Identification and characterization of novel HPV E6 interacting proteins

E6 is one of the major transforming proteins from Human Papillomavirus (HPV). The transforming properties of HPV E6 results in part from its ability to complex with and modulate the action of host cell proteins. We have used the yeast two-hybrid system to screen a cDNA library prepared from normal human epidermal keratinocytes and identified novel protein partners for HPV E6 proteins.; A clone that encoded Gps2 interacted with E6 proteins from high and low oncogenic risk HPVs. The specificity of these reactions was verified and the regions of E6 that were required for interaction were mapped. Steady-state and pulse chase analyses of cells cotransfected with DNAs expressing either ₆E6 or ₁₈E6 and Gps2 demonstrated that the E6 proteins induced the degradation of Gps2 in vivo, but not in vitro . Gps2 exhibited transcriptional activation activity and high risk E6 suppressed this activity.; Zyxin was identified from the library by using ₆E6 as bait. A focal adhesion molecule, zyxin interacted specifically with the E6 protein from HPV type 6. Zyxin did not interact significantly with E6 proteins from HPVs 11, 16 or 18. The interaction was confirmed by in vitro and in vivo analyses and it required the LIM domains [Lin-11, Islet-1 and Mec-3] found at the carboxyl terminus of zyxin. Zyxin could function as a transcriptional activator. When cotransfected with ₆E6, zyxin accumulated in the nucleus.; HINADL is the human homologue of a Drosophila INAD protein. It contains multiple PDZ domains, a protein binding motif. Identified from the library using ₁₈E6 as bait, hINADL was found to interact with ₁₈E6 but not ₆E6 in the in vitro assays. The 3rd PDZ domain in hINADL was necessary but not sufficient for the interaction. For ₁₈E6, the PDZ binding motif at the C-terminus was required.