Tumor necrosis factor-α-induced IKK phosphorylation of NF-κB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway
Tumor necrosis factor-α-induced IKK phosphorylation of NF-κB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway
复制标题
DOI：
10.1074/jbc.m301598200
复制
发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Saiki, I
中科院分区：
文献类型：
--
作者：
Sakurai, H;Suzuki, S;Saiki, I
NF-κ B的活化已被证明是由IkappaB和NF-κ B p65亚基的多重磷酸化调节的。在这里，我们的特点是使用一种新的抗磷酸化p65（Ser-536）抗体的细胞内信号通路导致磷酸化p65的Ser-536。内源性p65的Ser-536响应于包括细胞质中的TNF-α在内的多种NF-κ B刺激剂而迅速磷酸化，并在细胞核中迅速去磷酸化。在来自traf 2（-/-）traf 5（-/-）小鼠的鼠胚胎成纤维细胞中，TNF-α诱导的Ser-536磷酸化严重受损，但IL-1 β诱导的Ser-536磷酸化未受损。Bay 11-7082是IkappaB磷酸化的抑制剂，在体内抑制TNF-α诱导的磷酸化。此外，TGF-β激活的激酶1（TAK 1），IKK α和IKK β的过表达刺激磷酸化，其显性失活突变体阻断TNF-α诱导的磷酸化。此外，针对TAK 1、IKK α和IKK β的小干扰RNA（siRNA）阻断了内源性p65的磷酸化。另一方面，calyculin-A，一种蛋白磷酸酶抑制剂，在体内阻断了细胞核中的去磷酸化。这些结果表明，IkappaB α和p65的磷酸化利用了类似的信号传导途径，这进一步支持了IkappaB和NF-κ B都是NF-κ B活化中IKK复合物的底物的观点。
The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha- but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2(-/-) traf5(-/-) mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha- induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.