Positive regulation of TNFR1 signaling via SH3 recognition motif

Abstract

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. WhileTNFR1 induces apoptosis by activation of caspase-8 via the “death domain”, it also activates IKKα/β, MKK3/6, MKK4/7 by activation ofTAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very closeto the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codonsof P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A.Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminishedTNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.