Reduced Phosphorylation of p50 Is Responsible for Diminished NF-κB Binding to the Major Histocompatibility Complex Class I Enhancer in Adenovirus Type 12-Transformed Cells

Abstract

Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-κB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-κB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-κB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-κB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-κB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-κB binding was further demonstrated by showing that an NF-κB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-κB in adenovirus-transformed cells.