Cells in animals and other multi-cellular organisms need to know when and where they should grow and divide. Individual cells communicate with their surrounding environment and each other via signaling pathways such as the Hippo-YAP pathway, which stimulates cells to grow and therefore influences the size of organs. When the Hippo part of the pathway is active it causes a protein known as YAP to move out of a compartment in the cell called the nucleus. Inside the nucleus, YAP helps to activate genes that promote cell growth. If the Hippo pathway can no longer respond to cues from the environment, YAP becomes over-active and can contribute to the development of various cancers. Therefore researchers are trying to better understand how it is regulated.

Many signals both from inside and outside the cell influence YAP activity. For example, some signals block YAP from entering the nucleus, whereas others cause YAP to be broken down entirely. Several studies have recently identified a signal protein called angiomotin as a regulator of YAP. However, the studies provide conflicting reports as to whether angiomotin promotes or inhibits cell growth.

Like many other proteins, angiomotin can be tagged with a small molecule called a phosphate group that can alter its activity. Moleirinho, Hoxha et al. studied human cells containing versions of angiomotin that mimic different forms of the protein with or without the phosphate. The experiments indicate that when a phosphate is attached at a particular position (known as serine 176), angiomotin predominantly interacts with YAP and another protein called Merlin at the cell surface. On the other hand, when angiomotin does not have a phosphate attached to it, all three proteins can move into the nucleus, where YAP is able to activate genes and promote cell growth.

Overall, these findings indicate that adding a phosphate group to angiomotin can act as a switch to regulate where in the cell it and YAP are found and thus, whether YAP is active. Future experiments will investigate which enzymes add the phosphate group to serine 176, and when they are able to do so.

Angiomotin (Amot) was originally identified as an angiostatin-binding protein involved in the regulation of endothelial cell polarization, migration, proliferation, and angiogenesis (Kikuno et al., 1999; Levchenko et al., 2003; Troyanovsky et al., 2001). Amot is expressed as two isoforms generated by alternative splicing, full-length Amot-p130 (referred to as Amot in this manuscript) and truncated Amot-p80, which lacks the 409-amino acid N-terminus (Ernkvist et al., 2006). Amot, Angiomotin-like 1 (AmotL1) and Angiomotin-like 2 (AmotL2) compose the Motin family of proteins, which share an N-terminus with conserved glutamine-rich domains and PPxY motifs, and a C-terminus containing conserved coiled-coil (CC) and PDZ-binding domains (Bratt et al., 2002; Nishimura et al., 2002; Zheng et al., 2009). Although structurally similar, the functions of Motin family members appear to be distinctive, as divergent spatiotemporal and expression levels have been described across human and mouse tissues and cell lines for each of the family members (Moleirinho et al., 2014).

Functionally, Amot is required for endothelial cell migration, by binding to the Syx:Patj/Mupp1 polarity complex to localize RhoA activity to the leading edge of migratory cells (Ernkvist et al., 2009). It has also been implicated in epithelial cell polarity, as an inhibitor of the GTPase-Activating Protein (GAP) Rich1 and shown to compromise the integrity of tight junctions by promoting Rich1-mediated hydrolysis of Rho GTPases Rac1 and Cdc42 (Wells et al., 2006; Yi et al., 2011). Amot regulates collective migration of epithelial cells as part of a signaling axis composed of Merlin-Amot-Rich1 and the Rac1 small GTPase (Das et al., 2015). At tight junctions (TJ), TJ-associated Merlin inhibits Rac1 activation through the Amot-Rich1 axis, relocalizing Merlin to the cytoplasm during cell migration and releasing Rac1 from a suppressed state (Das et al., 2015; Yi et al., 2011). Amot also directly binds to YAP, a central effector of the Hippo signaling pathway, via ¹⁰⁶LPTY¹⁰⁹/²³⁹PPxY²⁴² motifs present in the N-terminal domain of Amot-p130 and WW domain present in YAP (Yi et al., 2013, 2011; Zhao et al., 2011).

The Hippo-YAP signaling pathway, originally characterized in Drosophila, is highly conserved in mammals and regulates organ size, cell contact inhibition, proliferation, apoptosis and polarity (Ramos and Camargo, 2012; Yi and Kissil, 2010; Yu et al., 2015). At the core of the mammalian pathway, MST1/2 promote phosphorylation of WW45 (also known as Sav1), Lats1/2, and MOB1 (MOBKL1A/B). Once activated, Lats1/2 phosphorylates the primary downstream effector YAP, promoting ubiquitination and proteosomal degradation by a SCF^beta-TRCP E3 ligase and sequestering YAP from the nucleus, where it functions as a transcriptional co-activator (Hao et al., 2008; Zhao et al., 2010b, 2007). Among the different transcription factors activated by YAP, the DNA-binding TEA domain (TEAD) transcription factors are thought to mediate a YAP-driven pro-proliferative gene expression program (Galli et al., 2015; Zhao et al., 2010b, 2007).

