The arrowhead indicates the position of CRAG mRNA. (d) NIs of CRAG in UV-irradiated hippocampal neurons. Cells were stimulated with or without a pulse of UV irradiation at 100 J/m2 and after 10 min fixed and immunostained with anti-CRAG antibody (green) and Hoechst 33258 (blue). The arrowheads indicate CRAG inclusions in the nucleus. (e) NLS-dependent and GTPase-independent NIs of CRAG. HeLa cells expressing HA-CRAG of WT, NLS, or GTPase mutants were treated with or without a pulse of UV irradiation at 200 J/m2. After 2 h, cells were fixed and immunostained with anti-HA antibody (green) and Hoechst 33258 (blue). (f) Spontaneous NIs of GFP-CRAG. HeLa cells expressing GFP-CRAG WT and NLS- and GTPase-deficient mutants were stained with Hoechst 33258 (blue). Bars, 20 μm.
Figure 2.
Figure 2.
CRAG modulates PML function and dynamics. (a) Active CRAG colocalized with and induced a large ring-like structure of PML body. UV-stimulated DRG neurons were immunostained with anti-PML (red), anti-CRAG (green), and Hoechst 33258 (blue). HeLa cells expressing HA-CRAG WT or GTPase mutants were immunostained with anti-PML (green), anti-HA (red), and Hoechst 33258 (blue). Likewise, unstimulated HeLa cells coexpressing HA-PML with GFP-CRAG WT or GTPase mutants were immunostained with anti-HA (red) and Hoechst 33258 (blue). Measurement of colocalization was performed by counting the cells showing colocalization of GFP-CRAG to PML on 100 cells. The percentages are calculated from three independent experiments. Error bars represent SD. n = 3.(b) Association of endogenous CRAG with PML in response to UV stimulation. Lysates of control or UV-irradiated hippocampal neurons (100 J/m2 for 10 min) were immunoprecipitated with anti-CRAG in the presence or absence of 10 μg/ml antigen peptide against anti-CRAG or with anti-PML antibody. Immunoprecipitates (IP) were immunoblotted with the indicated antibodies. (c) GFP-CRAG associates with PML in a GTPase-dependent manner. Lysates or immunoprecipitates with anti-GFP antibody from HeLa cells transfected with indicated vectors were immunoblotted with anti-HA (top) or anti-GFP antibody (bottom). (d) Colocalization of GFP-CRAG WT, but not GTPase mutants, with ubiquitin signals. HeLa cells expressing GFP-CRAG WT or GTPase mutants were immunostained with anti-ubiquitin antibody (red). (e) GFP-CRAG induced ubiquitin ligase activity in PML immunoprecipitates. Immunoprecipitates of HA-PML WT or HA-PML RING-finger (RF) mutants from HeLa cells cotransfected with or without GFP-CRAG were used for an in vitro ubiquitin ligase assay containing His-tagged ubiquitin (described in Materials and methods) and immunoblotted with the indicated antibodies. (f) GFP-CRAG enhanced in vivo ubiquitination. Ubiquitinated proteins were immunoprecipitated from HeLa cells coexpressing HA-PML and Flag-ubiquitin with or without GFP-CRAG and analyzed by immunoblot with anti-HA and anti-Flag. Bars, 20 μm.
Figure 3.
Figure 3.
Association of CRAG with polyQ requires ROS generation. (a) Accumulation of CRAG NI in the brain of an MJD patient. A brain slice (pons) obtained from an MJD patient was immunostained with anti-CRAG antibody. The arrowhead indicates the NI of CRAG. (b) Colocalization of CRAG with polyQ in the NIs. HeLa cells expressing GFP-Q12 or -Q69 are shown as control. HeLa cells coexpressing GFP-Q12/HA-CRAG or GFP-Q69/HA-CRAG were stained with anti-HA antibody (red) and Hoechst 33258 (blue). (c) Association of CRAG with GFP-Q69 but not -Q12. HeLa cells transfected with the indicated constructs were immunoprecipitated with anti-HA antibody (left) or anti-GFP antibody (right) and immunoblotted with anti-GFP or anti-HA antibodies. (d) Association of endogenous CRAG with GFP-Q69 but not -Q12. DRG neuronal cells transfected with the indicated constructs were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-CRAG antibodies and anti-GFP antibodies. (e) ROS scavenger blocked Q69-mediated CRAG activation. COS-7 cells were transfected with the indicated vectors. After 24 h of incubation with or without ROS scavenger Radicut (100 μg/ml), CRAG was immunoprecipitated with anti-HA antibody and an in vitro GTPase assay was performed. (f) ROS scavenger blocked colocalization of CRAG with Q69. COS-7 cells were cotransfected with GFP-Q69 and HA-CRAG in the presence of 100 μg/ml Radicut. After 24 h, an immunofluorescence assay was performed with anti-HA (red) and Hoechst 33258 (blue). (g) ROS scavenger blocked the interaction of CRAG with GFP-Q69. COS-7 cells were cotransfected with GFP-Q69 and HA-CRAG in the presence of the indicated concentration of Radicut. After 24 h, CRAG was immunoprecipitated with anti-HA and immunoblotted with anti-GFP and anti-HA antibodies. Bars, 20 μm.
