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. 2008 Jul;10(7):866-73.
doi: 10.1038/ncb1747. Epub 2008 Jun 15.

Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis

Nilkantha Sen  1 , Makoto R Hara, Michael D Kornberg, Matthew B Cascio, Byoung-Il Bae, Neelam Shahani, Bobby Thomas, Ted M Dawson, Valina L Dawson, Solomon H Snyder, Akira Sawa
Affiliations

Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis

Nilkantha Sen et al. Nat Cell Biol. 2008 Jul.

Abstract

Besides its role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) initiates a cell death cascade. Diverse apoptotic stimuli activate inducible nitric oxide synthase (iNOS) or neuronal NOS (nNOS), with the generated nitric oxide (NO) S-nitrosylating GAPDH, abolishing its catalytic activity and conferring on it the ability to bind to Siah1, an E3-ubiquitin-ligase with a nuclear localization signal (NLS). The GAPDH-Siah1 protein complex, in turn, translocates to the nucleus and mediates cell death; these processes are blocked by procedures that interfere with GAPDH-Siah1 binding. Nuclear events induced by GAPDH to kill cells have been obscure. Here we show that nuclear GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB binding protein (CBP) through direct protein interaction, which in turn stimulates the acetylation and catalytic activity of p300/CBP. Consequently, downstream targets of p300/CBP, such as p53 (Refs 10,11,12,13,14,15), are activated and cause cell death. A dominant-negative mutant GAPDH with the substitution of Lys 160 to Arg (GAPDH-K160R) prevents activation of p300/CBP, blocks induction of apoptotic genes and decreases cell death. Our findings reveal a pathway in which NO-induced nuclear GAPDH mediates cell death through p300/CBP.

