Ku80 as a novel receptor for thymosin β4 that mediates its intracellular activity different from G-actin sequestering
Ku80 as a novel receptor for thymosin β4 that mediates its intracellular activity different from G-actin sequestering
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DOI：
10.1074/jbc.m707539200
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发表时间：
2008-01-18
影响因子：
4.8
通讯作者：
Cierniewski, Czeslaw S.
中科院分区：
文献类型：
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作者：
Bednarek, Radoslaw;Boncela, Joanna;Cierniewski, Czeslaw S.
我们的数据表明，胸腺素β 4 （T β 4）的细胞内表达增加是诱导内皮细胞纤溶酶原激活物抑制剂1型（PAI-1）基因表达的必要和充分条件。为了描述这种作用的机制，我们制造了功能基序受损的T β 4突变体，并测试了它们在细胞内的位置和活性。与绿色荧光蛋白融合的T β 4（(AcSDKPT/4A)）、T β 4（(KLKKTET/7A)）和T β 4（(K16A)）突变体的细胞质分布与野生型T β 4没有显著差异。T β 4、T β 4（(AcSDKPT/4A)）和T β 4（(K16A)）的过表达影响肌动蛋白丝的细胞内形成。正如预期的那样，细胞核对T β 4（K16A）的摄取受损。另一方面，T β 4（(KLKKTET/7A)）的过表达导致形成典型黏附细胞的肌动蛋白丝网络，表明突变体缺乏肌动蛋白结合位点。细胞内T β 4诱导PAI-1基因的机制不依赖于n端四肽AcSDKP，仅部分依赖于其结合g -肌动蛋白或进入细胞核的能力。T β 4和T β 4（(AcSDKPT/4A)）对PAI-1基因的诱导程度相同，而突变体T β 4（(KLKKTET/7A)）和T β 4（(K16A)）保留了约60%的原始活性。通过蛋白质组学分析，发现atp依赖性DNA解旋酶II的Ku80亚基与T β 4相关。Ku80和T β 4一致地在内皮细胞的复合物中共同免疫沉淀。内皮细胞与Ku80缺失突变体和T β 4共转染表明，Ku80的c端臂结构域直接参与这种相互作用。此外，特异性短干扰RNA下调Ku80可导致PAI-1在mRNA和蛋白合成水平上的表达显著降低。这些数据表明Ku80作为T β 4的新受体并介导其细胞内活性。
Our data demonstrate that increased intracellular expression of thymosin beta 4 (T beta 4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced T beta 4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of T beta 4((AcSDKPT/4A)), T beta 4((KLKKTET/7A)), and T beta 4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type T beta 4. Overexpression of T beta 4, T beta 4((AcSDKPT/4A)), and T beta 4((K16A)) affected intracellular formation of actin filaments. As expected, T beta 4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of T beta 4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular T beta 4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both T beta 4 and T beta 4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants T beta 4((KLKKTET/7A)) and T beta 4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with T beta 4. Ku80 and T beta 4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and T beta 4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for T beta 4 and mediates its intracellular activity.