In this study, we have shown that the HPV16 E6 binding protein Tip-1 (Hampson et al, 2004
) interacts with the Dbl family member GEF16 via its carboxyl PDZ binding sequence and that ectopic expression of high-risk, but not low-risk, E6 is associated with a modest upregulation of GEF16 in both transformed and non-transformed cells. It is significant that overexpression of GEFs can promote malignant transformation and progression (Rossman et al, 2005
; Fritz and Kaina, 2006
), and we have previously shown that ectopic expression of GEF16 transforms NIH3T3 cells (Hampson et al, 2009
Guanidine exchange factors promote the GTP activation of Rho GTPase's by catalysing the exchange of GDP for GTP. The 48-kDa GEF16 protein reported here is one of the smallest Dbl-related GEFs containing one src
homology 3 domain in addition to the minimal structural unit (PH/DH domains) and a PDZ binding motif at the C-terminal (accession no. NM_014448.3). Bioinformatic analysis indicated that GEF16 has a putative Cdc42 binding sequence QRTLQKL located in its DH domain (Blanke and Jackle, 2006
), which prompted us to investigate its ability to activate this particular Rho protein.
Initial evidence supporting this hypothesis was provided by the observation that GEF16 co-immunoprecipitated with both Tip-1 and Cdc42, although this was only found when high-risk E6 was also present and when cells were pretreated with the proteasome inhibitor MG132. In the absence of MG132, GEF16 did not co-precipitate with Tip-1 in either control or E6-expressing cells, which suggests that proteasomal degradation may influence this interaction. Indeed, MG132 is known to stabilise the interaction of the Rho-GEF Pbl/ECT2 with the E3 ligase E6AP (Reiter et al, 2006
), and the suggestion that GEF levels may be regulated by the proteasome has been previously reported by others. Examples include the Cdc42-specific GEF h-PEM2, which is subject to proteasomal degradation via the HECT-type E3 ligase Smurf1 (Yamaguchi et al, 2008
) and the Cdc42-specific GEF FWD1, which is stabilized in the presence of MG132 (Hayakawa et al, 2005
). Indeed, we have shown that the full-length GEF16 protein is unstable in yeast cells and it contains PEST sequences, which have previously been shown to signal rapid degradation in yeast (Marchal et al, 1998
In light of these stability problems, we initially opted to analyse the ability of recombinant GEF16 to activate Rho proteins using a kinetic in vitro
system. This showed that GEF16 did not activate Rho A, had a modest ability to activate Rac-1, but showed the largest activity with Cdc42. These experiments were then extended to evaluate the effects of adding recombinant GEF16 plus Tip-1, and GEF16 plus Tip-1 and HPV16 E6 to the Cdc42 assay system, with the result that the greatest activation was observed when all three proteins were present. This in vitro
assay system has been used very successfully by others (Guilluy et al, 2010
), but, as it is based on the use of recombinant proteins, we employed two additional in-cell test systems to establish whether these effects occurred with native proteins in situ
. Both of these in-cell assays showed that high-risk E6-expressing cells had the greater levels of Cdc42 activation when compared with vector control or low-risk T6 E6-expressing cells. Furthermore, since silencing expression of either Tip-1 or GEF16 produced a highly significant (P
=0.001) reduction in Cdc42 activation in T16 E6-expressing cells, this provides clear evidence that these two proteins are involved with the ability of T16 E6 to upregulate Cdc42 activation in cells. The observation that silencing Tip-1 has little effect on Cdc42 activation in T6 E6 and vector control cells reinforces the conclusion that T16 E6 upregulates Cdc42 activation through both Tip-1 and GEF16.
While the interplay between GEF16, Tip-1 and E6 and the subsequent alterations in activated Cdc42 levels are clearly complex and still require further elucidation, the results presented suggest that GEF16-dependent activation of Cdc42 may be upregulated by high-risk E6. One potential explanation for these findings may be the uniqueness of Tip-1 as a PDZ protein in that it contains only a single class 1 PDZ domain. It is known that the majority of PDZ proteins contain multiple protein–protein interaction domains, which act as molecular scaffolds important for a variety of cellular functions including cell signalling. The Tip-1 protein is known to form dimers in solution (Aledo et al, 2001
) and it has been reported to be a negative regulator of PDZ-based scaffolding assemblies (Alewine et al, 2006
). Therefore, it is possible that the binding of HPV16 E6 to the Tip-1 PDZ domain may affect its ability to interact with and regulate a signalling pathway involving GEF16 and Cdc42, resulting in an increase in activated Cdc42 levels. Indeed, this is supported by the previously reported interactions of Scrib, β
PIX and Cdc42. Scrib is a multidomain LRR/PDZ scaffold protein, which controls the localisation and activation of Cdc42 via its interaction with β
PIX, which is a Cdc42-activating GEF (Osmani et al, 2006
). Furthermore, it is highly significant that HPV16 E6 has also been shown to interact with Scrib, yet, unlike Tip-1, this results in its proteasomal degradation (Nakagawa and Huibregtse, 2000
). Thus, it is hypothesised that the interaction of high-risk E6 with PDZ proteins, such as Scrib and Tip-1, may cooperate to perturb the delicate balance of interactions between GEFs (β
PIX and/or GEF16) with Cdc42. This coordinated assault by E6 ultimately leads to deregulated control of Cdc42 activation.
Our data are not without precedent since it is well known that other viral oncoproteins can affect Rho protein function. For example, HTLV1 Tax, which interestingly like HPV high-risk E6 also interacts with Tip-1, has been shown to interact directly with Rac, Rho and most significantly Cdc42 (Wu et al, 2004
). Similarly, the Epstein–Barr virus LMP1 oncoprotein can promote the activation of Cdc42 signalling (Eliopoulos and Young, 2001
Therefore, in summary our data provide evidence that there are pathways connecting the expression of the high-risk HPV type 16 E6 protein to enhanced Cdc42 activation and Tip-1 and GEF16 function as components of this system. The exact nature of the interplay between these proteins and the precise molecular mechanisms by which they elicit Cdc42 activation are worthy of further investigation.