Exploring the Role of Phosphorylation of FADD in TNFa‐Induced Apoptosis of Mouse Hepatocytes

Abstract

Fas-associated death domain (FADD) is a crucial adaptor protein mediating death signals triggered by death receptors. It was reported that FADD protein level are upregulated 20 fold after TNFa/Actinomycin D (ActD) treatment in cultured hepatocytes. In contrast, FADD mRNA level are reduced under the same conditions. This suggests a post-translational mechanism may be responsible for upregulation of FADD during hepatocyte apoptosis. We addressed whether phosphorylation of FADD plays a role in mouse hepatocytes (MHC) apoptosis induced by TNFa/ActD treatment. TNFa and ActD, but not TNFa alone, strongly upregulated a 28kDa FADD protein in MHCs 6h after treatment. Surprisingly, an extra band at ~66 kDa was consistently detected in MHCs even when using two different anti-FADD monoclonal antibodies. We show that the ~66kDa band was specific for phosphorylated FADD (pFADD). We confirmed this finding by ectopically expressing mouse FADD fused with a myc tagged protein in 293T cells and immunoblotting with both anti-myc and anti-pFADD antibodies. Mutation of Serine 191, a known FADD phosphorylation site, completely abolished the ~66 kDa band. We used immunofluorescence to show that pFADD is exclusively located in the nucleus of mouse hepatocytes. Our results indicate that this novel ~66 kDa protein is part of a complex that contains pFADD. This suggests that phosphorylation plays an important role in FADD nuclear localization and may prove to be important in FADD protein upregulation in TNFa stimulated MHCs.