Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Rajee Antony,Walter J Lukiw,Nicolas G Bazan

doi:10.1074/jbc.m109.095232

Abstract

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x(L) is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x(L) at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x(L) phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x(L) is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit alpha of PP2A (PP2A/Calpha) by small interfering RNA enhanced Bcl-x(L) phosphorylation when activated with hydrogen peroxide and tumor necrosis factor alpha-induced oxidative stress. Disruption of PP2A/Calpha exacerbated oxidative stress-induced apoptosis. PP2A/Calpha colocalized and interacted with S62Bcl-x(L) in cells stressed with H(2)O(2)/tumor necrosis factor alpha. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x(L) by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Calpha and increased the association of PP2A/Calpha with S62Bcl-x(L) as well as total Bcl-x(L). NPD1 also enhanced the heterodimerization of Bcl-x(L) with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.

Highlights

Grant R01 EY005121 through the NEI, United States Public Health Service. 1 To whom correspondence should be addressed: Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, 2020 Gravier St., Ste
The present study was undertaken to examine the effect of neuroprotectin D1 (NPD1) in modulating the phosphorylation of Bcl-xL and to identify the specific species of protein phosphatase involved during induced oxidative stress in ARPE-19 cells
To test whether phosphatase 2A (PP2A)-mediated Bcl-xL dephosphorylation is directly involved in ARPE-19 cell survival, cells were transfected with control siRNA and PP2A/C siRNA and treated with H2O2/TNF␣ in the presence and absence of NPD1

Summary

Introduction

Grant R01 EY005121 through the NEI, United States Public Health Service. 1 To whom correspondence should be addressed: Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, 2020 Gravier St., Ste. The present study was undertaken to examine the effect of NPD1 in modulating the phosphorylation of Bcl-xL and to identify the specific species of protein phosphatase involved during induced oxidative stress in ARPE-19 cells. The knockdown of endogenous PP2A/C␣ protein (Fig. 2B) in ARPE-19 cells abolishes the Ser-62 dephosphorylation in response to oxidative stress (Fig. 2C).

Results

Conclusion