Differential regulation of human papillomavirus E6 by protein kinase A: conditional degradation of human discs large protein by oncogenic E6

Abstract

The protein Kinase A (PKA) pathway was found to selectively regulate the function of oncogenic but not non-oncogenic E6 proteins. High risk E6 proteins are phosphorylated at their Dlg/PDZ binding motif at the C-terminus by a PKA like activity. This PKA and PDZ binding module is found only for human PV, is strictly conserved in all the transforming HPVs and is absent in all the low risk HPV types. We present evidence of a conditional regulation of E6 induced degradation of Dlg. HPV18E6 positive but not HPV negative keratinocytes exhibit increased Dlg steady state levels under conditions of high PKA activity, with a concomitant increase in the presence of Dlg at tight junctions. In vitro binding experiments show that E6 phosphorylation by PKA reduces its binding to Dlg and molecular modelling can explain this observation in a structural context. E6 dependent degradation of Dlg in cells with high PKA levels is inhibited and this is dependent on phosphorylation of the PDZ binding site in E6. In contrast, the degradation of p53 induced by E6 is not a ected by PKA. We propose a di erential regulation of E6 for the ubiquitin mediated degradation of speci®c E6 target proteins. Oncogene (2000) 19, 5884 ± 5891.

Differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus–associated lesions as a marker for progression to malignancy

“…E6 phosphorylation by PKA reduces its capacity for binding and degrading Dlg. 22 It is possible that in the epithelial cells of SIL specimens E6 phosphorylation by PKA reduces its ability to bind Dlg and induce its degradation, explaining the high levels of Dlg observed in these samples. We investigated biopsies from other HPV-associated, intraepithelial, noninvasive pathologies.…”

“…6 In addition, this E6 function was specifically regulated by PKA phosphorylation of the E6 carboxy-terminal PDZ-binding motif. 22 E6 oncoproteins promote the degradation of other PDZ proteins, such as the human homologue of the Drosophila scribble oncosuppressor, h-scrib. 7 The ability of E6 to degrade 2 proteins with similar cellular functions emphasizes the relevance of these E6 activities to HPV-associated carcinogenesis.…”

Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation

“…The PDZ domains are stretches of 80 -90 amino acids (Ponting and Phillips, 1995;Fanning and Anderson, 1999) that bind with high affinity to specific sequences (T/SXV) (Songyang et al, 1997), usually found in the extreme carboxy termini of their target proteins. The binding of certain of these sequences has been reported to be modified by phosphorylation (Ku¨hne et al, 2000;Tolkacheva et al, 2001;Choi et al, 2002) and it seems likely that this may be a more general mechanism for the temporal or spatial control of such protein interactions. PDZ binding motifs have been identified in a number of virus proteins that are associated with oncogenesis, including the HTLV-1 Tax protein, Adenovirus 9 E4-ORF1 protein and the E6 proteins of high-risk HPV types (Lee et al, 1997).…”

“…The high risk HPV E6 proteins have been shown to be involved in de-regulating aspects of cell homeostasis related to cell growth and polarity in response to cell contact, through the degradation of hDlg (Gardiol et al, 1999;Ku¨hne et al, 2000;Pim et al, 2000; and MAGI-1 (Glaunsinger et al, 2000;Thomas et al, 2001). Given that MAGI-2 and MAGI-3 are highly homologous to MAGI-1, and that they are associated with such fundamental growth control pathways, it was obviously of interest to determine whether the high-risk HPV E6 proteins are able to interact with them.…”

Crosstalk between the human papillomavirus E2 transcriptional activator and the E6 oncoprotein

“…For GST fusion protein production, HPV-16 E1, HPV-16 E2, HPV-16 E6, HPV-18 E6, HPV-18 E6*I, HPV-18 E6*I DM, HPV-16 E7 and Dlg fusion proteins were used as described previously Piccini et al, 1995;Massimi et al, 1996;Lee et al, 1997;Pim et al, 1997;Pim and Banks, 1999;Kuhne et al, 2000). GST-11 E2 was generated by cloning into pGEX-2T (BamHI/EcoRI) PCR products obtained by amplification with the following primers: 5 0 -AGCGGATCCATGGAAGCAATA-3 0 (F), 5 0 -AGC-GAATTCTTACAATAAATGTAATGA-3 0 (R).…”