Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity
Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity
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DOI：
10.1074/jbc.m110.173781
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发表时间：
2011-02-25
影响因子：
4.8
通讯作者：
Cierniewski, Czeslaw S.
中科院分区：
文献类型：
--
作者：
Boncela, Joanna;Przygodzka, Patrycja;Cierniewski, Czeslaw S.
纤溶酶原激活物抑制剂-1（派-1）是一种多功能蛋白，是纤溶、细胞外基质稳态和细胞运动的重要生理调节因子。最近的观察表明派-1也可能涉及维持细胞的完整性，特别是关于细胞增殖或凋亡。在本研究中，我们提供的证据表明，派-1与蛋白酶体相互作用，并影响其活性。首先，利用酵母双杂交系统，我们发现蛋白酶体的α 3亚基直接与派-1相互作用。然后，为了确保派-1-蛋白酶体复合物在体内形成，从内皮细胞共免疫沉淀两种蛋白质并用特异性抗体鉴定。在用pCMV-PAI-1转染HeLa细胞并将两种蛋白质与抗PAI-1抗体共免疫沉淀后，证明了这种相互作用的特异性。随后，派-1-蛋白酶体复合物的细胞分布通过免疫金染色和电子显微镜分析建立。这两种蛋白质出现在一个弥漫的胞质模式，但也可以发现在一个致密的核周和核的位置。此外，派-1诱导形成的侵略者自由位于内皮细胞质中。共聚焦显微镜和Western免疫印迹证实，派-1表达的增加消除了用pCMV-PAI-1和pd 2 EGFP-N1共转染HeLa细胞后分析的降解决定子的降解，并阻止了p53和I κ B α的降解。
Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the alpha 3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as I kappa B alpha, as evidenced both by confocal microscopy and Western immunoblotting.