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Figures

Figure 1
Figure 1
GAPDH is acetylated in the nucleus at Lys 160 following NO stimulation. (a) GAPDH acetylation in RAW264.7 cells treated with LPS/IFNγ for 16 h is abolished by the iNOS inhibitor 1400W. Cell lysates were immunoprecipitated with an anti-acetyl Lys antibody, and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. (b) Both p300 and CBP contribute to acetylation of GAPDH in U2OS cells treated with the NO donor, GSNO. Depletion of p300 or CBP by RNAi leads to diminished acetylation of GAPDH. (c) GAPDH mutation at Lys 160 abolishes its acetylation in the presence of 200 μM GSNO in HEK293 cells. (d) Acetylation of sulphonated GAPDH (sGAPDH) is observed only in the nucleus where it requires intact Lys 160. Cytosolic or nuclear fractions of HEK293 cells were immnoprecipiated with an anti-acetyl Lys antibody, and the immunoprecipitates were analysed by western blotting with a specific anti-sulphonated-GAPDH antibody. (e), R-(−)-Deprenyl (Dep) inhibits the acetylation of GAPDH in brains of mice treated with MPTP.
Figure 2
Figure 2
GAPDH interacts with p300/CBP and GAPDH-K160R acts as a dominant-negative mutant. (a) GAPDH, native or NO-modified, binds similarly in vitro to a fragment of human p300 (F-p300) (amino acids 1135−2414). GST or GST–GAPDH was pre-treated with 50 μM GSH or GSNO for 30 min at 37 °C. F-p300 was added and binding assessed by a GSH-agarose pulldown assay. (b) GAPDH–p300 binding occurs in RAW264.7 cells treated with LPS/IFNγ for 16 h and is abolished by the iNOS inhibitor 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p300 antibody and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. (c) GAPDH and p300 are colocalized in the nucleus of RAW264.7 cells after exposure to LPS/IFNγ. Cells were stained with immunofluorescent anti-GAPDH and anti-p300 antibodies (green, p300; red, GAPDH). Scale bar, 10 μm. (d) R-(−)-Deprenyl (Dep) inhibits the enhancement of GAPDH–p300/CBP binding in mouse brain elicited by MPTP treatment. (e) K160R mutation of GAPDH abolishes GAPDH–p300 interactions in HEK293 cells. (f) GAPDH-K160R expression prevents acetylation of endogenous GAPDH and its binding to p300/CBP in a concentration-dependent manner, suggesting that GAPDH-K160R functions as a dominant-negative mutant. Forty-eight hours after transfection with 0.1, 0.5, 2, 3, or 4.5 μg of HA–GAPDH-K160R, HEK293 cells were treated with 200 μM GSNO for 24 h. Cell lysates were immunoprecipitated with anti-GAPDH, anti-p300, anti-CBP or anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with anti-HA or anti-GAPDH antibodies. Arrows indicate endogenous GAPDH and arrowheads indicate exogenous HA–GAPDH-K160R. (g) Nuclear localization of GAPDH augments its binding to p300/CBP and acetylation at Lys 160. Peritoneal macrophages from wild-type and iNOS knockout mice were transfected with various GAPDH constructs. Cell lysates were immunoprecipitated with anti-p300, anti-CBP or anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with anti-HA antibody. Both HA–NLS–GAPDH and HA–NLS–GAPDH–C150S, but not HA–NLS–GAPDH-K160R, augmented GAPDH–p300/CBP binding, as well as GAPDH acetylation in both iNOS knockout and wild-type cells.
Figure 3
Figure 3
GAPDH increases the catalytic activity of p300. (a) Increased acetylation of p300 occurs in RAW264.7 cells treated with LPS/IFNγ for 16 h; this was abolished by the iNOS inhibitor 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p300 antibody, and the immunoprecipitates were analysed by western blotting with an acetylation-specific p300/CBP antibody. (b) Genetic deletion of iNOS abolished the increased acetylation of p300/CBP and diminishes p300–GAPDH binding in peritoneal macrophages treated with LPS/IFNγ for 16 h. (c) Increased acetylation of p300 in RAW264.7 cells treated with LPS/IFNγ is abolished with depletion of GAPDH by RNAi. (d) GAPDH-K160R dominant-negative mutant diminished the increased acetylation of p300 elicited by GSNO in HEK293 cells. Forty-eight hours after transfection with wild-type HA–GAPDH or HA–GAPDH-K160R construct, cells were treated with 200 μM GSH or GSNO. (e) GAPDH augments auto-acetylation of a fragment of p300 (F-p300) in vitro. Auto-acetylation of F-p300 was assessed with 14C-acetyl-CoA in the presence or absence of purified GAPDH. (f) GAPDH, native or NO-modified, augments auto-acetylation of a fragment of p300 (F-p300) in vitro. Auto-acetylation of p300 was analysed by western blotting with anti-acetyl Lys antibody. SNO-GAPDH, S-nitrosylated GAPDH.
Figure 4
Figure 4
GAPDH–p300 activates downstream targets, such as p53 and PUMA. (a) p53 is acetylated in GSNO-treated HEK293 cells; this is abolished by expression of GAPDH-K160R. Cell lysates were immunoprecipitated with anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with an anti-p53 antibody. (b) GAPDH–p53 binding was augmented by treatment with LPS/IFNγ for 16 h in RAW 264.7 cells. This was blocked by 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p53 antibody and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. (c) GAPDH–p300–p53 forms a complex in vitro, with p300 required for the interaction. Binding was examined by GSH-agarose pulldown assay. (d) Formation of a p53–p300-sulphonated GAPDH (sGAPDH) complex at the PUMA promoter region in U2OS cells treated with GSNO, assayed by ChIP assay. Cells were treated with 200 μM GSH or GSNO for 24 h. (e) Formation of p53–p300–sGAPDH complex at the PUMA promoter region in U2OS cells with GSNO was blocked by expression of GAPDH-K160R. Forty-eight hours after transfection with HA–GAPDH or HA–GAPDH-K160R, cells were treated with 200 μM GSNO for 24 h. (f) RNAi depletion of GAPDH diminishes the formation of the p53–p300– sGAPDH complex at the PUMA promoter in U2OS cells. Forty-eight hours after transfection with control or GAPDH siRNA, cells were treated with 200 μM GSH or GSNO for 24 h. *P < 0.001, n = 3, mean ± s.e.m., one-way ANOVA. (g) Overexpression of GAPDH, but not of GAPDH-K160R, in U2OS cells increases PUMA, BAX and p21 protein levels in the presence of GSNO. Forty-eight hours after transfection with mock, HA–GAPDH or HA–GAPDH-K160R constructs, cells were treated with 200 μM GSH or GSNO. (h) Acetylation of p53 was increased in brains of mice treated with MPTP, which was decreased by pre-treatment with R-(−)-deprenyl (Dep). (i) PUMA levels were increased in brains of mice treated with MPTP, effects prevented by pre-treatment with R-(−)-deprenyl (Dep).
Figure 5
Figure 5
Influence of GAPDH and GAPDH-K160R on cell death. (a) Increased apoptosis in peritoneal macrophages with LPS/IFNγ or in dopaminergic neuroblastoma SH-SY5Y cells with GSNO is blocked by GAPDH-K160R. Forty-eight hours after transfection with either mock, HA–GAPDH or HA–GAPDH-K160R, peritoneal macrophages isolated from wild-type mice were treated with LPS/IFNγ for 16 h. SH-SY5Y cells were treated with 200 μM GSH or GSNO for 24 h, after transfection with mock, HA–GAPDH- or HA–GAPDH-K160R. Apoptosis was assessed by counting condensed nuclei in transfected cells. **P < 0.001, n = 3, mean ± s.e.m., one-way ANOVA. (b) PUMA levels are increased in SH-SY5Y cells stably overexpressing GAPDH but decreased in cells overexpressing GAPDH-K160R, in comparision with cells with the empty vector, in the presence of 200 μM GSNO for 24 h. Depletion of endogenous p53 by RNAi significantly decreased the levels of PUMA. (c) Knockdown of p53 expression in SH-SY5Y cells stably overexpressing wild-type GAPDH or GAPDH-K160R in the presence of 200 μM GSNO for 24 h leads to decrease apoptosis, suggesting a major role of p53 in NO–GAPDH–p300/CBP death cascade. Cell death was measured by TUNEL assay. *P < 0.01, **P < 0.001, ***P < 0.0001, n = 4, mean ± s.e.m., one-way ANOVA.
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References

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