Ku80 as a novel receptor for thymosin β4 that mediates its intracellular activity different from G-actin sequestering

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.