YAP’s involvement in cancer has been demonstrated in several tissues, including liver, intestine, heart, pancreas, and brain (Yu et al., 2015; Guerrant et al., 2016). Importantly, recent studies revealed YAP plays a key role in developing resistance to RAF- and MEK-targeted therapies in lung and colon cancer cells (Lin et al., 2015) and cancer relapse in KRAS-driven colon and pancreatic cancers (Kapoor et al., 2014; Shao et al., 2014). Upstream of the core kinase cassette is the tumor suppressor Merlin (moesin-ezrin-radixin-like protein), which is inactivated in Neurofibromatosis type 2 (NF2) (McClatchey et al., 1998). Functions for Merlin have been described in the nucleus (Li et al., 2014, 2010) as well as at the plasma membrane (Yin et al., 2013; Zhang et al., 2010).

In the mouse-liver, homozygous deletion of Nf2 results in tumor formation. However, heterozygous deletion of Yap significantly suppresses the loss-of-Nf2 phenotype, thus implicating YAP as a major downstream effector of NF2 (Zhang et al., 2010). Analysis of liver-specific Nf2 knockout mice and Nf2:Amot double knockout (DKO) mice showed Amot is required for hepatic ductal cell proliferation and tumor formation in the context of either Nf2 loss or DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-induced injury. Additionally, substantially increased expression of Amot was observed in NF2-null human schwannomas samples, which primarily displayed localization of Amot in the nucleus (Yi et al., 2013). Further evidence demonstrated that in renal cell carcinoma (RCC), Amot promotes the proliferation of renal epithelial and RCC cells, and is crucial for YAP transcriptional activity by promoting its nuclear localization (Lv et al., 2016). However, it has also been reported that Amot functions as a negative regulator of YAP activity, as direct association of Amot and YAP results in translocation to cytoplasm/cell junctions (Leung and Zernicka-Goetz, 2013; Zhao et al., 2011). Moreover, a number of studies suggest that Amot acts as a scaffold protein to promote localization of YAP, as well as other Hippo/YAP core kinases, to the cytoplasm/cell junctions/actin cytoskeleton and promote Lats1/2-mediated phosphorylation of YAP (Chan et al., 2011; Dai et al., 2013; Paramasivam et al., 2011; Wang et al., 2011). One possible explanation for these opposing regulatory functions of Amot could be attributed to post-translational modifications. Phosphorylation of Ser175 on human Amot (corresponding to Ser176 in mouse) at the conserved HVRSLS motif by Lats1/2 has been reported as a key post-translational modification controlling Amot function and association with YAP (Hirate et al., 2013; Moleirinho et al., 2014). Here we show that Amot-p130 functions as a scaffold protein mediating YAP and Merlin association and that phosphorylation of Amot at Ser176 induces translocation of the complex from the cytoplasm and nucleus to the plasma membrane. This relocalization of the Amot/YAP/Merlin complex significantly impacts the activity of YAP and regulates YAP’s ability to promote cell proliferation and tumorigenesis. These results uncover an unrecognized layer of regulation in the Hippo-YAP pathway and resolve questions regarding the seemingly opposing functions of Amot.

The role of Angiomotin regulating the Hippo/YAP pathway has so far been elusive, mainly due to conflicting reports suggesting that YAP regulation by Angiomotin is either positive or negative. To gain a deeper understanding of Amot function, we employed multiple cell types originating from tissues in which dysregulation of the Hippo-YAP pathway is observed under pathological conditions. Our study reveals that Angiomotin phosphorylation at serine 176 mediates YAP localization. This post-translational modification is the key event determining a promoting or repressing regulation of YAP by Angiomotin. We found that phosphorylation at serine 176 does not mediate Angiomotin’s scaffolding functions but does impact the localization of a tertiary complex composed by Amot, YAP and Merlin, which is present in both the cytoplasm and nucleus for the wild type protein. The serine 176 phosphomimetic mutant (Amot^S176E) is recruited to the plasma membrane where it is found co-localized and associated with Patj, Pals1 and E-cadherin at junctional structures. Our previous studies indicate that Amot is required for YAP function in the nucleus (Yi et al., 2013). Thus, the relocation of Amot out of the nucleus and sequestration of Amot/YAP complex to the plasma membrane, function to prevent YAP from operating as a growth-promoting transcriptional activator. The non-phosphorylated mutant (Amot^S176A) is preferentially localized to the nucleus, where it facilitates YAP interactions with TEADs and concomitant activation of target gene transcription.

