Reduced phosphorylation of p50 is responsible for diminished NF-kappaB 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-kappaB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-kappaB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-kappaB 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-kappaB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-kappaB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-kappaB binding was further demonstrated by showing that an NF-kappaB 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-kappaB in adenovirus-transformed cells.

FIG. 1

Regulation of the MHC class I promoter in adenovirus-transformed cells. Transcription of class I genes is greatly reduced in Ad12- versus Ad5-transformed cells because of increased binding of the COUP-TF repressor to the R2 site and decreased binding of the NF-κB activator to the R1 site of the enhancer. Diminished MHC class I levels correlate with tumorigenic potential. Arrow, transcriptional start site; closed circle, TATA box; gray rectangle, interferon response element (IRS); black rectangle, H-2K^b class I enhancer.

FIG. 2

A freely dissociable inhibitor of NF-κB is not present in Ad12-transformed cell nuclear extracts. One microgram of Ad5-transformed cell nuclear extracts (NE) was incubated in an EMSA reaction alone (lane 2), with 1 ng of purified recombinant IκBα (rIκBα) (lane 3), or with increasing amounts (1, 2, 4, 8, 12, or 16 μg) of Ad12-transformed cell nuclear extracts (lanes 4 to 9), followed by electrophoresis on a nondenaturing gel. The increased binding activity in the titration curve (lanes 4 to 9) was largely contributed by the increasing amounts of Ad12-transformed cell nuclear extracts (compare lanes 1 and 10 [1 and 16 μg, respectively]). Identical results were obtained when the total protein in each reaction mixture was adjusted to 17 μg with BSA (data not shown). 50-50 homodimer and 50-65 heterodimer species are indicated. F, free R1 site probe.

FIG. 3

Denatured-renatured NF-κB from Ad12-transformed cells retains diminished binding activity. (A) Nuclear extracts (NE) from Ad5- and Ad12-transformed cells were analyzed by EMSA either directly (lanes 1 and 2) or following denaturation-renaturation (Den-Ren) of gel-isolated 50- and 65-kDa proteins (lanes 3 and 4). (Lower panel) Western analysis indicating retention of p50 and p65 during the denaturation-renaturation procedure. (B) Nucleoprotein complexes from denatured-renatured NF-κB (lanes 7 to 12) mirrored those from nuclear extracts (lanes 1 to 6) in a supershift analysis with p50 (1613) and p65 (1226) antibodies (Ab). Overexposures of nucleoprotein complexes from lanes 4 to 6 and 10 to 12 are shown in lanes 13 to 15 and 16 to 18, respectively. 50-50 homodimer, 50-65 heterodimer, and supershift (SS) species are indicated. F, free R1 site probe.

FIG. 3

Denatured-renatured NF-κB from Ad12-transformed cells retains diminished binding activity. (A) Nuclear extracts (NE) from Ad5- and Ad12-transformed cells were analyzed by EMSA either directly (lanes 1 and 2) or following denaturation-renaturation (Den-Ren) of gel-isolated 50- and 65-kDa proteins (lanes 3 and 4). (Lower panel) Western analysis indicating retention of p50 and p65 during the denaturation-renaturation procedure. (B) Nucleoprotein complexes from denatured-renatured NF-κB (lanes 7 to 12) mirrored those from nuclear extracts (lanes 1 to 6) in a supershift analysis with p50 (1613) and p65 (1226) antibodies (Ab). Overexposures of nucleoprotein complexes from lanes 4 to 6 and 10 to 12 are shown in lanes 13 to 15 and 16 to 18, respectively. 50-50 homodimer, 50-65 heterodimer, and supershift (SS) species are indicated. F, free R1 site probe.

FIG. 3

Denatured-renatured NF-κB from Ad12-transformed cells retains diminished binding activity. (A) Nuclear extracts (NE) from Ad5- and Ad12-transformed cells were analyzed by EMSA either directly (lanes 1 and 2) or following denaturation-renaturation (Den-Ren) of gel-isolated 50- and 65-kDa proteins (lanes 3 and 4). (Lower panel) Western analysis indicating retention of p50 and p65 during the denaturation-renaturation procedure. (B) Nucleoprotein complexes from denatured-renatured NF-κB (lanes 7 to 12) mirrored those from nuclear extracts (lanes 1 to 6) in a supershift analysis with p50 (1613) and p65 (1226) antibodies (Ab). Overexposures of nucleoprotein complexes from lanes 4 to 6 and 10 to 12 are shown in lanes 13 to 15 and 16 to 18, respectively. 50-50 homodimer, 50-65 heterodimer, and supershift (SS) species are indicated. F, free R1 site probe.

