The TAZ transcription coactivator has been implicated in cell differentiation and organ development (10
). The physiological regulation of the TAZ protein is not clear. Our study demonstrates that TAZ is regulated by the Hippo pathway, which is a novel tumor suppressor pathway initially defined by Drosophila
genetic studies. Our data also indicate a role for TAZ in cell proliferation and EMT.
During the course of this study, it was reported that YAP is phosphorylated and inhibited by the Hippo pathway (6
). TAZ shares approximately 50% sequence identity with YAP. We have determined that serine 89 in the HXRXXS motif is the major Lats2 phosphorylation site in TAZ, while serine 311 is also a functionally important site. Interestingly, the major Lats-dependent phosphorylation site in TAZ (Ser89) identified in this study corresponds to the major Lats-dependent phosphorylation site in YAP (Ser127), reported by Dong et al. (6
). We also have supporting data indicating that YAP Ser127 is the major Lats phosphorylation site.
Several lines of evidence support the phosphorylation of TAZ by Lats2. First, TAZ contains four conserved HXRXXS motifs, which likely represent the Lats recognition consensus. Second, the coexpression of Lats2 induces a mobility shift of TAZ, and this mobility shift can be completely reversed by lambda phosphatase treatment. Third, Lats2 coexpression increases the phosphorylation of TAZ Ser89, as indicated by Western blotting with the phosphoYAP(Ser127) antibody. Fourth, Lats2 directly phosphorylates TAZ in an in vitro kinase assay. Fifth, a knockdown of endogenous Lats significantly decreases TAZ Ser89 phosphorylation. Sixth, Lats2 promotes cytoplasmic localization of the wild-type TAZ but not the S89A mutant. Furthermore, the mutation of Ser89 or Ser311 resulted in a TAZ mutant that is partially resistant to inhibition by Lats2.
It has been reported that Akt/PKB was identified as a kinase that phosphorylated YAP at its 14-3-3 binding site and inhibited its transactivation function (1
). However, the reported YAP inhibition by Akt-dependent phosphorylation was inconsistent with the findings in our studies and those of Dong et al. that Lats2 was responsible for YAP2 Ser127 phosphorylation but not that of AKT (6
). This raises the question of whether Akt also phosphorylates TAZ at its 14-3-3 binding site (Ser89). Interestingly, Hong et al. have also reported that Akt does not phosphorylate TAZ at its 14-3-3 binding site (Ser89) (10
). Therefore, we propose that TAZ is phosphorylated and inhibited by Lats but not by AKT. Matallanas et al. have recently reported that RASSF1A alleviated YAP1 cytoplasmic retention, thereby culminating in p73-mediated apoptosis, indicating that Yap1 might play a critical role in tumor suppression (18
). These observations are inconsistent with the prevailing model in which YAP functions as an oncogene and is inhibited by the Hippo tumor suppressor pathway. Future studies are required to fully understand the cellular function of YAP and TAZ.
TAZ shares approximately 50% sequence identity with YAP1 and YAP2, which are alternative splicing products of the YAP gene. Both YAP and TAZ are phosphorylated and inhibited by the Lats kinase. YAP controls organ size and has been implicated in cancer development. Consistently we found that TAZ also promotes cell growth. Furthermore, Yap has been reported to induce EMT, while our study also indicates a role for TAZ in promoting EMT. Therefore, YAP and TAZ likely will have overlapping and distinct functions.
TAZ knockout mice have been independently reported by two groups (9
). Hossain et al. showed that Wwtr1-null mice have only minor skeletal defects but develop renal cysts (11
), and Makita et al. reported that TAZ inactivation in mice results in pathological changes in the kidney and lung, resembling polycystic kidney disease and pulmonary emphysema, respectively (17
). Whether the TAZ knockout animals are more resistant to tumor development has not been investigated.
We propose a model of TAZ regulation by the Hippo pathway via the following sequence. Lats phosphorylates TAZ on Ser89. This phosphorylation creates a 14-3-3 binding site. The phosphorylated TAZ then binds to 14-3-3 and is retained in cytoplasm, resulting in the depletion of TAZ from the nucleus. Therefore, TAZ fails to bind nuclear transcription factors and cannot activate target genes when it is phosphorylated by Lats. This model suggests that TAZ inactivation is an important downstream signaling output of the Hippo pathway.
Our study indicates a function for TAZ in cell proliferation and EMT, which are both important steps for cancer development. The expression of TAZ in MCF10A cells significantly increases the growth rate of the cells. Furthermore, the TAZ-expressing cells continue to proliferate even when cells reach confluence. This finding is supported by a high fraction of TAZ-expressing cells that continue to synthesize DNA in a confluent culture. Our study suggests the growth-stimulating activity of TAZ.
Another interesting finding from this study is that TAZ promotes EMT. The expression of TAZ, especially the phosphorylation-defective S89A and 4SA mutants, alters the morphology of MCF10A cells. Moreover, the TAZ-expressing cells showed a dramatic decrease in epithelial markers with a concomitant increase in mesenchymal markers. Therefore, TAZ may play a role in cancer development by promoting proliferation and EMT. This notion is consistent with the fact that TAZ is inhibited by the Hippo tumor suppressor pathway. More interestingly, the expression of the TAZ phosphorylation-defective 4SA mutant not only significantly promotes cell proliferation but also dramatically alters the morphology of MCF10A cells, indicating that the functional role of TAZ in cancer development may result from the dysregulation of the Hippo pathway. Dysregulation of the upstream region of the Hippo pathway such as NF2, Mst/Sav, and Lats/Mob would lead to the inhibition of the Hippo pathway and subsequent activation of TAZ by the loss of inhibitory phosphorylation. Mer is a tumor suppressor, and Sav and Mob mutation have also been reported in tumor cell lines (14
). Therefore, the mutation or dysregulation of the components of the Hippo pathway may result in promoting cell proliferation and inducing EMT via activation of TAZ.
Studies of Drosophila
have shown that the Hippo pathway inhibits cell proliferation and induces apoptosis (12
). Dysregulation of the Hippo pathway results in organ and tissue hypertrophy (7
). Consistently, mutations of the tumor suppressor NF2
gene coding for Mer provide direct evidence supporting the tumor suppressor function of this pathway in humans (19
). Our discovery that TAZ promotes proliferation and EMT provides a molecular basis for and additional support to the function of the Hippo pathway in tumor suppression. We speculate that the phosphorylation and inactivation of TAZ and YAP may represent the major downstream effects of the Hippo pathway in tumor suppression. TAZ and YAP may be the key physiological substrates of Lats, which is also a candidate of a tumor suppressor gene. In summary, our study has connected TAZ to the Hippo pathway and suggests a potential function for TAZ in tumorigenesis.