Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes

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Abstract
Human papillomavirus E6 oncoproteins induce the proteasomal degradation of several multi-PDZ (PSD95/Dlg/ZO-1) domain-containing proteins such as the human homologue of Drosophila discs large. Binding to PDZ domain-containing proteins is mediated by a PDZ-binding motif contained within the C-terminus of E6. The ability of E6 proteins to induce degradation of PDZ domain-containing proteins correlates with their oncogenic potential. Here we examined the biological effect of this region of the human papillomavirus type 18 E6 oncoprotein on keratinocyte morphology. Our results show that in simian virus 40-immortalized human keratinocytes, stable expression of E6 correlated with the induction of an exaggerated mesenchymal-like morphology and actin cytoskeleton disorganization compared with parental cells. The altered phenotype was accentuated in cells expressing an E6 protein containing a mutation (Arg153Leu) within a protein kinase A recognition motif that abrogates protein kinase A's negative regulation of the activity of the PDZ-binding domain. The E6-induced changes indicated an epithelial-mesenchymal transition and were supported by the finding that E6-expressing cells contained vimentin. Changes to the epithelial phenotype of cells expressing a mutant E6 protein (Thr156Glu) that is unable to degrade discs large was significantly less marked, although they did show evidence of epithelial-mesenchymal transition. These observations imply that the activity of the E6 PDZ-binding motif contributes only to a part of the transition. Further analysis of the E6 cell lines showed a decrease in adherens junction and desmosome formation. Cells expressing a functional PDZ-binding motif showed the greatest disruption of intercellular junction formation, but this did not correlate with a decrease in total cellular levels of the individual components of adhesion junctions. This suggests that the activity of the PDZ-binding motif may have influenced either the assembly or integrity of functional adhesion complexes. An E6-mediated decrease in peripheral membrane levels of PDZ proteins like discs large could be the basis for the enhanced morphological transformation of immortalized keratinocytes.