EBV-encoded EBNA-5 binds to p18ARF and prevents p53-induced growth arrest and apoptosis
E. Kashuba, PhD, Associated Professor
Institute of Experimental Pathology, Oncology and Radiobiology of National Academy of Sciences of Ukraine, Kyiv, Ukraine
MTC, Karolinska Institute, 171 77, Stockholm, Sweden
The EBV transformed LCLs maintain wild type p53 during serial culturing. We addressed the question why the p53 downstream growth arrest and apoptotic pathways are non-functional in p53 expressing LCLs, unless activated by genotoxic agents, by examining p53 containing protein complexes in LCLs.
We have shown that trimolecular complexes were formed where MDM2 served as a bridge between EBNA-5 and p53. All three proteins colocalized in the nucleus of lymphoblastoid cells with a high p53 expression.
To explore the functional consequences of the MDM2–EBNA-5 binding, p53 polyubiquitination and degradation assays were performed in vitro. GST-EBNA-5 inhibited MDM2-dependent polyubiquitination (but not monoubiquitination) of p53, similarly to GST-p14ARF. The p53 degradation on commercially purified 26S proteasome subunits was inhibited by EBNA-5 as well.
These findings indicate that the high p53 levels in LCL are due to EBNA-5 dependent inhibition of p53 degradation. Chromatin immunoprecipitation showed that p53 could bind to the p21 promoter in mitogen stimulated but not the EBV activated B-cells. Taking together, these findings are consistent with the interpretation that the trimolecular EBNA-5-MDM-2-p53 complex inhibits the binding of p53 to DNA.
We have cloned full length of human p14ARF protein (p18ARF) from the EST clone. We have shown by GST pull-down and transfections experiments that p18ARF binds to MDM2, MDMX, and EBNA-5. We have found that an enhanced expression of p18ARF in cells bearing wt p53 resulted in the cell death, similarly to mouse p19ARF. Simultaneous expression of p18ARF and EBNA-5 rescued these cells. Biological significance of the binding between MDMX, p18ARF and EBNA-5 is currently under investigation.
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