Hapil Zevkliler Fatma Zehra, Çopuroğlu Fatma Ece, Ertosun Mustafa Gökhan, Mert Ufuk, Özeş Derya, Özeş Osman Nidai
Department of Medical Biology and Genetics, Akdeniz University, Antalya, Turkiye.
Department of Reconstructive Surgery, Akdeniz University, Antalya, Turkiye.
Turk J Biol. 2023 Nov 6;48(1):1-12. doi: 10.55730/1300-0152.2677. eCollection 2024.
BACKGROUND/AIM: Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation.
Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed.
In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation.
TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.
背景/目的：肿瘤坏死因子α（TNFα，又称TNF）是一种多效性细胞因子，其大部分作用通过1型TNF受体（TNFR1）发挥。TNF结合后，TNFR1招募TRADD（肿瘤坏死因子受体1相关死亡结构域）。这种相互作用触发信号体复合物的形成，据称该复合物可诱导细胞凋亡（通过下游半胱天冬酶激活）、炎症（通过核因子κB）和应激途径（JNK和p38）。然而，TNF诱导ERK和AKT激活的机制尚未完全阐明。已知TNFR1组成性结合c-Src和JAK2，并且这些酶先前已被证明可调节TNF信号传导。因此，我们推测TNFR1可能被JAK2和/或c-Src酪氨酸磷酸化，并且TNF诱导的ERK和Akt激活可能由这种磷酸化介导。
进行定点诱变（SDM），用丙氨酸（A）或天冬氨酸（D）替代TNFR1上的两个假定酪氨酸磷酸化位点（Y360和Y401），分别抑制或模拟组成性磷酸化。在转染了突变型或野生型TNFR1的293T细胞中，通过蛋白质印迹法测定ERK和Akt的激活情况。通过免疫共沉淀检测TNFR1与c-Src、JAK2、p85和Grb2的相互作用。通过荧光素酶测定法测量核因子κB的激活，通过MTT法测量细胞增殖，并通过比色法半胱天冬酶8/3测定法评估细胞凋亡。为了测定坏死率，进行细胞DNA片段化ELISA。
在本报告中，我们表明TNFR1在Y401处被JAK2酪氨酸激酶磷酸化，在Y360和Y401处被c-Src磷酸化。Y360和Y401的磷酸化增强了Grb2和PI3Kp85与TNFR1的相互作用。我们还证明Y360D和Y401D的磷酸模拟突变增强了ERK和Akt的激活。
TNFR1被c-Src和JAK2酪氨酸磷酸化，触发一条“非经典”途径，激活ERK和Akt。
