■肿瘤坏死因子α(TNFα,TNF)是一种多效性细胞因子,通过1型TNF受体(TNFR1)发挥其大部分作用。TNF结合后,TNFR1招募TRADD(肿瘤坏死因子受体1型相关死亡结构域)。这种相互作用引发信号体复合物的形成,这些复合物被声称诱导细胞凋亡(通过下游胱天蛋白酶激活),炎症(通过NF-κB)和应激途径(JNK&p38)。然而,TNF诱导的ERK和AKT激活的潜在机制尚未完全揭示.已知TNFR1组成型结合c-Src和JAK2,并且这些酶先前被证明调节TNF信号传导。因此,我们假设TNFR1可能被JAK2和/或c-Src酪氨酸磷酸化,而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),以丙氨酸(A)或天冬氨酸(D)取代TNFR1上的两个推定酪氨酸磷酸化位点(Y360和Y401),抑制或模拟组成型磷酸化,分别。在用突变或野生型TNFR1转染的293T细胞中,通过蛋白质印迹测定ERK和Akt激活。TNFR1与c-Src相互作用,通过co-IP检查JAK2、p85和Grb2。NF-kB激活通过荧光素酶测定法测量,而增殖通过MTT测量,凋亡通过比色caspase8/3测定法评估。为了确定坏死率,进行细胞DNA片段化ELISA。
■在本报告中,我们显示TNFR1在Y401被JAK2酪氨酸激酶磷酸化,在Y360和Y401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的磷模拟突变增强ERK和Akt活化。
■TNFR1是由c-Src和JAK2磷酸化的酪氨酸,触发“非规范”途径,激活ERK和Akt.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a \"noncanonical\" pathway, that activates ERK and Akt.