Tumor necrosis factor alpha (TNF-α) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this “canonical TNF-α signaling” does not explain how ERK, AKT, and STAT3 can be activated by TNF-α. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-α signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT₄₁₁ and REAT₄₁₇ within the death domain of TNFR1, and investigated whether “canonical” and “noncanonical” TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-α stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-α-induced activations of ERKs, AKT, STAT3, p38α, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-α-induced phosphorylations of ERKs, AKT, Stat3, p38α, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both “canonical” and “noncanonical” TNF-α signaling.

中文翻译：

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通过PKA介导的TNFR1磷酸化对TNFR1信号的负调控。

肿瘤坏死因子α（TNF-α）通过诱导肿瘤细胞毒性和激活免疫系统，在体内平衡中起着至关重要的作用。由TNFR1激活JNK，p38和核因子-κB途径并随后诱导细胞凋亡和坏死病形成的信号复合物是众所周知的。但是，这种“经典的TNF-α信号传导”不能解释TNF-α如何激活ERK，AKT和STAT3。另外，关于TNFR1信号转导的负调控了解甚少。因为环状AMP激活激酶（PKA）显示抗TNF和抗炎活性，所以我们推测PKA可能通过直接磷酸化TNFR1影响TNF-α信号传导。与此相符，我们确定了两个推定的PKA磷酸化基序RRRT ₄₁₁和REAT ₄₁₇在TNFR1的死亡结构域中，研究“正常”和“非典型” TNFR1信号是否受T411和T417修饰的调控。在这项研究中，我们首次证明PKA在TNF-α刺激后直接结合并磷酸化TNFR1。为了进一步支持我们的假设，我们在位置T411和T417上生成了丙氨酸和拟膦酸（天冬氨酸）突变体TNFR1，异位表达这些突变体，并确定了它们对TNF-α诱导的ERKs，AKT，STAT3，p38α和ERKs活化的影响。 JNK1 / 2。我们的结果清楚地表明，拟磷酸酶突变体显着抑制并且丙氨酸突变体增强了TNF-α诱导的ERK，AKT，Stat3，p38α和JNKs的磷酸化。