Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein

Abstract

The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. Screening a lambdagt11 cDNA expression library with a 9ORF1 protein probe yielded a novel cellular PDZ domain-containing protein, 9BP-1, which binds to wild-type, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly, in vitro and in vivo, with the PDZ domain-containing protein hDlg/SAP97 (DLG), which is a mammalian homolog of the Drosophila discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domain-binding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG in vitro. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.

Figure 1

Nucleotide and predicted amino acid sequences of the 2.7-kb partial murine 9BP-1 cDNA recovered from the 9BP-1 phage. The underlined and bold 82–84 amino acid residue segments designate four protein regions having sequence similarity to PDZ domains. Underlined nucleotides denote the putative 9BP-1 polyadenylylation signal.

Figure 2

Association of the 9ORF1 oncoprotein with the cellular factor DLG in vitro. (A) Binding of a wild-type GST-9ORF1 protein probe to bacterially expressed membrane-immobilized GST-DLG proteins (1 μg) in a protein blotting assay. Arrowheads show locations of GST-NT and GST-SH3/GuK proteins that did not bind to the GST-9ORF1 probe. The multiple bands seen with full length DLG fusion protein are probably proteolytic products of this large fusion protein. (B) An illustration of the DLG protein fragments used in A and summary of their corresponding 9ORF1 protein-binding activities. ++++, strong binding activity; ++, moderate binding activity; +, weak binding activity; −, no detectable binding activity. (C) Interaction of wild-type and transformation-defective C-terminal mutant 9ORF1 proteins with cellular DLG in vitro. GST pull-down reactions with the indicated GST fusion proteins were performed with 1 mg of protein from CREF cell lysates, and recovered cellular proteins were immunoblotted with rabbit polyclonal antiserum to DLG (9). Protein bands were visualized with an enhanced chemiluminescence detection system. (D) Interaction of C-terminal mutant 9ORF1 proteins and C-terminal 9ORF1 fragments with cellular DLG in vitro. GST pull-down reactions were performed as described in C. See Table 1 for description of mutant 9ORF1 proteins.

Figure 3

Association of the 9ORF1 oncoprotein with DLG in vivo. (A) Coimmunoprecipitation of cellular DLG with the 9ORF1 protein. Two milligrams of protein from cell lysates prepared from the indicated CREF cell pool were subjected to immunoprecipitation with 2.5 μl of 9ORF1 rabbit polyclonal antiserum. Precipitated proteins were separated by SDS/PAGE, transferred to a poly(vinylidene fluoride) membrane, and immunoblotted with either DLG rabbit polyclonal antiserum (9) (upper three panels) or 9ORF1 antiserum (lower three panels). (B) Coimmunoprecipitation of the 9ORF1 protein with cellular DLG. These experiments were performed as described in A except that cellular lysates were immunoprecipitated with 0.4 μg of affinity-purified DLG rabbit polyclonal antibodies (34). Membranes were immunoblotted with either 9ORF1 antiserum (upper three panels) or DLG antiserum (lower three panels). IP, immunoprecipitation.

Figure 4

Association of HTLV-1 Tax and HPV E6 oncoproteins with DLG in vitro. GST pull-down reactions with the indicated GST fusion protein were performed as described in the legend to Fig. 2 except that 500 μg of protein from human TE85 cell lysate was used per reaction.