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

Abstract

Our data demonstrate that increased intracellular expression of thymosin β4 (Tβ4) 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 Tβ4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tβ4(AcSDKPT/4A), Tβ4 (KLKKTET/7A), and Tβ4(K16A) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tβ4. Overexpression of Tβ4, Tβ4(AcSDKPT/4A), and Tβ4(K16A) affected intracellular formation of actin filaments. As expected, Tβ4(K16A) uptake by nuclei was impaired. On the other hand, overexpression of Tβ4(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 Tβ4 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 Tβ4 and Tβ4(AcSDKPT/4A) induced the PAI-1 gene to the same extent, whereas mutants Tβ4(KLKKTET/7A) and Tβ4(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 Tβ4. Ku80 and Tβ4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tβ4 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 Tβ4 and mediates its intracellular activity. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.