A PROTEASOME INHIBITOR PREVENTS ACTIVATION OF NF-KAPPA-B AND STABILIZES A NEWLY PHOSPHORYLATED FORM OF I-KAPPA-B-ALPHA THAT IS STILL BOUND TO NF-KAPPA-B
A PROTEASOME INHIBITOR PREVENTS ACTIVATION OF NF-KAPPA-B AND STABILIZES A NEWLY PHOSPHORYLATED FORM OF I-KAPPA-B-ALPHA THAT IS STILL BOUND TO NF-KAPPA-B
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DOI：
10.1002/j.1460-2075.1994.tb06878.x
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发表时间：
1994-11-15
期刊：
影响因子：
11.4
通讯作者：
BAEUERLE, PA
中科院分区：
文献类型：
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作者：
TRAENCKNER, EBM;WILK, S;BAEUERLE, PA
诱导型转录因子 NF-kappa B 的激活涉及从潜在的细胞质复合物中去除抑制性亚基 I kappa B-alpha。据报道，I kappa B-alpha 在 NF-kappa B 激活过程中既经历磷酸化又经历蛋白水解。在这项研究中，我们提供的证据表明多催化胞质蛋白酶（蛋白酶体）参与 I kappa B-alpha 的降解。微摩尔量的肽 Cbz-Ile-Glu(O-t-Bu)-Ala-亮氨酸 (PSI) 是蛋白酶体胰凝乳蛋白酶样活性的特异性抑制剂，可通过抑制 I kappa B-alpha 降解来阻止 NF-kappa B 响应肿瘤坏死因子-α (TNF) 和冈田酸 (OA) 的激活。 m-钙蛋白酶抑制剂 Cbz-Leu-亮氨酸无效。在 PSI 存在的情况下，新磷酸化形式的 Iκ B-α 在 TNF 和 OA 刺激的细胞中积累。然而，I kappa B-alpha 的共价修饰不足以激活 NF-kappa B：细胞中没有出现实质性的 NF-kappa B DNA 结合活性，因为新磷酸化形式的 I kappa B-alpha 仍与 p65 NF-kappa B 紧密结合。吡咯烷二硫代氨基甲酸酯是 NF-kappa B 激活的抗氧化剂抑制剂，不干扰蛋白酶体活性，可防止 I kappa B-alpha 从头磷酸化。 kappa B-alpha 及其随后的降解。这表明 I kappa B-alpha 的磷酸化对于 NF-kappa B 的激活同样必要。与无细胞实验相反，在完整细胞中，激酶反应并未从 NF-kappa B 中释放 I kappa B-alpha，但似乎标记了抑制剂，以便随后被蛋白酶体的胰凝乳蛋白酶样亚基快速降解。
Activation of the inducible transcription factor NF-kappa B involves removal of the inhibitory subunit I kappa B-alpha from a latent cytoplasmic complex, It has been reported that I kappa B-alpha is subject to both phosphorylation and proteolysis in the process of NF-kappa B activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of I kappa B-alpha. Micromolar amounts of the peptide Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), a specific inhibitor of the chymotrypsin-like activity of the proteasome, prevented activation of NF-kappa B in response to tumor necrosis factor-alpha (TNF) and okadaic acid (OA) through inhibition of I kappa B-alpha degradation. The m-calpain inhibitor Cbz-Leu-leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B-alpha accumulated in TNF- and OA-stimulated cells. However, the covalent modification of I kappa B-alpha was not sufficient for activation of NF-kappa B: no substantial NF-kappa B DNA binding activity appeared in cells because the newly phosphorylated form of I kappa B-alpha was still tightly bound to p65 NF-kappa B. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF-kappa B activation which did not interfere with proteasome activities, prevented de novo phosphorylation of I kappa B-alpha as well as its subsequent degradation. This suggests that phosphorylation of I kappa B-alpha is equally necessary for the activation of NF-kappa B. In contrast to cell-free experiments, in intact cells the kinase reaction did not release I kappa B-alpha from NF-kappa B, but appeared to tag the inhibitor for subsequent and rapid degradation by a chymotrypsin-like subunit of the proteasome.