HPV 16 is one of the subtypes of the human papilloma virus that causes cervical cancer; together with HPV 18, it is responsible for about 70% of cases of this disease. Not all HPV 16 variants are equally carcinogenic, however. In high-risk variants of the HPV 16 subtype, a protein known as E6 oncoprotein has been implicated in the process of malignant transformation of human cells, but relatively little is yet known about the mechanism of action of this protein.

In order to gain further understanding of this process, Ian Hampson and his colleagues at the University of Manchester Gynaecological Oncology Laboratories, Manchester, UK, probed the network of proteins that bind to and interact with this protein using a variety of molecular biology techniques.

Previous work by the same group had identified Tax-interacting protein 1 (Tip-1), a class 1 PDZ-domain protein, as interacting with E6, and found that the interaction between these proteins gave rise to increased cell motility. Tip-1 is also known to be involved in several signalling pathways that are important in neoplasia. Hampson and his team used yeast two-hybrid, in vitro pulldown, site directed mutagenesis, PCR, co-immunoprecipitation and Western blotting to probe and identify proteins that interact with Tip-1.

These experiments first identified, and then confirmed, the guanine nucleotide exchange factor 16 (here abbreviated to GEF16) as an interacting partner of Tip-1 and proved that this interaction occurs via the Tip-1 PDZ domain.

When E6 oncoprotein from high-risk, but not from low-risk HPV 16 was expressed in C33A cervical cancer cells, GEF16 mRNA and protein levels were slightly but significantly up-regulated. Following treatment with a proteasome inhibitor to prevent protein degradation, GEF16 was then found to co-immunoprecipitate with Tip-1 and the cell cycle GTPase Cdc42 in the presence of E6 from high-risk HPV16 only.

This finding, and the presence of a Cdc42 binding motif in the GEF16 protein sequence, indicated that GEF16 might activate Cdc42, pushing cells through the cell cycle. Both ELISA and an in vitro fluorescence kinetic assay were used to prove that this Cdc42 activation took place, and ELISA was further used to show that activation could be reduced by siRNA silencing of GEF16 or Tip-1.

Together, these results show that Tip-1, already known to bind to HPV 16 oncoprotein E6, also interacts with GEF16 and that this complex is likely to play a role in the activation of the cell cycle GTPase Cdc42. This interaction and activation, which only takes place with high-risk variants of HPV 16, is likely to be an important factor in viral carcinogenesis. Hampson and his colleagues note further that investigations of the exact nature of the molecular mechanisms of these interactions should prove useful.

Reference

Oliver, A.W., He, X., Borthwick, K., Donne, A.J., Hampson, L. and Hampson, I.N. (2010) The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42 British Journal of Cancer published online ahead of print 7 Dec 2010 doi:10.1038/sj.bjc.6606026