In this study, we showed that the WW domain of TAZ is important for its oncogenic capability, as well as for its ability to activate transcription of genes such as ITGB2
that are known to be upregulated by TAZ. Mutation of the WW domain of TAZ reduced its transforming ability as assessed by anchorage-independent cell growth in soft agar. Our results are consistent with the finding that the YAP WW domains are required for YAP to transform NIH3T3 cells and for YAP's ability to induce ITGB2 (Zhao et al., 2009
; Zhang et al., 2009b
). We observed no clear role for the TAZ WW domain in mediating interactions with negative regulatory proteins, although these could have been masked if the observed positive regulatory interactions were epistatic with respect to their influence on TAZ activity. Increasing evidence shows that the WW domain of Yorkie, YAP and TAZ interacts with multiple different proteins containing PPXY motifs. Evidence from flies and from mammalian tissue culture studies suggest that the WW domains of Yorkie and YAP interact with both positive and negative regulatory proteins, and therefore it is conceivable that some WW domain-interacting proteins may also execute inhibitory effects on the transforming ability of TAZ and YAP. As such, the regulatory role of the Yorkie, YAP and TAZ WW domains may depend on the cellular context and experimental conditions that may alter the relative abundance of PPXY-containing proteins that interact with Yorkie, YAP and TAZ.
Among the proteins that were pulled down by TAZ and S89A, but not by WWm, were PPXY motif-containing Wbp2, Amot and AmotL1, implying that these proteins interact with the WW domain of TAZ. As WWm has reduced transforming ability, we explored the possibility that TAZ may engage Wbp2 via its WW domain for transforming ability and gene regulation. Consistent with this hypothesis, we provided evidence showing that Wbp2 interacts directly with TAZ and this interaction is likely mediated by the WW domain of TAZ and the second PPXY motif of Wbp2. The functional consequence of this interaction was then established by our observations that the potent transforming ability of S89A is suppressed by knockdown of endogenous Wbp2 and the moderate transforming ability of wild-type TAZ is noticeably enhanced by exogenous Wbp2, supporting a role of Wbp2 in positively regulating the transforming ability of TAZ. Further support for a role of Wbp2 came from the results that the defect of transforming ability of WWm can be rescued by fusing Wbp2 or its C-terminal region to the C-terminus of WWm. In addition, these fusion proteins displayed enhanced transforming ability, suggesting that the physical interaction of Wbp2 with TAZ may significantly empower the transforming potential of TAZ. Furthermore, mutation of the WW domain of the Hippo refractory S89A also reduced its transforming activity, whereas fusion of full-length Wbp2 to the S89A-WWm restored the activity (Supplementary Figure S2). This suggests that Wbp2 interaction with the WW domain is also involved in the much enhanced transforming activity of S89A. The enhanced transforming ability could possibly be explained by the fact that TAZ and Wbp2 would now be constitutively associated and, therefore, Wbp2 would not be subject to competition with other PPXY motif-containing proteins (especially negatively regulatory proteins) for the TAZ WW domain.
As Wbp2 is also a YAP interacting factor (Chen and Sudol, 1995
), we have examined whether it can enhance the transforming ability of YAP. We first created mutant YAP having the first (YAP-WW1m), the second (YAP-WW2m) or both (YAP-WW(1+2)m) WW domains mutated and examined their transforming ability (Supplementary Figure S3). In our study, mutation of WW1, WW2 or both WW domains enhanced the transforming ability of YAP in both NIH3T3 and MCF10A cells as compared with wild-type YAP. These results are different from those reported by Guan's group, where mutations of WW domains of YAP reduced NIH3T3 cell growth in soft agar (Zhao et al., 2009
), and those by Harvey's group, where YAP WW domain mutants reduce NIH3T3 cell growth but enhance MCF10A cell growth in soft agar (Zhang et al., 2009b
). Although this issue remains to be further investigated, one possible explanation is that the WW domains of YAP are primarily interacting with negative factors under our experimental conditions in both NIH3T3 and MCF10A cells. Importantly, when Wbp2 was fused directly to the C terminus of these YAP mutants to create YAP-WW1m-Wbp2, YAP-WW2m-Wbp2 and YAP-WW(1+2)m-Wbp2, these fusions induced more NIH3T3 and MCF10A cell growth in soft agar as compared with their respective WW domain mutants (Supplementary Figure S3), suggesting that Wbp2 potentiates YAP transforming activity. These results suggest that physical interaction of Wbp2 with YAP also has a positive effect on its transforming ability.
