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. While TNFR1 induces apoptosis by activation of caspase-8 via the "death domain", it also activates IKKα/β, 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, (P448PAP451, 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 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 diminished TNFα-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.

Keywords: AKT; ERK; Grb2; TNFR1; TNF-α.

Figure 1

A proline-rich PPAP region, “P448PAP451”, a binding site for proteins containing the SH3 domain very close to the Cterminus, was discovered in the translation map of the TNFR1. The SH3 domain recognition sequence PPAP of TNFR1 is underlined in red. (A) The mutant TNFR1 was generated using site-directed mutagenesis. The Sanger sequence analysis was performed via Sanger sequencing, and the sequences of antisense strand of wild type and reverse strand of mutant TNFR1 were confirmed. (B) The wild type and mutant TNFR1 (TNFR1-SH3-P/A) can be ectopically expressed in 293T cells. (C) To determine ectopic expression of wild type and mutant TNFR1, both vectors were transfected into 293T cells (30 μg/10 cm plate) via calcium phosphate protocol for 48 h 100 μg of total cell lysates were used for western blotting. Fold changes of wild type and mutant proteins were determined by normalizing GAPDHcorrected values to that of background TNFR1.

Figure 2

The mutant TNFR1 (TNFR1-SH3-P/A) interferes with TNFα-mediated phosphorylations of ERK1/2 and AKT kinases. Pattern of TNF-α-induced phosphorylations ERK1/2 (A) and AKT (B) were explored by western blot of 100 μg lysates obtained from 293T cells treated with TNF-α for the indicated time points. To determine the fold changes in phosphorylations, the band intensities of the images were determined using ImageJ, and the numerical values of phospho bands were divided into that of nonphospho bands.

Figure 3

The mutant TNFR1 (TNFR1-SH3-P/A) interferes with TNFα-mediated phosphorylations of p38 and JNK kinases. Pattern of TNF-α-induced phosphorylations p38 (A) and JNK1 (B) and JNK2 (C) were explored by Western blot of 100 μg lysates obtained from 293T cells treated with TNF-α for the indicated time points. To determine the fold changes in phosphorylations, the band intensities of the images were determined using ImageJ, and the numerical values of phospho bands were divided into that of nonphospho bands.

Figure 4

The mutant TNFR1 (TNFR1-SH3-P/A) augments TNFα-mediated induction of NF-kB, but does not affect caspase-8 activation. TNFR1-SH3-P/A mutant differentially affects TNFα-induced NF-kB activation. (A) To determine the NF-kB activity, 293T cells were transfected with 150 ng of NF-kB luciferase reporter plasmid along with 150 ng of PC-3, TNFR1, or TNFR1-SH3-P/A for 48 h, then cells were treated with 10 ng/mL TNFα for 6 h and luciferase activity was determined using Luminometer (Thermo Fisher Scientific). TNFR1-SH3-P/A mutant differentially affects TNFα-induced caspase-8 activation. (B) To determine the impact of mutant TNFR1 on caspase 8 activation, 293T cells were transfected with 150 ng of PC-3, TNFR1, or TNFR1-SH3-P/A for 48 h, then cells were treated with 10 ng/mL TNFα for 24 h and caspase-8 activation was measured by colorimetric caspase-8 activation assay kit.

Figure 5

The adaptor protein GRB2 is essential for TNFR1 signaling. The GRB2 constitutively bind to PPAP sequence of TNFR1 via its SH3 domain. After TNFα binding to TNFR1, cSRC and JAK2 (which constitutively bind to TNFR1) phosphorylate Y360 and Y401. The phosphorylated tyrosines were represented as single (P) on TNFR1. GRB2 binds to these tyrosine phosphorylated amino acids via its SH2 domain. TNFR1-bound GRB2 binds to RAS and initiate RAS-induced RAS-RAF-ERK and RAS-p110α -AKT pathways.