Phosphorylation of serine 468 by GSK-3β negatively regulates basal p65 NF-κB activity
Phosphorylation of serine 468 by GSK-3β negatively regulates basal p65 NF-κB activity
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DOI：
10.1074/jbc.c400442200
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发表时间：
2004-11-26
影响因子：
4.8
通讯作者：
Kracht, M
中科院分区：
文献类型：
--
作者：
Buss, H;Dörrie, A;Kracht, M
NF-κ B的活性在几个水平上受到控制，包括强反式激活p65（RelA）亚基的磷酸化。然而，磷酸化位点的总数、靶向p65 NF-κ B的信号通路和蛋白激酶以及这些磷酸化的功能作用仍未被揭示。使用肽阵列与体外激酶测定的组合，我们确定丝氨酸468作为p65 NF-κ B的一个新的磷酸化位点。丝氨酸468位于GSK-3 β共有位点内，并且重组GSK-3 β在体外特异性磷酸化GST-p65-（354-551）融合蛋白的Ser（468）。在完整细胞中，P65的内源性Ser（468）的磷酸化由PP 1/PP 2A磷酸酶抑制剂calyculin A诱导，并且该作用由GSK-3 β抑制剂LiCl抑制。用p65蛋白重建p65缺陷细胞，其中丝氨酸468突变为丙氨酸，揭示了丝氨酸468对NF-κ B活化的负调节作用。总的来说，我们的研究结果表明，GSK-3 β-PP 1依赖性机制调节p65 NF-κ B在未刺激细胞中Ser（468）的磷酸化，从而控制NF-κ B的基础活性。
The activity of NF-kappaB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-kappaB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-kappaB. Serine 468 lies within a GSK-3beta consensus site, and recombinant GSK-3beta specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser(468) in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3beta inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-kappaB activation. Collectively our results suggest that a GSK-3beta-PP1-dependent mechanism regulates phosphorylation of p65 NF-kappaB at Ser(468) in unstimulated cells and thereby controls the basal activity of NF-kappaB.