Thrombin induces the release of the Y-box protein dbpB from mRNA: a mechanism of transcriptional activation.
We have recently demonstrated that thrombin induces expression of the platelet-derived growth factor B-chain gene in endothelial cells (EC) through activation of the Y-box binding protein DNA-binding protein B (dbpB). We now present evidence that dbpB is activated by a novel mechanism: proteolytic cleavage leading to release from mRNA, nuclear translocation, and induction of thrombin-responsive genes. Cytosolic, full-length dbpB (50 kDa) was rapidly cleaved to a 30-kDa species upon thrombin stimulation of EC. This truncated, "active" dbpB exhibited nuclear localization and binding affinity for the thrombin response element sequence, which is distinct from the Y-box sequence. Oligo(dT) affinity chromatography revealed that cytosolic dbpB from control EC, but not active dbpB from thrombin-treated EC, was bound to mRNA. Latent dbpB immunoprecipitated from cytosolic extracts of control EC was activated by ribonuclease treatment. Furthermore, when EC cytosolic extracts were subjected to Nycodenz gradient centrifugation, latent dbpB fractionated with mRNA, whereas active dbpB fractionated with free proteins. The cytosolic retention domain of dbpB, which we localized to the region 247-267, was proteolytically cleaved during its activation. In contrast to full-length dbpB, truncated dbpB stimulated platelet-derived growth factor B-chain and tissue factor promoter activity by over 5-fold when transiently cotransfected with reporter constructs. These results suggest a novel mode of transcription factor activation in which an agonist causes release from mRNA of a latent transcription factor leading to its transport to the nucleus and its regulation of target gene expression.
Stenina
O I
OI
Department of Cell Biology, Lerner Research Institute of The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Shaneyfelt
K M
KM
DiCorleto
P E
PE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011132:Polyribosomes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013917:Thrombin / Q000494:pharmacology*; D014157:Transcription Factors; D015533:Transcriptional Activation; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D013917:Thrombin