Figure 4.
Figure 4.
CRAG promotes nuclear translocation and degradation of polyQ through the ubiquitin–proteasome pathway. (a) Establishment of DOX-inducible Tet-On HeLa cell lines expressing HA-CRAG WT and NLS mutants. Two clones each were treated with or without 1 μM DOX for 24 h and immunoblotted with anti-HA or anti-tubulin antibodies. Cells transiently transfected with control vector, CRAG WT, or NLS mutants were indicated in the first two lanes. (b) NLS-dependent nuclear translocation of Q69. At 24 h after transient transfection of Tet-On HeLa cells with Q69-myc (DOX + 0 h), cells were treated with DOX for the indicated times and immunostained with anti-myc (green), anti-HA antibodies (red), and Hoechst 33258 (blue). Merged images are shown in insets. (c) Biochemical analysis of nuclear translocation of Q69 by CRAG. Cytosolic (C) and nuclear (N) fractions from HeLa cells transfected with the indicated vectors were separated and immunoblotted with anti-HA, anti-myc, and anti–nuclear mitotic apparatus protein (NuMA; nuclear marker) antibodies. The optical density of Q69 bands (bottom) was analyzed by the NIH Image software. (d) Effect of CRAG WT and NLS mutants on the nuclear translocation of Q69. The percentage of cells showing nuclear translocation of Q69 was calculated from 100 HeLa cells transfected with the indicated vectors. (e) CRAG-dependent disappearance of Q69. HA-CRAG–inducible Tet-On HeLa cells were transfected with Q69-myc. At 24 h after transient transfection, cells were treated with or without 1 μM DOX for the indicated times and immunoblotted with anti-HA, anti-myc, and anti-tubulin antibodies. DOX alone did not affect the Q69 protein level in Tet-On HeLa cells without CRAG (WT; right). Note that CRAG expression did not affect the Q12 protein level. (f) MG132 blocked the CRAG-induced Q69 degradation. COS-7 cells expressing Q69-myc or Q69-myc/HA-CRAG were treated with or without 10 μM MG132 for 6 h and immunoblotted with anti-HA and anti-myc antibodies. (g) CRAG suppressed Q69-induced cell toxicity. Apoptosis in Tet-On HeLa cells without (left) or with (right) CRAG in the presence or absence of DOX was determined by FACS analysis using annexin-V/propidium iodide staining (right). Apoptotic cells were collected in the top right box. (h) GFP-CRAG enhanced in vivo ubiquitination of Q69. GFP-Q69 was immunoprecipitated from cells coexpressing GFP-Q69 and Flag-ubiquitin with or without GFP-CRAG and analyzed by immunoblot with the indicated antibodies. Bars, 20 μm.
Figure 5.
Figure 5.
Knockdown of CRAG in neuronal cells blocked nuclear translocation of polyQ and polyQ-mediated cell death. (a) Effect of siRNA oligonucleotides on CRAG expression in COS-7 cells and neurons. Three different siRNA oligonucleotides against CRAG sequence (411–429, 525–543, and 698–716) were synthesized and transfected with HA-CRAG into COS-7 cells. Scramble oligonucleotide (SC) was used as a negative control. (left) The arrowhead indicates the position of CRAG. SC and siRNA (No. 1) were transfected into hippocampal neurons (HN). Inhibitory effect on CRAG expression was estimated by immunoblot with the indicated antibodies. The optical density of CRAG bands was analyzed by the NIH Image software. (b) Knockdown of endogenous CRAG by siRNA. Hippocampal neurons were fixed at 24 h after transfection of siRNA (No.1) plus pEGFP vector and immunostained with anti-CRAG antibody (red). Asterisks and arrowheads indicate CRAG-deficient cell body and neurite, respectively. (c) Effect of siRNA-mediated CRAG depletion on nuclear translocation of Q69. Mouse DRG neurons were cotransfected with Q69-myc and scramble or CRAG siRNA. After 24 h of incubation, an immunofluorescence assay was performed with anti-CRAG (red), anti-myc antibodies (green), and Hoechst 33258 (blue). Arrows and arrowheads indicate CRAG-deficient and not deficient cells, respectively. (d) Knockdown of CRAG blocks nuclear translocation of Q69 and promotes Q69-induced cell death. The percentage of cells exhibiting nuclear translocation of Q69 was measured from 100 DRG neurons expressing Q69 (top). An immunofluorescence assay was performed as described in panel c and the percentage of cell death at 48 h after transfection was measured from 100 DRG neurons expressing Q69, judged by chromatin condensation with Hoechst 33258 staining (bottom). Error bars represent SD. n = 3. (e) Schematic model of CRAG action on polyQ. Bars, 20 μm.

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