YAP and TAZ are able to induce EMT and increase cell proliferation (Overholtzer et al., 2006
; Lei et al., 2008
; Zhang et al., 2009a
). Moreover, WW domains of YAP are essential for the increased cell proliferation, but dispensable for EMT (Zhao et al., 2009
). We have also examined the relevance of the WW domain of TAZ and its interaction with Wbp2 in EMT and cell proliferation. The expression of EMT markers was examined to investigate the role of TAZ WW domain and its interaction with Wbp2 (Supplementary Figure S4A). In our study, the alterations of EMT markers are not as clear-cut as reported, although the general trend holds true. The expression of epithelial marker E-cadherin was suppressed mostly in MCF10A cells expressing S89A. E-cadherin downregulation was noticeable but only moderate in cells expressing TAZ, WWm, WWm-Wbp2 and WWm-Wbp2-C-ter, whereas cells expressing WWm-Wbp2-N-ter had similar levels of E-cadherin as vector-transduced cells. Consistently, vimentin is mostly upregulated in cells expressing WWm-Wbp2-C-ter and S89A. Significant vimentin upregulation was also observed upon overexpression of TAZ, WWm and WWm-Wbp2, but not WWm-Wbp2-N-ter. Furthermore, S89A and WWm-Wbp2-C-ter are relatively more potent in inducing the expression of fibronectin and, to a lesser extent, N-cadherin. The ability of WWm to induce the expression of some EMT markers is similar to TAZ and WWm-Wbp2. These results indicate that WW domain is not very crucial for EMT, although S89A and Wwm-Wbp2-C-ter have enhanced ability to induce EMT. Our results are consistent with the earlier observation that the WW domain of YAP is not very crucial for EMT (Zhao et al., 2009
). Furthermore, cell proliferation assays show that the WW domain of TAZ (Supplementary Figure S4B) and cellular Wbp2 (Supplementary Figure S4C) are both positively involved in cell proliferation.
Consistent with the notion that the role of the WW domains of TAZ and YAP depends on the relative abundance and interacting affinity of various PPXY motif-containing proteins, our preliminary analysis indicates that Amot also interacts with the WW domain of TAZ and YAP and that this interaction seems to suppress the transforming ability of TAZ and YAP. As such, if the positive PPXY motif-containing proteins such as Wbp2 are more dominant in the cell under a certain condition, then the WW domain is important for the transforming ability. On the other hand, if the negative PPXY motif-containing proteins such as Amot are dominant, then the WW domain is involved in suppressing the transforming ability of TAZ and YAP. Future studies will provide better understanding about this hypothesis. The mechanism by which Wbp2 regulates TAZ-mediated transformation and gene expression is currently unknown. HA-Wbp2 is distributed both in the cytoplasm and in the nucleus (Supplementary Figure S5A) as assessed by immunofluorescence microscopy. When Flag-TAZ was expressed alone or together with Wbp2, TAZ is preferentially distributed in the nucleus and coexpressed Wbp2 does not have a significant effect on the localization of TAZ (Supplementary Figure S5B). Furthermore, overexpression of Wbp2 does not affect the phosphorylation or abundance of TAZ (Supplementary Figure S5C) and increase in the amount of Wbp2 does not affect the interaction of TAZ with TEAD4 (Supplementary Figure S6). Therefore, the mechanism underlying Wbp2 action needs to be further defined. A previous study has suggested that it interacts with E6-AP to promote transcriptional activation of estrogen and progesterone receptors (Dhananjayan et al., 2006
). The transcriptional activation of progesterone receptor by Wbp2 may be mediated by the recruitment of YAP. It was shown that the third Wbp2 PPXY motif is most important. One possibility is that via Wbp2, other proteins such as E6-AP may be recruited to TAZ-containing complexes in such a way that transcription of selected genes such as ITGB2
is positively regulated. How the interaction of Wbp2 with TAZ is regulated is another interesting issue because it was reported that Wbp2 is tyrosine-phosphorylated in response to the stimulation by epidermal growth factor in MCF10A cells (Chen et al., 2007
). It will be interesting to examine whether Tyr-phosphorylation of Wbp2 can regulate its interaction with TAZ and transcriptional outcome. Future studies addressing these issues will provide additional insights into the molecular mechanism by which TAZ and Wbp2 coordinately control transcription and cell transformation.