MAML1/2 promote YAP/TAZ nuclear localization and tumorigenesis Jiyoung Kima,1, Hyeryun Kwona,1, You Keun Shinb, Gahyeon Songa, Taebok Leec, Youngeun Kima, Wonyoung Jeonga, Ukjin Leea, Xianglan Zhangd, Gilyeong Namb, Hei-Cheul Jeungb, Wantae Kime,2, and Eek-hoon Jhoa,2 aDepartment of Life Science, University of Seoul, 02504 Seoul, Republic of Korea; bDepartment of Internal Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine, 06230 Seoul, Republic of Korea; cConfocal Core Facility, Center for Medical Innovation, Seoul National University Hospital, 03082 Seoul, Republic of Korea; dOral Cancer Research Institute, College of Dentistry, Yonsei University, 03722 Seoul, Republic of Korea; and eDepartment of Biochemistry, Chungnam National University, 34134 Daejeon, Republic of Korea Edited by Filippo G. Giancotti, The University of Texas MD Anderson Cancer Center, Houston, TX, and accepted by Editorial Board Member Anton Berns April 22, 2020 (received for review October 14, 2019) The Hippo pathway plays a pivotal role in tissue homeostasis and YAP/TAZ subcellular localization by a variety of upstream regulators tumor suppression. YAP and TAZ are downstream effectors of the is considered a critical regulatory step in the Hippo pathway (20–22). Hippo pathway, and their activities are tightly suppressed by In addition to cytoplasmic sequestration of YAP/TAZ through phosphorylation-dependent cytoplasmic retention. However, the interaction with 14-3-3 (23, 24), a number of studies have molecular mechanisms governing YAP/TAZ nuclear localization have reported alternative modulators of YAP/TAZ subcellular local- not been fully elucidated. Here, we report that Mastermind-like 1 and ization (3). Merlin, Kibra, and Angiomotin have all been shown 2 (MAML1/2) are indispensable for YAP/TAZ nuclear localization and to physically bind to and exclude YAP/TAZ from the nucleus transcriptional activities. Ectopic expression or depletion of MAML1/2 (20, 21, 25). Additionally, protein tyrosine phosphatase PTPN14 induces nuclear translocation or cytoplasmic retention of YAP/TAZ, induces cell-density-dependent nuclear export of YAP, thereby respectively. Additionally, mutation of the MAML nuclear localization suppressing YAP oncogenic function (26, 27). This inhibitory signal, as well as its YAP/TAZ interacting region, both abolish nuclear role of PTPN14 on YAP activity depends on a physical in- localization and transcriptional activity of YAP/TAZ. Importantly, we teraction between PTPN14 and YAP1 and is independent of MAML1 demonstrate that the level of messenger RNA (mRNA) is PTPN14’s enzymatic activity (26). More recently, SET1A- regulated by microRNA-30c (miR-30c) in a cell-density-dependent mediated monomethylation at the K342 residue of YAP (28), CELL BIOLOGY manner. In vivo and clinical results suggest that MAML potentiates binding of Dishevelled (a positive regulator of canonical Wnt YAP/TAZ oncogenic function and positively correlates with YAP/TAZ signaling) to phosphorylated YAP (29), and the interaction be- activation in human cancer patients, suggesting pathological rele- tween TEAD and NES of YAP/TAZ (30) have also been shown vance in the context of cancer development. Overall, our study not to be involved in the nuclear export of YAP. To date, the reg- only provides mechanistic insight into the regulation of YAP/TAZ sub- ulation of cytoplasmic sequestration of YAP/TAZ has been ex- cellular localization, but it also strongly suggests that the miR30c– tensively studied; however, the molecular effectors as well as the MAML–YAP/TAZ axis is a potential therapeutic target for developing underlying mechanisms governing nuclear translocation and re- novel cancer treatments. tention of YAP/TAZ are unclear. In addition, unlike its Dro- sophila ortholog Yorkie, a functional nuclear localization signal Hippo signaling | MAML1/2 | nuclear localization | TEAD | YAP/TAZ Significance he Hippo pathway is an evolutionarily conserved regulator of Torgan size that functions by coordinating cell proliferation and apoptosis (1–5). Diverse upstream inputs, such as cell den- The Hippo-YAP/TAZ pathway plays pivotal roles in controlling sity, mechanical stress, and soluble factors, tightly regulate the tissue growth, and its abnormal regulation is frequently im- Hippo pathway, which, in response to these extracellular signals, plicated in a variety of cancers. We have identified MAML1/2 as controls cell proliferation and organ growth (6–9). Dysregulation critical regulators for YAP/TAZ nuclear retention and tran- of the Hippo pathway induces massive overgrowth in diverse scription activities. Clinical analysis with specimens of a human organs due to uncontrolled cell proliferation (10–12). Hippo cancer patient and a public cancer