Nuclear GAPDH Triggers Apoptosis

Abstract

In addition to its functions in cellular metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is nitrosylated and translocated to the nucleus in response to stimuli that activate inducible nitric oxide synthase (iNOS). Sen et al . show that when nitrosylated GAPDH is translocated to the nucleus, it interacts with and is acetylated by the nuclear protein acetyltransferase p300 or its related homolog CBP (collectively p300/CBP). Lys 160 was the acetylated residue on GAPDH, and a K160R mutant was not acetylated and acted as a dominant-negative protein preventing acetylation of wild-type protein, but not inhibiting S -nitrosylation or nuclear translocation. When a nuclear localized version of GAPDH (NLS-GAPDH) was expressed in wild-type or iNOS -null macrophages, NLS-GAPDH bound to p300/CBP and was acetylated in response to lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treatment, which stimulates iNOS. A nuclear-localized GAPDH that had a mutation at the nitrosylation site also interacted with p300/CBP and was acetylated. Thus, nitrosylation does not appear to be required for the interaction with p300/CBP; instead, it seems to promote the nuclear localization of GAPDH. When p300 and CBP are autoacetylated, their acetylating activities are increased. LPS/IFN-γ stimulated p300 acetylation, and this was blocked by inhibition of iNOS or if GAPDH was depleted from the cells by RNA interference (RNAi). In vitro GAPDH also increased the acetylation of p300, and this did not require nitrosylation of GAPDH and was not enhanced by nitrosylation of GAPDH. To gain insight into how the interaction between GAPDH and p300/CBP may contribute to apoptosis, Sen et al . showed that NO stimulated the acetylation of p53 in human embryonic kidney (HEK) 293 cells, and this was prevented if the cells were expressing the K160R mutant, iNOS was inhibited, or GAPDH was depleted by RNAi. Chromatin immunoprecipitation experiments revealed that p53, GAPDH, and p300 were all present at the promoter of PUMA , which encodes a p53-regulated protein implicated in apoptosis. When peritoneal macrophages or the neuroblastoma cell line SH-SY5Y were transfected with GAPDH, cell death induced by LPS/IFN-γ or an NO donor, respectively, was increased, and when the cells were transfected with the K160R, mutant cell death was prevented. Thus, GAPDH appears to contribute to cell death triggered by an NO cascade by functioning in the nucleus to stimulate the acetyltransferase activity of p300/CBP, leading to the activation of p53 and proapoptotic gene expression. N. Sen, M. R. Hara, M. D. Kornberg, M. B. Cascio, B.-I. Bae, N. Shahani, B. Thomas, T. M. Dawson, V. L. Dawson, S. H. Snyder, A. Sawa, Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis. Nat. Cell Biol. 10 , 866-873 (2008). [PubMed]