E3 ligase EDD1/UBR5 is utilized by the HPV E6 oncogene to destabilize tumor suppressor TIP60.

Tat-interacting protein of 60 kDa (TIP60) is an essential lysine acetyltransferase implicated in transcription, DNA damage response and apoptosis. TIP60 protein expression is reduced in cancers. In cervical cancers, human papillomavirus (HPV) E6 oncogene targets cellular p53, Bak and some of the PDZ domain-containing proteins for proteasome-mediated degradation through E6AP ligase. Recently, E6 oncogene from high-risk and low-risk categories was also shown to target TIP60. However, the molecular mechanisms and whether destabilization of TIP60 contributes to HPV E6-mediated transformation remain unanswered. Our proteomic analyses revealed EDD1 (E3 identified by differential display), an E3 ligase generally overexpressed in cancers as a novel interacting partner of TIP60. By investigating protein turnover and ubiquitination assays, we show that EDD1 negatively regulates TIP60's stability through the proteasome pathway. Strikingly, HPV E6 uses this function of EDD1 to destabilize TIP60. Colony-formation assays and soft agar assays show that gain of function of TIP60 or depletion of EDD1 in HPV-positive cervical cancer cells significantly inhibits cell growth in vitro. This phenotype is strongly supported by the in-vivo studies where re-activation of TIP60 in cervical cancer cells dramatically reduces tumor formation. In summary, we have discovered a novel ligase through which E6 destabilizes TIP60. Currently, in the absence of an effective therapeutic vaccine for malignant cervical cancers, cervical cancer still remains to be a major disease burden. Hence, our studies implying a distinct tumor suppressor role for TIP60 in cervical cancers show that reactivation of TIP60 could be of therapeutic value.

Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome.

Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology.

The cell polarity regulator hScrib controls ERK activation through a KIM site-dependent interaction.

The cell polarity regulator, human Scribble (hScrib), is a potential tumour suppressor whose loss is a frequent event in late-stage cancer development. Little is yet known about the mode of action of hScrib, although recent reports suggest its role in the regulation of cell signalling. In this study we show that hScrib is a direct regulator of extracellular signal-regulated kinase (ERK). In human keratinocytes, loss of hScrib results in elevated phospho-ERK levels and concomitant increased nuclear translocation of phospho-ERK. We also show that hScrib interacts with ERK through two well-conserved kinase interaction motif (KIM) docking sites, both of which are also required for ERK-induced phosphorylation of hScrib on two distinct residues. Although wild-type hScrib can downregulate activation of ERK and oncogenic Ras co-transforming activity, an hScrib mutant that lacks the carboxy terminal KIM docking site has no such effects. These results provide a clear mechanistic explanation of how hScrib can regulate ERK signalling and begin to explain how loss of hScrib during cancer development can contribute to disease progression.

Inhibition of HPV-16 E7 oncogenic activity by HPV-16 E2.

Human papillomavirus (HPV) E7 is essential in inducing S-phase progression in differentiating epithelial cells. We have previously shown that HPV-16 E7 activity can be controlled by a direct interaction with the viral transcriptional activator E2, thereby inhibiting transforming potential of E7. We have extended these analyses to show that E2 induces a generalized re-localization of E7 within the cell nucleus, one potential consequence of which is the inhibition of E7-induced degradation of pRb. Most importantly, we show that E2 can also inhibit the ability of E7 to induce centrosome abnormalities, thus preventing aberrant mitoses. Taken together, these studies highlight the central importance of E2 in controlling the functions of E7, independently of the ability of E2 to regulate transcription.

Human and primate tumour viruses use PDZ binding as an evolutionarily conserved mechanism of targeting cell polarity regulators.

A unique feature of the cancer-causing mucosotropic human papillomaviruses (HPVs) is the ability of their E6 proteins to interact with a number of PDZ domain-containing cellular substrates, including the cell polarity regulators hDlg and hScrib. These interactions are essential for the ability of these viruses to induce malignant progression. Rhesus papillomaviruses (RhPV) are similar to their human counterparts in that they also cause anogenital malignancy in their host, the Rhesus Macaque. However, unlike HPV E6, the RhPV E6 has no PDZ-binding motif. We now show that such a motif is present on the RhPV E7 oncoprotein. This motif specifically confers PDZ-binding activity and directs the interaction of RhPV E7 with the cell polarity regulator Par3, which it targets for proteasome-mediated degradation. These results demonstrate an amazing evolutionary conservation of function between the RhPV and the HPV oncoproteins, where both target proteins of the same cell polarity control network, although through different components and pathways.

Human papillomaviruses, cervical cancer and cell polarity.

Human papillomaviruses (HPVs) are the causative agents of a number of human cancers, of which cervical cancer is the most important. This occurs following persistent infection with a limited number of viral subtypes and is characterized by continued expression of the viral E6 and E7 oncoproteins. A unique characteristic of the cancer-causing HPV types is the presence of a PDZ recognition motif on the carboxy terminus of the E6 oncoprotein. Through this motif, E6 directs the proteasome-mediated degradation of cellular proteins involved in the regulation of cell polarity and in cell proliferation control. These include components of the Scrib and Par polarity complexes, as well as a number of other PDZ domain-containing substrates. Thus, PVs are now providing novel insights into the functioning of many of these cellular proteins, and into which of these functions, in particular, are relevant for maintaining normal cellular homeostasis. In this review, we discuss the biological consequences of papillomaviral targeting of these cell polarity regulators, both with respect to the viral life cycle and, most importantly, to the development of HPV-induced malignancy.