Angiomotin’s involvement in regulation of Hippo-YAP signaling arises from studies showing that Amot functions as a scaffold protein for several components of this signaling cascade including YAP, Merlin, Kibra, Lats, and F-actin (Moleirinho et al., 2014). Our findings extend Amot scaffolding functions to the formation of the YAP-Merlin complex independent of Amot Ser176 phosphorylation, although it is possible that additional PTMs might regulate the complex specifically at different sub-cellular localizations. Multiple reports describe the role of Amot^S176 phosphorylation on Amot interactions with binding partners and function (Adler et al., 2013b; Chan et al., 2013; Hirate et al., 2013; Mana-Capelli et al., 2014). While these reports converge towards the idea that Amot serine 176 phosphorylation is mediated by Lats1/2 and that this impacts Amot’s binding to F-actin, they differ significantly when examining the influence on Amot localization and function. Whilst the discussion of these differences merits a separate review, we highlight a few points that agree or conflict with our current findings. In regards to the effect of Amot phosphorylation on binding to F-actin or YAP, previous reports conclude that serine 176 phosphorylation impairs the interaction between Amot and F-actin and suggest that this can favor binding to YAP (Chan et al., 2013; Mana-Capelli et al., 2014; Dai et al., 2013). Our findings suggest that serine 176 phosphorylation does not impact association with F-actin. However, in agreement with one of these studies (Dai et al., 2013) our findings suggest that phosphorylation on Amot has little impact on the Amot-YAP association. Possible explanations for the differences between our current findings and previous reports could be attributed to the use of HEK293 cells expressing high levels of endogenous Amot, which might mask some of the interactions with the mutated alleles. To circumvent this possibility, our studies incorporated HEK293 cells in which endogenous Amot expression was knocked-down and the different mutant alleles reintroduced. Additionally, previous studies used different cell types, which not only could impact the stability of the Amot/YAP interaction but also explain the observed differences in Amot localization. In MCF10A, MCF7 and MDA-MB-468 cells, hypophosphorylated Amot showed junctional localization and co-localization with F-actin (Adler et al., 2013b; Chan et al., 2013). Although we cannot exclude differences in the actin cytoskeleton between the different cell lines used in those studies, we observe enrichment of Amot^S176E to the plasma membrane, in agreement with Hirate et al. (2013), and enrichment of Amot^S176A to the nucleus. Furthermore, our studies confirm previous findings and show that Amot is required for YAP activity and that the Amot^S176A mutant displays prominent nuclear function by means of increased levels of YAP target gene expression, increased cell proliferation rates and higher colony formation capacity (Adler et al., 2013b; Chan et al., 2013; Hirate et al., 2013). Importantly, in vivo studies are needed to clarify the role of Amot in different cell and tissue types. We further extended the analysis of Amot phosphorylation on the Amot-YAP-Merlin complex, by determining whether its formation depends on YAP^S127 phosphorylation site. In agreement with previous reports showing that the Amot-YAP interaction is independent of phosphorylation at YAP^S127 (Wang et al., 2011; Zhao et al., 2011), we observed that the YAP-Merlin-Angiomotin complex also occurs independently of YAP^S127 (Figure 3—figure supplement 3A and B). Interestingly, mutation of serine in each of YAP’s five HxRxxS consensus sites (S5A, Zhao et al., 2010b) precluded formation of the complex (Figure 3—figure supplement 3C). The molecular mechanisms underlying the reduced association between the S5A and the Amot-merlin complex remain to be elucidated. However, this is not likely as a result of increased nuclear localization of YAP-S5A, since wild-type Amot can also be found in the nucleus in complex with wild type YAP. Therefore, it is likely that at least one additional post-translational modification of YAP could regulate the formation of the Amot-YAP-Merlin complex and future studies are needed to identify this modification.

