Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering.
Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) 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 Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((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 Tbeta4 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 Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((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 Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 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 Tbeta4 and mediates its intracellular activity.
Bednarek
Radoslaw
R
Center of Medical Biology, Polish Academy of Sciences, Lodz 93-232, Poland.
Boncela
Joanna
J
Center of Medical Biology, Polish Academy of Sciences, Lodz 93-232, Poland.
Smolarczyk
Katarzyna
K
Center of Medical Biology, Polish Academy of Sciences, Lodz 93-232, Poland.
Cierniewska-Cieslak
Aleksandra
A
Department of Molecular and Medical Biophysics, Medical University of Lodz, Lodz 92-215, Poland.
Wyroba
Elzbieta
E
The Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw 02-093, Poland.
Cierniewski
Czeslaw S
CS
Center of Medical Biology, Polish Academy of Sciences, Lodz 93-232, Poland; Department of Molecular and Medical Biophysics, Medical University of Lodz, Lodz 92-215, Poland. Electronic address: cciern@zdn.am.lodz.pl.
D000199:Actins / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D042783:Endothelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000378:metabolism; D013947:Thymosin / Q000235:genetics / Q000378:metabolism*; D000099201:Thymosin beta(4)
D000199:Actins; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D000072200:Ku Autoantigen; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C000606509:Xrcc6 protein, human