Singh S, Darnay B G, Aggarwal B B
Cytokine Research Laboratory, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 1996 Dec 6;271(49):31049-54. doi: 10.1074/jbc.271.49.31049.
The transcription factor NF-kappaB is retained in the cytoplasm by its interaction with the inhibitory subunit known as IkappaB. Signal-induced serine phosphorylation and subsequent ubiquitination of IkappaBalpha target it for degradation by the 26 S proteasome. Recently, pervanadate, a protein-tyrosine phosphatase inhibitor, was shown to block the degradation of IkappaBalpha, thus inhibiting NF-kappaB activation. We investigated the mechanism by which pervanadate inhibits the degradation of IkappaBalpha. Western blot analysis of IkappaBalpha from tumor necrosis factor-treated cells revealed a slower migrating IkappaBalpha species that was subsequently degraded. However, pervanadate-treated cells also revealed a slower migrating species of IkappaBalpha that appeared in a time- and dose-dependent manner and was not degraded by tumor necrosis factor. The slower migrating species of IkappaBalpha from pervanadate-treated cells was tyrosine-phosphorylated as revealed by cross-reactivity with anti-phosphotyrosine antibodies, by the ability of the specific tyrosine phosphatase PTP1B to dephosphorylate it, and by phosphoamino acid analysis of IkappaBalpha immunoprecipitated from 32P-labeled cells. By site-specific mutagenesis and deletion analysis, we identified Tyr-42 on IkappaBalpha as the phosphoacceptor site. Furthermore, in an in vitro reconstitution system, tyrosine-phosphorylated IkappaBalpha was protected from degradation. Our results demonstrate that inducible phosphorylation and degradation of IkappaBalpha are negatively regulated by phosphorylation at Tyr-42, thus preventing NF-kappaB activation.
转录因子NF-κB通过与称为IκB的抑制亚基相互作用而保留在细胞质中。信号诱导的IκBα丝氨酸磷酸化及随后的泛素化使其成为26S蛋白酶体降解的靶标。最近，蛋白酪氨酸磷酸酶抑制剂过钒酸钠被证明可阻断IκBα的降解，从而抑制NF-κB激活。我们研究了过钒酸钠抑制IκBα降解的机制。对经肿瘤坏死因子处理的细胞中的IκBα进行蛋白质印迹分析，发现一种迁移较慢的IκBα物种，随后其被降解。然而，用过钒酸钠处理的细胞也显示出一种迁移较慢的IκBα物种，其以时间和剂量依赖性方式出现，且不被肿瘤坏死因子降解。用过钒酸钠处理的细胞中迁移较慢的IκBα物种被酪氨酸磷酸化，这通过与抗磷酸酪氨酸抗体的交叉反应、特异性酪氨酸磷酸酶PTP1B使其去磷酸化的能力以及对从32P标记细胞中免疫沉淀的IκBα进行磷酸氨基酸分析得以揭示。通过位点特异性诱变和缺失分析，我们确定IκBα上的Tyr-42为磷酸化位点。此外，在体外重组系统中，酪氨酸磷酸化的IκBα受到保护而不被降解。我们的结果表明，IκBα的诱导性磷酸化和降解受到Tyr-42磷酸化的负调控，从而阻止NF-κB激活。