We found that Amot^S176 phosphorylation induces association of the YAP-Merlin complex with Patj, Pals1 and E-cadherin, and that plasma membrane sequestration inhibits YAP activity. Several reports have described the localization of Amot at junctions in epithelial and endothelial cells (Bratt et al., 2005; Ernkvist et al., 2008; Patrie, 2005; Wells et al., 2006) and its colocalization with Patj/Pals1 via its C-terminal PDZ-binding motifs (Wells et al., 2006). However, binding to Patj is not required for Amot’s localization to the plasma membrane, as a mutant lacking the PDZ binding motifs still localize to the cell cortex (Sugihara-Mizuno et al., 2007). This suggests that an additional mechanism can regulate Amot membrane localization, which is in agreement with our findings where phosphorylation of Amot^S176 induces localization of Amot-YAP-Merlin complex to the cell cortex. In HeLa, HEK293 and MDCK cells, Amot mediates YAP localization at the plasma membrane resulting in suppression of cell proliferation (Chan et al., 2011; Paramasivam et al., 2011; Zhao et al., 2011). Thus, we propose that phosphorylation of Amot triggers a shift of the YAP-Merlin complex from the nucleus to the plasma membrane, thus suppressing the nuclear activity of YAP and exerting cell growth and tumor suppressive functions (Figure 7C).

Amot associates with Merlin and Rich1 at junctional structures and inhibits Rac1 and downstream signaling into the MAPK pathway (Yi et al., 2011). Our results suggest that Amot^S176 phosphorylation does not impact the ability to modulate Rac1 activity at the cell membrane. Yet, it is possible that Amot^S176 phosphorylation modulates Merlin’s nuclear function. Previous studies showed that nuclear accumulation of Merlin results in inhibition of the CRL4^DCAF1 E3 ubiquitin ligase. In NF2-mutant tumors, CRL4^DCAF1 ubiquitinates Lats, suppressing phosphorylation and activating YAP (Li et al., 2014, 2010). In light of our findings, we can speculate that in the nucleus, Lats is inactivated by CRL4^DCAF1-driven ubiquitylation and becomes unable to phosphorylate Amot, resulting in an increase of YAP transcriptional activity. However, if Lats remains active and Amot phosphorylation occurs, Amot-YAP-Merlin tertiary complex translocates from the nucleus to the cytoplasm and plasma membrane, resulting in suppression of YAP nuclear functions. Future studies will be required to shed light on this possibility.

Another open question pertains to what mechanism/s regulate Angiomotin/YAP localization. Lats1/2 are obvious candidates, as they were shown to phosphorylate both YAP and Amot (Adler et al., 2013b; Chan et al., 2013; Hirate et al., 2013; Mana-Capelli et al., 2014). However, the role of Lats1/2 in regulation of these proteins and the relationship between Lats, YAP and Angiomotin is complex. For example, a number of reports suggest that Lats phosphorylates Amot, leading to reduced binding to F-actin and increased YAP binding and inhibition (Chan et al., 2013; Mana-Capelli et al., 2014). Moreover, Amot has been proposed to activate Lats2 and increase phosphorylation of YAP (Paramasivam et al., 2011; Chan et al., 2013). In contrast, we have previously shown that in multiple systems, Amot antagonizes the association of Lats and YAP and inhibits phosphorylation of YAP by Lats (Yi et al., 2013). Given the complexity of the interactions between Amot and YAP, addressing the role of Lats1/2 in regulating the translocation and activity of the Amot-YAP complex will require future studies. Another potential regulatory mechanism stems from the lack of interaction between Amot and YAP-S5A mentioned above. This finding suggests that additional phosphorylation events could regulate the complex. As a number of other kinases such as AMPK and CK1delta/epsilon have been shown to phosphorylate YAP (Wang et al., 2015; Zhao et al., 2010a). Further work will be required to determine the relevant phosphorylation sites and responsible kinases

In conclusion, our studies suggest a mechanism to explain the previous conflicting observations regarding the role of Amot by demonstrating that Amot^S176 phosphorylation state is a key event that dictates a positive or negative regulation of YAP by Amot by targeting the Amot-YAP-Merlin complex to the plasma membrane, sequestration in the cytoplasm or translocation to the nucleus.

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Author details

1. Susana Moleirinho

   Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   SM, Conceptualization, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Sany Hoxha

   Competing interests

   The authors declare that no competing interests exist.

2. Sany Hoxha

   Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   SH, Formal analysis, Investigation, Methodology

   Contributed equally with

   Susana Moleirinho

   Competing interests

   The authors declare that no competing interests exist.

3. Vinay Mandati

   Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   VM, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

4. Graziella Curtale

   Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   GC, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

5. Scott Troutman

   Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   ST, Investigation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

6. Ursula Ehmer

   Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany

   Contribution

   UE, Investigation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

7. Joseph L Kissil

   Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   JLK, Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   jkissil@scripps.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6471-346X

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