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. 2011 Sep 30;286(39):33879-89.
doi: 10.1074/jbc.M111.234997. Epub 2011 Aug 8.

CRMP5-associated GTPase (CRAG) Protein Protects Neuronal Cells Against Cytotoxicity of Expanded Polyglutamine Protein Partially via c-Fos-dependent Activator protein-1 Activation

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CRMP5-associated GTPase (CRAG) Protein Protects Neuronal Cells Against Cytotoxicity of Expanded Polyglutamine Protein Partially via c-Fos-dependent Activator protein-1 Activation

Shun Nagashima et al. J Biol Chem. .
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Abstract

We previously demonstrated that CRAM (CRMP5)-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, facilitated degradation of expanded polyglutamine protein (polyQ) via the nuclear ubiquitin-proteasome pathway. Taking advantage of this feature, we also showed that lentivirus-mediated CRAG expression in the Purkinje cells of mice expressing polyQ resulted in clearance of the polyQ aggregates and rescue from ataxia. However, the molecular basis of the function of CRAG in cell survival against polyQ remains unclear. Here we report that CRAG, but not centaurin-γ3, induces transcriptional activation of c-Fos-dependent activator protein-1 (AP-1) via serum response factor (SRF). Mutation analysis indicated that the nuclear localization signal and both the N- and C-terminal regions of CRAG are critical for SRF-dependent c-Fos activation. CRAG knockdown by siRNA or expression of a dominant negative mutant of CRAG significantly attenuated the c-Fos activation triggered by either polyQ or the proteasome inhibitor MG132. Importantly, c-Fos expression partially rescued the enhanced cytotoxicity of CRAG knockdown in polyQ-expressing or MG132-treated cells. Finally, we suggest the possible involvement of CRAG in the sulfiredoxin-mediated antioxidant pathway via AP-1. Taken together, these results demonstrated that CRAG enhances the cell survival signal against the accumulation of unfolded proteins, including polyQ, through not only proteasome activation, but also the activation of c-Fos-dependent AP-1.