FIG. 4

Low binding activity of denatured-renatured NF-κB from Ad12-transformed cells is restored by DOC treatment. Nuclear extracts (NE) (lanes 1 and 2) and denatured-renatured (Den-Ren) NF-κB (lanes 3 and 4) from Ad12-transformed cells were subjected to EMSA following treatment with DOC (lanes 2 and 4). Migration of the NF-κB complex is indicated (50/65). F, free R1 site probe.

FIG. 5

The p50 subunit contributes to the decreased DNA binding activity of NF-κB in Ad12-transformed cells. Lanes 1 and 2, denatured-renatured (Den-Ren) NF-κB from Ad5- and Ad12-transformed cell nuclear extracts, respectively. Lane 3, hybrid denatured-renatured NF-κB with p50 from Ad5-transformed cell nuclear extracts and p65 from Ad12-transformed cell nuclear extracts. Lane 4, hybrid denatured-renatured NF-κB with p50 from Ad12-transformed cell nuclear extracts and p65 from Ad5-transformed cell nuclear extracts. 50-50 homodimers and 50-65 heterodimers are indicated. F, free R1 site probe.

FIG. 6

DOC enhances DNA binding by affecting p65. Denatured-renatured homodimers of p50 (lanes 1 to 4) or p65 (lanes 5 to 8) from Ad5 (lanes 1, 2, 5, and 6)- and Ad12 (lanes 3, 4, 7, and 8)-transformed cell nuclear extracts were subjected to EMSA following treatment with DOC (even-numbered lanes). The reduced signals in lanes 7 and 8 compared to those in lanes 5 and 6 were due to less denatured-renatured p65 homodimer from Ad12 than from Ad5, respectively, used in the EMSA as determined by quantitative Western analysis. Migration of homodimers is indicated (50/50 and 65/65). F, free R1 site probe.

FIG. 7

Two-dimensional gel analysis of charged p50 species from Ad12- and Ad5-transformed cell nuclear extracts. Equivalent amounts of protein from nuclear extracts (NE) of Ad5- and Ad12-transformed cells were separated by two-dimensional gel electrophoresis, transferred to polyvinylidene difluoride paper, and probed with p50 antibody (1157). A to D, four major charged species of p50; the vertical arrow points to species A. The horizontal arrow pointing to the oval near the negative pole indicates the migration of the tropomyosin internal control, visualized by Coomassie blue staining prior to immunoblotting. The pH in the Western blots ranged from 4.8 (positive pole) to 8.9 (negative pole).

FIG. 8

p50 is less phosphorylated in Ad12- than in Ad5-transformed cells. Normalized lysates from ³²P_i-labeled cells were subjected to sequential immunoprecipitation (IP) with antibodies 1613 and 1157 (reactive to native and denatured p50 epitopes, respectively) to detect phosphorylated p50. The equal amounts of phosphorylated YY1 that were immunoprecipitated from the two cell types verified that the normalization of counts was accurate. The positions of labeled protein species are indicated. The identity of the band (marked with an asterisk) that cross-reacted with the YY1 antisera is unknown.

FIG. 9

Phosphorylation of p50 is critical for NF-κB binding activity. (A) Dephosphorylation of NF-κB ablates binding activity. Nuclear extracts were untreated (lane 1) or treated with CIP in the absence (lane 2) or presence (lane 3) of phosphatase inhibitors prior to EMSA. (B) Phosphorylation of p50 specifically contributes to DNA binding of NF-κB. The denaturation-renaturation procedure included a CIP treatment step after elution of the 50-kDa proteins from the polyacrylamide gel. EMSA reaction mixtures contained NF-κB with CIP-treated p50 (lane 1) or CIP-treated p50 in the presence of phosphatase inhibitors (lane 2) and untreated (phosphorylated) p65. F, free R1 site probe.

FIG. 10

Model showing that NF-κB containing hypophosphorylated p50 has a reduced ability to bind to DNA in Ad12-transformed cells. In contrast, in Ad5-transformed cells, NF-κB containing hyperphosphorylated p50 actively binds to DNA. The black oval in p65 represents its transactivation domain.