database reveals patholog- signaling inactivation and subsequent Yes-associated protein ical association between MAML expression and YAP signature. (YAP) activation induce rapid tumor progression in various Furthermore, our results suggest that the cell density govern- animal models and are strongly correlated with several types of ing YAP/TAZ subcellular localization can also function as an human cancers such as lung, breast, and liver cancer (10, 13–15). important intrinsic mechanism for miR30c-dependent regula- Activation of the Hippo pathway is triggered by a core kinase cas- tion of MAML1 level. Our findings provide mechanistic insights sette that comprises the mammalian sterile 20-like kinases (MST1/ of YAP/TAZ-dependent growth control pathway and tumorigenesis. 2), the large tumor suppressor (LATS1/2), and the scaffold protein Author contributions: J.K., H.K., Y.K.S., Y.K., H.-C.J., W.K., and E.-h.J. designed research; Salvador1 (SAV1) (16). Sequential phosphorylation and activation J.K., H.K., Y.K.S., G.S., T.L., Y.K., W.J., U.L., X.Z., G.N., and W.K. performed research; J.K., of MST1/2 and LATS1/2 by upstream signals such as high cell H.K., Y.K.S., T.L., Y.K., W.J., U.L., X.Z., G.N., H.-C.J., W.K., and E.-h.J. analyzed data; and density result in phosphorylation-dependent cytoplasmic retention J.K., H.K., and E.-h.J. wrote the paper. of the transcriptional coactivator YAP and its paralogue TAZ The authors declare no competing interest. (transcriptional coactivator with PDZ-binding motif). This occurs This article is a PNAS Direct Submission. F.G.G. is a guest editor invited by the through interaction with 14-3-3, and the retained YAP/TAZ are Editorial Board. down-regulated by β-TrCP–mediated proteolytic degradation (6, 9, Published under the PNAS license. 17, 18). LATS1/2-mediated phosphorylation of YAP/TAZ can be 1J.K. and H.K. contributed equally to this work. suppressed by low cell density or cell-proliferative conditions, both 2To whom correspondence may be addressed. Email: [email protected] or ej70@uos. of which induce the nuclear translocation of YAP/TAZ. Once in the ac.kr. nucleus, YAP/TAZ interacts with TEA domain transcription factor This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (TEAD), resulting in the activation of genes involved in cell pro- doi:10.1073/pnas.1917969117/-/DCSupplemental. liferation, antiapoptosis, and survival (19). Overall, the regulation of First published June 1, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1917969117 PNAS | June 16, 2020 | vol. 117 | no. 24 | 13529–13540 Downloaded by guest on September 25, 2021 (NLS) sequence for YAP/TAZ has not been reported in mam- target gene transcription by directly interacting with downstream mals, suggesting the existence of a potential novel molecular effectors of these pathways (31–33). Therefore, there is strong mediator of YAP/TAZ nuclear localization. evidence demonstrating Notch-independent functions of MAML Mastermind-like (MAML) was originally identified as a piv- proteins in regulating various biological processes (34). How- otal coactivator of Notch-dependent transcription, and three ever, despite its importance as a transcriptional coactivator in distinct MAML genes (MAML1 to 3) are expressed in mammals. various signaling pathways, the overall physiological relevance In addition to Notch signaling, numerous studies have reported of MAML in homeostasis and disease has not been well that MAML promotes Wnt, Shh, and nuclear factor-κB signaling studied. Fig. 1. MAML1/2 promote the nuclear localization and transcriptional activities of YAP/TAZ. (A) Ectopically expressed MAML1 and YAP in HEK293 cells colocalized in the nucleus. (Scale bars, 10 μm.) (B) Co-IP experiments showed that endogenous MAML1 interacts with YAP/TAZ. (C) The fluorescence signal generated by proximity ligation assay demonstrates that endogenous MAML1 and YAP are closely located in the nucleus. (Scale bars, 10 μm.) (D) MAML1 and MAML2, but not MAML3, increased YAP reporter activity in HCT116 cells. (E) Knockdown of MAML1/2 in HEK293A reduced nuclear localization of YAP. The areas marked with white rectangles were magnified and shown in E, Right. (Scale bars, 50 μm.) (F) Knockdown of MAML1/2 in HEK293T cells reduced YAP reporter activity. (G) Quantitative real-time PCR showed that knockdown of MAML1/2 in HeLa cells reduced mRNA levels of Hippo and Notch signaling target genes. (H) A wound-healing assay showed that knockdown of MAML1/2 in HeLa cells reduced migration rate, which was induced by the ectopic expression of YAP. In D and F–H, the statistical analyses represent average values from a representative experiment performed in triplicate. Error bars represent SDs of triplicate assays. **P < 0.01. n.s., not significant.