Figures

FIGURE 1.
FIGURE 1.
CRAG induces AP-1 activity. A, structural comparison of CRAG with centaurin-γ3/AGAP3 (G3) and centaurin-γ2/AGAP1 short form (G2-s). NLS, nuclear localization signal; ANK, ankyrin repeat; PH, pleckstrin homology domain. B, CRAG activates AP-1. Neuro2A cells were transfected with both pAP-1-Luc and pRL-CMV together with either empty expression vector or indicated vector. Luciferase activities were assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 3). *, p < 0.05 (Student's t test). C, CRAG induces c-Fos activation. Lysates of Neuro2A cells as described above were immunoblotted with the indicated antibodies. Arrowheads indicate the positions of phosphorylated c-Fos and c-Jun. The protein levels of c-Fos and c-Jun normalized with tubulin are shown in the right panel when the control value was arbitrarily set 1.0. Error bars indicate ±S.D. (n = 4). *, p < 0.05; **, p < 0.01. D, subcellular localizations of CRAG, G3, and G2-s. Neuro2A cells transfected with either the empty vector, HA-CRAG, HA-G3, or HA-G2-s were immunostained with anti-HA (green) and Hoechst 33258 (blue). Scale bar, 5 μm.
FIGURE 2.
FIGURE 2.
Identification of CRAG domains required for AP-1 activation. A, structural comparison of CRAG wild-type (WT) with various CRAG mutants. WT, CRAG WT; ΔC, CRAGΔC 1–374 mutant; NLSm, CRAG NLS mutant K386E R369E; ΔN, CRAGΔN 60–375 mutant; GTPm, CRAG GTPase mutant. B, AP-1 activities in cells expressing CRAG WT and mutants. Neuro2A cells were transfected with both pAP-1-Luc and pRL-CMV together with either empty expression vector or each indicated vector. Luciferase activities were assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 3). *, p < 0.05; ***, p < 0.005 (Student's t test). C, effects of CRAG mutants on activations of c-Fos and c-Jun. Lysates of Neuro2A cells as described above were immunoblotted with the indicated antibodies. Arrowheads indicate the positions of phosphorylated c-Fos and c-Jun. The protein levels of c-Fos and c-Jun normalized with tubulin are shown in the right panel when the control value was arbitrarily set 1.0. Error bars indicate ±S.D. (n = 4). *, p < 0.05; **, p < 0.01. D, CRAG induces the formation of c-Fos/c-Jun heterodimer. Co-immunoprecipitation assay was performed on cells expressing vector, CRAG WT, or CRAGΔC. Cell lysates were immunoprecipitated with anti-c-Jun antibody, and the immunoprecipitates were immunoblotted with the indicated antibodies. E, subcellular localizations of CRAG WT and mutants. Neuro2A cells as described above were immunostained with anti-HA (green) antibody and Hoechst 33258 (blue). Scale bar, 5 μm.
FIGURE 3.
FIGURE 3.
CRAG mediates c-Fos-dependent AP-1 activation via SRF. A, CRAG activates SRF. G3, centaurin-γ3; G2-s, centaurin-γ2 short form. B, identification of CRAG domains required for SRF activation. WT, CRAG WT; ΔC, CRAGΔC 1–374 mutant; NLSm, CRAG NLS mutant K386E R369E; ΔN, CRAGΔN 60–375 mutant; GTPm, CRAG GTPase mutant. C, inhibitory effects of two SRF mutants and SRF cofactor MAL mutant on CRAG-induced SRF activation. D, effects of SRF inhibitions on CRAG-induced AP1 activities. (C–E) SRF cofactor MAL mutant (C471) and two SRF mutants (Δ413 and Δ338) are described under “Experimental Procedures.” A–D, Neuro2A cells were transfected with both pSRF-Luc and pRL-CMV together with either empty vector or the indicated vector. Luciferase activities were assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Student's t test). E, effects of SRF inhibitions on CRAG-induced c-Fos activations. Lysates of Neuro2A cells as described above were immunoblotted with the indicated antibodies.
FIGURE 4.
FIGURE 4.
CRAG knockdown and CRAG dominant negative mutant reduce polyQ- and MG132-induced c-Fos and AP-1 activities. A and B, effect of CRAG knockdown on polyQ-induced AP-1 (A) and c-Fos/c-Jun activations (B). C and D, effect of CRAG knockdown on MG132-induced AP-1 (C) and c-Fos/c-Jun activations (D). E and F, effect of expression of CRAGΔC (ΔC) on polyQ-induced AP-1 (E) and c-Fos/c-Jun activations (F). G and H, effect of expression of CRAGΔC on MG132-induced AP-1 (G) and c-Fos/c-Jun activations (H). Luciferase assay was performed with Neuro2A cells transfected with both pAP-1-Luc and pRL-CMV with indicated vector and/or siRNA (sc: scramble siRNA, siCRAG: CRAG-specific siRNA). For MG132 treatment, Neuro2A cells were treated with either DMSO or 10 μm MG132 for 24 h. Lysates of Neuro2A cells transfected with the indicated vector and/or siRNA were immunoblotted with indicated antibodies. The protein levels of c-Fos and c-Jun normalized with tubulin are shown in the right panel when the control value was arbitrarily set at 1.0. Error bars indicate ±S.D. (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Student's t test).
FIGURE 5.
FIGURE 5.
Expression of c-Fos partially rescued from polyQ- and MG132-induced cytotoxicity enhanced by CRAG knockdown. Effect of c-Fos on cytotoxicity enhanced by CRAG knockdown in cells expressing polyQ (A and B) and with MG132 treatment (C and D). Lysates of Neuro2A cells transfected with the indicated vector(s) were immunoblotted with the indicated antibodies. The protein levels of cleaved caspase3 normalized are shown in the upper panel when the control value was arbitrarily set at 1.0 (A and C). For MG132 treatment, Neuro2A cells were treated with either DMSO or 10 μm MG132 for 24 h. An ATP reduction assay was performed on Neuro2A cells as described above (B and D). Error bars indicate ±S.D. (n = 5). *, p < 0.05; ***, p < 0.005 (Student's t test).
FIGURE 6.
FIGURE 6.
CRAG activates antioxidative pathway via AP-1-mediated Srxn-1 activation. A, CRAG activates Srxn-1. Neuro2A cells were transfected with both Srxn-1-Luc and pRL-CMV together with either empty vector or indicated vector. Luciferase activities were assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 3). *, p < 0.05 (Student's t test). B, both AP-1 sites in the Srxn-1 promoter are required for CRAG-dependent Srxn-1 activation. There are two AP-1 sites in the Srxn-1 promoter, and three different mutants were generated as indicated in the figure. The effect of CRAG expression on luciferase activity of Srxn-1 promoter mutants was assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 4). *, p < 0.05; **, p < 0.01 (Student's t test). C, induction of Srxn-1 mRNA by CRAG. Neruro2A cells were transfected with either control vector or CRAG. At 48 h after transfection, quantitative RT-PCR was performed to quantify Srxn-1 mRNA in control and CRAG-transfected cells. D, CRAG reduces hydrogen peroxide-induced Prx-SO2/3H. Neruro2A cells were transfected with either control vector or CRAG. At 48 h after transfection, cells were treated with 200 μm hydrogen peroxide for 1 h, following incubation in the fresh medium. Cells were harvested at the indicated time. Lysates of Neuro2A cells were immunoblotted with anti-Prx-SO2/3H antibody. E, CRAG knockdown enhanced hydrogen peroxide-induced Prx-SO2/3H accumulation. Neruro2A cells were transfected with either control or CRAG-specific siRNA. At 48 h after transfection, cells were treated with 100 μm hydrogen peroxide for 1 h, following incubation in the fresh medium. Cells were harvested at the indicated time. Lysates of Neuro2A cells were immunoblotted with anti-Prx-SO2/3H antibody. F, a schematic model for the CRAG-mediated AP-1 signaling pathway.

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