DNA-Dependent Protein Kinase Mediates YB-1 (Y-Box Binding Protein)-Induced Double Strand Break Repair

Abstract

BACKGROUND: DNA-PK (DNA-dependent protein kinase) is a stress-activated serine/threonine kinase that plays a central role in vascular smooth muscle cell proliferation and vascular proliferative disease processes such as neointimal formation. In this study, we link the activation of DNA-PK to the function of the transcription factor YB-1 (Y-box-binding protein). METHODS: To identify YB-1 phosphorylation by DNA-PK, we generated different YB-1-expressing vectors. YB-1 nuclear translocation was investigated using immunoblotting and immunofluorescence staining. For YB-1 activity, luciferase assays were performed. RESULTS: We show by mutational analysis and kinase assay that the transcriptional regulator YB-1 is a substrate of DNA-PK. Blockade of DNA-PK by specific inhibitors revealed its critical involvement in YB-1phosphorylation as demonstrated by inhibition of an overexpressed YB-1 reporter construct. Using DNA-PK-deficient cells, we demonstrate that the shuttling of YB-1 from the cytoplasm to the nucleus is dependent on DNA-PK and that the N-terminal domain of YB-1 is phosphorylated at threonine 89. Point mutation of YB-1 at this residue abrogated the translocation of YB-1 into the nucleus. The phosphorylation of YB-1 by DNA-PK increased cellular DNA repair after exposure to ionizing radiation. Atherosclerotic tissue specimens were analyzed by immunohistochemistry. The DNA-PK subunits and YB-1 phosphorylated at T89 were found colocalized suggesting their in vivo interaction. In mice, the local application of the specific DNA-PK inhibitor NU7026 via thermosensitive Pluronic F-127 gel around dilated arteries significantly reduced the phosphorylation of YB-1. CONCLUSIONS: DNA-PK directly phosphorylates YB-1 and, this way, modulates YB-1 function. This interaction could be demonstrated in vivo, and colocalization in human atherosclerotic plaques suggests clinical relevance of our finding. Phosphorylation of YB-1 by DNA-PK may represent a novel mechanism governing atherosclerotic plaque progression.

YB1 modulates the DNA damage response in medulloblastoma

“…YB1 was shown to mediate strand separation of cisplatin-bound DNA in addition to driving expression of the MDR1 receptor, resulting in cisplatin efflux 10 – 13 . Additionally, following etoposide or doxorubicin treatment of NIH3T3 cells, the proteolytic YB1 fragment was found to interact with proteins Mre-11 and Rad50 that are responsible for homologous recombination; and, following radiation, YB1 can be phosphorylated by DNA-PKcs to accelerate repair 14 , 15 . As the more deleterious effects of IR involve repair of double strand breaks (DSBs) through homologous recombination (HR) or non-homologous end joining (NHEJ), any role for YB1 in this process could lead to synergism.…”

Inhibition of protein translational machinery in triple-negative breast cancer as a promising therapeutic strategy

“…YB-1 functions as a transcription factor that regulates the transcription and translation of genes involved in proliferation, apoptosis, stress response, DNA repair, and drug resistance. 34 It is well established that ribosomal proteins are not only limited to ribosome complex and protein biosynthesis but are also involved in the development of cancer. 35 , 36 The continued progression of treated cancers demands increased protein synthesis capacity, both to sustain cell proliferation and to counteract the effects of ongoing treatments.…”

Y RNA-derived fragments in a complex with YBX1 modulate PARP1 residency at DNA double strand breaks

“…While it is a known contributor to DNA repair, the exact mechanism by which YBX1 becomes recruited to DNA damage sites remains unknown. It has been suggested by multiple studies to be dependent on RNA molecules(Liu et al, 2016; Nothen et al, 2023), although YBX1 is also known to interact with many other DDR-associated proteins which may influence its chromatin recruitment(Kim et al ., 2013). Moreover, YBX1 contains an intrinsically disordered domain at its C-terminus and has been reported to undergo liquid-liquid phase separation(Liu et al ., 2021).…”