Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein

Abstract

In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). In this study, we demonstrate that these E6 proteins can bind to the second PDZ domain of the human homologue of the Drosophila discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers.

Figure 1

An XS/TXV/L motif conserved specifically among E6 proteins of high-risk mucosotropic HPVs. HPV16 E6 polypeptides and the C-terminal region sequence alignment of several HPV E6 proteins are represented schematically. The two zinc-finger domains (ZF1 and ZF2) are indicated with the position numbers of the amino acid residues. The whole sequences of HPV E6 proteins were aligned by clustalw (version 1.6; Human Genome Center, Baylor College of Medicine), but only the results for the C-terminal region are presented. The sequences used as the C-terminal oligopeptides (CTs) are underlined. Arabic numbers prior to the amino acid sequences shown as single letter abbreviations indicate the HPV types.

Figure 2

Interactions of E6 proteins of high-risk mucosotropic HPVs as well as APC with hDLG, and their inhibition by C-terminal oligopeptides (CTs) corresponding to high-risk mucosotropic HPV E6 proteins (see Fig. 1). (A) Radiolabeled IVT-hDLG was incubated with various MBP-E6 proteins or with MBP-βgal (β-galatosidase of Escherichia coli), and the captured proteins together with 5% equivalents of the input IVT-hDLG were resolved by SDS/PAGE. Autoradiographic electropherograms of the areas where IVT-hDLG proteins are expected to migrate are shown. (B) IVT-E6 proteins of various HPVs were mixed with GST or GST-hDLG protein-Sepharose (Upper), and the captured proteins were treated as for A. (Lower) Autoradiogram of 5% equivalents of the input IVT-E6s. (C) Mixtures of IVT-hDLG and MBP-16E6-Sepharose were coincubated with the indicated amounts (μg) of various CTs, and the captured proteins were treated as in A. (D) Same as for C except that mixtures of GST-APC-C369 protein-Sepharose and IVT-hDLG were used.

Figure 3

Determination of the hDLG protein segment necessary for binding to HPV16 E6 protein. (A) Schematic representation of hDLG and its deletion mutants (33). The locations of the three repeats (R1, R2, and R3) of the PDZ domain (PDZ), a Src homology 3 (SH3) domain, and a putative guanylate kinase domain (GK) are indicated. (B) Various segments of hDLG shown in A were prepared as GST-fused forms immobilized to glutathione-Sepharose, and they or GST were mixed with IVT-16E6. The captured proteins were treated as in Fig. 2A. (C) Mixtures of MBP-16E6-Sepharose and various segments of hDLG (see A) as IVT-forms were treated as in Fig. 2A Upper. The lower panel shows 5% equivalents of IVT products similarly treated. Arrowheads indicate positions of the bands corresponding to the specific IVT products.

Figure 4

Role of the C-terminal region of HPV16 E6 protein in its interaction with hDLG. (A) Mutant 16E6s whose 151st (C-end) amino acid residue was modified, and 11E6 as a control, were prepared as IVT forms mixed with GST-hDLG-Sepharose, and the captured proteins were treated as for Fig. 2A. The autoradiographic profiles of the areas where IVT-E6 mutant proteins were expected to migrate are shown. Five percent equivalents of the input IVT-E6s are also illustrated. (B) The mutant 16E6s examined in A were tagged with a myc-epitope and expressed in COS cells because high-avidity antibodies against 16E6 are not available. Cell lysates were then subjected to immunoprecipitation with anti-myc epitope antibody (9E10), and the precipitates were fractionated by SDS/PAGE and immunoblotted with anti-hDLG (33) and rabbit anti myc tag antibodies. Five percent equivalents of the lysates used for the immunoprecipitation were also similarly treated with the rabbit anti-myc tag antibodies for comparison.