Regulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis

doi:10.1016/S1044-7431(03)00203-3

Molecular and Cellular Neuroscience

Volume 24, Issue 2, October 2003, Pages 451-459

https://doi.org/10.1016/S1044-7431(03)00203-3 Get rights and content

Abstract

The molecular mechanism of Bcl-2 phosphorylation and its relationship to Bax is largely unknown. Here we show that the phosphorylation of Bcl-2 is involved in the intracellular translocation of Bax from cytosol to mitochondria in NO-induced neuronal apoptosis. We examined how the phosphorylation of Bcl-2 is regulated during the apoptosis and found it to be mediated by the activation of p38 and ERK, members of the MAPK superfamily. Furthermore, we investigated whether Bcl-2 phosphorylation affected Bax translocation, using mutant Bcl-2 expression vectors. Cortical neuronal cells overexpressing the Bcl-2 mutant S70A (which cannot be phosphorylated) prevented the translocation of Bax. In contrast, transfection with Bcl-2 (S70D), a constitutively active Bcl-2 mutant, enhanced the translocation. Our results suggested that Bcl-2 phosphorylated at Ser-70 plays a critial role in the translocation of Bax from the cytosol to the mitochondria, and this may regulate NO-induced neuronal apoptosis.

Introduction

Nitric oxide (NO)⁴ is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity Beal, 1996, Dawson and Dawson, 1996, Heales et al., 1999. Excitotoxic injury causes an increase in the formation of NO, which leads to activation of the MAPK superfamily in neurons (i.e., extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38 MAPK). The balance between MAPK pathways is thought central to a diverse array of cellular responses, including cell growth, differentiation, survival, and death in the nervous system Boulton et al., 1991, Qui and Green, 1992, Loeb et al., 1992, Cobb, 1999, Han et al., 1994, Kyriakis et al., 1994, Rouse et al., 1994.

The Bcl-2 family plays a central role in mammalian programmed cell death pathways by suppressing (e.g., Bcl-2 and Bcl-X_L) or promoting (e.g., Bax, Bad, and Bcl-X_S) apoptosis (Yang and Korsmeyer, 1996). Bcl-2 has a protective effect on neuronal cell death by forming heterodimers with Bax to neutralize the proapoptotic activity of Bax (Oltavai et al., 1993). Recently it has been suggested that the antiapoptotic function of Bcl-2 is dependent on its phosphorylation state rather than its expression level. That is, several growth factors induce the phosphorylation of Bcl-2 at serine/threonine, which modifies the antiapoptotic effects of Bcl-2 (Ito et al., 1997). In this regard, signaling pathways and kinases that have been implicated in the phosphorylation of Bcl-2 include receptor tyrosine kinases, c-Jun N-terminal kinase/stress-activated protein kinase JNK/SAPK, Raf-1, protein kinase C (PKC), protein kinase A, and the cyclin B1-Cdc2 complex Maundrell et al., 1997, Blagosklonny et al., 1996, Blagosklonny et al., 1997, Yamamoto et al., 1999, May et al., 1994, Ruvolo et al., 1998, Ueno et al., 2000, Srivastava et al., 1998, Ling et al., 1998, Scatena et al., 1998.

Bax, a proapoptotic member of the Bcl-2 family, has been reported to have a predominantly cytosolic expression in healthy cells, despite having a membrane-targeting carboxyl terminus, but is translocated to the mitochondria in a number of cell types after the reception of a death signal, such as cytotoxic insult Hsu et al., 1997, Wolter et al., 1997, Goping et al., 1998, Gross et al., 1998, McGinnis et al., 1999. In fact, deletion of the carboxy-terminal tail prevents the translocation of Bax and thus suppresses apoptosis after a death stimulus, suggesting that the regulation of its association with the mitochondrial membrane is a key stage in the transduction of its apoptotic signal (Wolter et al., 1997).

Previous studies have been reported that NO caused the changing Bcl-2 and Bax protein levels (Tamatani et al. 1998) and the Bax translocation (Ghatan et al. 2000) during NO-induced neuronal apoptosis. In the present paper, we describe that Bcl-2 is phosphorylated during NO-induced neuronal apoptosis, an event that is followed by the translocation of Bax from the cytosol to mitochondria. Transfection experiments with Bcl-2 mutants bearing substitutions of the Ser-70 residue within the loop region showed increased resistance to Bax translocation induced by NO-induced neuronal apoptosis.

Section snippets

Activation of ERK and p38 MAPK and phosphorylation of Bcl-2 are involved in NO-induced apoptosis of cortical neurons

We reported previously that ERK and p38 MAPK are activated in the NO-induced apoptosis of cultured cortical neurons (Ishikawa et al., 2000). To further examine whether activated MAPK superfamily members are involved in NO-induced cell death, we tested the effects of U0126 and SB202190, specific inhibitors of ERK and p38 MAPK, respectively, and found that the cell death was effectively prevented by these inhibitors (Fig. 1). We also tested whether SB203580, another p38 MAPK inhibitor, inhibited

Discussion

Many reports have suggested that dephosphorylation enhances the antiapoptotic function of Bcl-2 and phosphorylation inactivates the antiapoptotic effect Blagosklonny et al., 1996, Yamamoto et al., 1999, Haldar et al., 1995, although there have also been reports to the contrary Ito et al., 1997, May et al., 1994, Ruvolo et al., 1998. Notably, the role of phosphorylation in the antiapoptotic function of Bcl-2 and the relationship between phosphorylated Bcl-2 and Bax translocation are still

Cell culture

Cortical tissues were dissected from brains of embryonic day 20 (E20) rats (Wistar ST, both sexes; Nippon SLC). The cultured cells were prepared as described previously (Yamada et al., 1995). Briefly, tissue fragments were suspended in 10 ml of fresh Ca²⁺, Mg²⁺ -free phosphate-buffered saline (PBS) containing papain (9 units/ml), DNase I (200 units/ml), dl-cysteine (0.2 mg/ml), recrystallized bovine serum albumin (0.2 mg/ml), and glucose (5 mg/ml), and then incubated for 30 min with rotation at

Acknowledgements

This study was supported in part by a Grant-in-aid for Scientific Research Priority Areas, Ministry of Education, Science and Culture, Japan and the Research Grant (12B-1) for Nervous and Mental Disorders from the Ministry of Health Labour and Welfare and a grant from the Fujisawa Foundation.

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¹: Present address: Division of Structural Cell Biology, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan.

²: Present address: Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

³: Present address: Laboratory of Neurobiology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan.

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Regular articleRegulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis

Abstract

Introduction

Section snippets

Activation of ERK and p38 MAPK and phosphorylation of Bcl-2 are involved in NO-induced apoptosis of cortical neurons

Discussion

Cell culture

Acknowledgements

Curr. Opin. Neurobiol.

Cell

Prog. Biophys. Mol. Biol.

J. Chem. Neuroanat.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Neuron

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

Blood

Raf-1/bcl-2 phosphorylationa step from microtubule damage to cell death

Cancer Res.

Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway

Cancer Res.

Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2

EMBO J.

The permeability transition pore signals apoptosis by directing Bax translocation and multimerization

FASEB J.

“Loop” domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis

Cancer Res.

p38 MAP kinase mediates bax translocation in nitric oxide-induced apoptosis in neurons

J. Cell. Biol.

Regular article
Regulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis