Investigation of the signal transduction mechanism of tumor necrosis factor receptor 1 (TNFR1)

Objective: TNF-α, which is a pleiotropic cytokine, exerts most of its effects through its receptor TNFR1. Although there are numerous studies on TNF-α-mediated inductions of NF-κB pathway, apoptosis and necroptosis; the mechanisms of TNFR1-mediated ERK, Akt and Stat3 activation pathways have not clearly been described yet. TNFR1 is known to interact with tyrosine kinases c-Src and JAK2, however, whether TNFR1 is phosphorylated on tyrosine residues by one of these kinases was not known. In this study, we aimed to reveal whether TNFR1-JAK2 interaction leads to tyrosine phosphorylation of TNFR1, and if so, to elucidate its effect on TNF-α mediated ERK, Akt, p38, JNK, Stat3 and CREB activations. Method: Following in vitro kinase reactions which revealed phosphorylation of TNFR1 by JAK2 and PKA; corresponding Y and T residues on putative phosphorylation motifs were substituted with phosphorylation-mimicking D and non-phosphorylatable A residues. The influence of these mutations on ERK, p38, JNK, Akt, Stat3, CREB and IRS-1 activation phosphorylations were explored by western blot and the impact on physical interactions between TNFR1 and Grb2, p85 and Stat3 were investigated by co-immunoprecipitation. NF-κB activation was measured by NF-Luc luciferase reporter system, influence on apoptosis was measured by colorimetric caspase-3 and caspase-8 activation assays, and impact on cell survival and TNFR1 shedding were determined by MTT and ELISA, respectively. Results: Our results indicate that TNFR1 is phosphorylated by JAK2 and PKA. However, PKA-mediated phosphorylation inhibits JAK2-mediated tyrosine phosphorylation. Y401 phosphorylation of TNFR1 strengthens Grb2, p85, and Stat3 binding; increases both ERK and Akt activation phosphorylations and DNA binding of Stat3. Y360 phosphorylation of TNFR1 increases CREB and Stat3 phosphorylations, while Y360A mutation leads to JNK and p38 activation. In cells transfected with Y401D mutants; caspase activation, NF-κB induction, and cell survival were found to be diminished. IRS-1 tyrosine phosphorylation was found to be negatively affected by both tyrosine and PKA phosphorylation of TNFR1. Conclusion: TNFR1 is phosphorylated by both JAK2 and PKA, and PKA phosphorylation attenuates TNFR1 tyrosine phosphorylation. TNFR1 phosphorylations differentially regulate TNF-α mediated ERK, Akt, Stat3, p38, JNK and CREB activations, as well as IRS-1 tyrosine phosphorylation.