The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the α Subunit of Eukaryotic Initiation Factor 2

Shiyong Wu,Csaba F László,Hao Chen,Zhixin Miao,Wei Lu

doi:10.1074/jbc.m109.008821

Abstract

UV light induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2alpha in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2alpha phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2alpha phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2alpha after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2alpha phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N(G)-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2alpha phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2alpha phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.

Highlights

We demonstrate that UV-induced activation of nitric-oxide synthase (NOS) mediates the activation of both protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), which coordinately regulate the phosphorylation of eIF2␣
Considering the possible matrix effect, the quantitative analysis of L-Arg in total lysate of HaCaT cells treated with 50 mM L-Arg was performed using a standard addition method [15]
Because keratinocytes comprise Ͼ90% of total skin cells, we first determined the dose-dependent effect of UVB on a human keratinocyte cell line: HaCaT cells

Summary

Introduction

Western blot analysis indicated that the UV-induced phosphorylation of eIF2␣ was reduced by 64% in the presence of LNMMA (Fig. 3, lane 4 versus 2) at 4 h post-UVB irradiation. Rapidly with NO1⁄7 ONOOϪ induce oxidative-stress, which leads to PERK activation [9, 27], we determined their roles in UVB-induced phosphorylation of eIF2␣.

Results

Conclusion