Restoration of MAGI-1 expression in human papillomavirus-positive tumor cells induces cell growth arrest and apoptosis.
The cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins target a number of cellular proteins that contain PDZ domains. However, the role of many of these interactions in either the HPV life cycle or in HPV-induced malignancy remains to be defined. Previous studies had shown that MAGI-1 was one of the most strongly bound PDZ domain-containing substrates of E6, and one consequence of this interaction appeared to facilitate the perturbation of tight junctions (TJs) by E6. In this study, we describe the generation of a mutation, K499E, within the MAGI-1 PDZ1 domain, which is resistant to E6 targeting. This mutant allows restoration of MAGI-1 expression in HPV-positive cells and defines additional activities of MAGI-1 that are overcome as a consequence of the association with E6. The reexpression of MAGI-1 in HPV-positive cells results in an increased recruitment of ZO-1 and PAR3 to sites of cell-cell contact, repression of cell proliferation, and induction of apoptosis. While the K499E mutation does not significantly affect these intrinsic activities of MAGI-1 in HPV-negative cells, its resistance to E6 targeting in an HPV-positive setting results in more cells expressing the mutant MAGI-1 than the wild-type MAGI-1, with a corresponding increase in TJ assembly, induction of apoptosis, and reduction in cell proliferation. These studies provide compelling evidence of a direct role for the perturbation of MAGI-1 function by E6 in the HPV life cycle and in HPV-induced malignancy.


OBJECTIVE
It is clear that the targeting of PDZ-containing substrates by E6 is important for the normal viral life cycle and for the progression to malignancy. Nevertheless, which of these PDZ domain-containing proteins is relevant for HPV pathology is still elusive. In a previous study, we provided evidence that MAGI-1 is a sensitive proteolytic substrate for both the HPV-16 and HPV-18 E6 oncoproteins; however, the biological consequences associated with loss of MAGI-1 expression in HPV-positive cervical cancer cells are still poorly understood. Using a mutant MAGI-1, resistant to E6-mediated degradation, we show that its expression in cervical cancer cells promotes membrane recruitment of the tight junction-associated proteins ZO-1 and PAR3, represses cell proliferation, and promotes apoptosis. These findings suggest that E6-mediated inhibition of MAGI-1 function contributes to HPV pathology by perturbing tight junction assembly with concomitant stimulation of proliferation and inhibition of apoptosis.
International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy.
Christian
C
Kranjec
International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy.
Paola
P
Massimi
International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy banks@icgeb.org.
Lawrence
L
Banks
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D017209:Apoptosis; D015153:Blotting, Western; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D051528:Guanylate Kinases; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D054731:PDZ Domains; D027383:Papillomaviridae / Q000502:physiology*; D011485:Protein Binding; D059748:Proteolysis; D012097:Repressor Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D051528:Guanylate Kinases; C113338:MAGI1 protein, human