Novel effect of the high risk-HPV E7 CKII phospho-acceptor site on polarity protein expression

Abstract

Background Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV. Methods We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7. Results We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis. Conclusions The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation.

Identification and characterisation of novel potential phospho-acceptor sites in HPV-16 E7

“…Future studies will be focused on determining which regions of these PDZ domain-containing proteins and other targets identified in this work, are important for the phospho-interaction with E7. Additionally, recent studies showed a regulation of DLG1 levels by HPV18 E7 and, moreover, phosphorylation of E7 by CKII modulates the redistribution and stabilization of DLG1 and hScrib [ 47 , 48 ]. These data, together with our results presented here, suggest a possible interplay of E6 and E7 with certain cellular targets.…”