A novel role for protein kinase Cdelta-mediated phosphorylation of acid sphingomyelinase in UV light-induced mitochondrial injury.
Multiple studies have addressed the mechanisms by which ultraviolet (UV) light induces cell death, and a few have focused on stress mediators such as acid sphingomyelinase (ASMase) or protein kinase Cdelta (PKCdelta). Based on a recent study that identified a novel mechanism of activation of ASMase through phosphorylation, the current study was undertaken to determine the upstream mechanisms regulating ASMase in response to UV and to investigate the role of ASMase and its phosphorylation at S508 as an integral event during UV light-induced cell death. Exposure of MCF-7 breast cancer cells to UV light type C (UVC) transiently activated ASMase with maximal activity detected at 10 min postirradiation. A significant increase in C16-ceramide was detected concomitant with a decrease in C16-sphingomyelin. In marked contrast, cells overexpressing the ASMase(S508A) mutant, which could not be phosphorylated, had no change in either ASMase activity or ceramide levels post-UV radiation. Loss of PKCdelta by RNA interference or its inhibition by rottlerin blocked ASMase phosphorylation and membrane targeting, thus implicating PKCdelta upstream of ASMase activation by UV light. Further investigations revealed that UV radiation altered mitochondrial morphology from elongated tubules to fragmented perinuclear organelles, consistent with the onset of the apoptotic cascade. Importantly, cells overexpressing ASMase(S508A) were protected (>50%) from UV light-induced mitochondrial fragmentation. Mechanistically, the results showed that ASMase(S508A) cells had 50% less active Bax than ASMase(WT) cells. These molecular differences culminated in resistance of ASMase(S508) cells to UVC-induced cell death (25%) as compared to ASMase(WT) cells (46%). Taken together, this study provides key molecular insights into activation of ASMase in response to UV light, the role of PKCdelta in this activation, and the role of ASMase in mediating apoptotic responses.
Zeidan
Youssef H
YH
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Wu
Bill X
BX
Jenkins
Russell W
RW
Obeid
Lina M
LM
Hannun
Yusuf A
YA
D017209:Apoptosis / Q000528:radiation effects; D001483:Base Sequence; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000528:radiation effects*; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000378:metabolism*; D034622:RNA Interference; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D014466:Ultraviolet Rays
D002518:Ceramides; D017931:DNA Primers; D013109:Sphingomyelins; D045304:Cytochromes c; D051745:Protein Kinase C-delta; D013108:Sphingomyelin Phosphodiesterase