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Br J Cancer. 2011 Jan 18;104(2):324-31. doi: 10.1038/sj.bjc.6606026. Epub 2010 Dec 7.

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42.

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  • 1University of Manchester Gynaecological Oncology Laboratories, School of Cancer and Enabling Sciences, St Mary's Hospital, Oxford Road, Manchester M13 9WL, UK.

Abstract

BACKGROUND:

Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF.

METHODS:

Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation.

RESULTS:

Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16.

CONCLUSION:

These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.

PMID:
21139582
[PubMed - indexed for MEDLINE]
PMCID:
PMC3031889
Free PMC Article
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