Positive regulation of TNFR1 signaling via SH3 recognition motif

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the "death domain", it also activates IKK alpha/ss, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P(448)PAP(451), a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P 448 and P 451 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-alpha-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNF alpha-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNF alpha-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.