Proteolytic activation of angiomotin by DDI2 promotes angiogenesis.

The scaffolding protein angiomotin (AMOT) is indispensable for vertebrate embryonic angiogenesis. Here, we report that AMOT undergoes cleavage in the presence of lysophosphatidic acid (LPA), a lipid growth factor also involved in angiogenesis. AMOT cleavage is mediated by aspartic protease DNA damage-inducible 1 homolog 2 (DDI2), and the process is tightly regulated by a signaling axis including neurofibromin 2 (NF2), tankyrase 1/2 (TNKS1/2), and RING finger protein 146 (RNF146), which induce AMOT membrane localization, poly ADP ribosylation, and ubiquitination, respectively. In both zebrafish and mice, the genetic inactivation of AMOT cleavage regulators leads to defective angiogenesis, and the phenotype is rescued by the overexpression of AMOT-CT, a C-terminal AMOT cleavage product. In either physiological or pathological angiogenesis, AMOT-CT is required for vascular expansion, whereas uncleavable AMOT represses this process. Thus, our work uncovers a signaling pathway that regulates angiogenesis by modulating a cleavage-dependent activation of AMOT.

Suppression of YAP safeguards human naïve pluripotency.

Propagation of human naïve pluripotent stem cells (nPSCs) relies on the inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also promotes differentiation into trophectoderm (TE). Therefore, robust self-renewal requires suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs and is implicated in TE suppression. Here, we dissect the mechanism of this effect. Tankyrase inhibition is known to block canonical Wnt/ß-catenin signalling. However, we show that nPSCs depleted of ß-catenin remain dependent on XAV939. Rather than inhibiting Wnt, we found that XAV939 prevents TE induction by reducing activation of YAP, a co-factor of TE-inducing TEAD transcription factors. Tankyrase inhibition stabilises angiomotin, which limits nuclear accumulation of YAP. Upon deletion of angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Expression of constitutively active YAP similarly precipitates TE differentiation. Conversely, nPSCs lacking YAP1 or its paralog TAZ (WWTR1) resist TE differentiation and self-renewal efficiently without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not in mouse nPSCs and highlight the pivotal role of YAP activity in human naïve pluripotency and TE differentiation. This article has an associated 'The people behind the papers' interview.

Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at primary cilia and regulates ciliary length and signaling.

The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to the MAGUK-binding stalk domain of KIF13B relieves motor autoinhibition and promotes microtubule plus-end-directed cargo transport. Here, we characterize angiomotin (AMOT) isoform 2 (p80, referred to as Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live-cell imaging analysis indicated that Ap80 is concentrated at and recruits PALS1 to the base of the primary cilium, but is not a cargo of KIF13B itself. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia and reduced agonist-induced ciliary accumulation of SMO, a key component of the Hedgehog signaling pathway, whereas Ap80 overexpression caused ciliary shortening. Our results suggest that Ap80 activates KIF13B cargo binding at the base of the primary cilium to regulate ciliary length, composition and signaling.

WWOX-Mediated Degradation of AMOTp130 Negatively Affects Egress of Filovirus VP40 Virus-Like Particles.

Ebola virus (EBOV) and Marburg virus (MARV) continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW domain-containing host interactors via its conserved PPxY late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ, and WWOX. In addition to host WW domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 virus-like particles (VLPs) and recombinant vesicular stomatitis virus (VSV) M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOT's scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY-WW domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCE Filoviruses (Ebola virus [EBOV] and Marburg virus [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the filovirus matrix protein VP40 to regulate VP40-mediated egress of virus-like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus life cycle and the host cell.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

Fluid shear stress promotes periodontal ligament cells proliferation via p38-AMOT-YAP.

Periodontal ligament (PDL) cells are a promising tool for periodontal regeneration therapy. Achieving a sufficient number of PDL cells is essential to PDL regeneration. In our study, appropriate flow shear stress (FSS, 1-6 dyn/cm2) promotes the proliferation of PDL cells. FSS remodels cytoskeleton and focal adhesion in a duration-dependent manner. FSS induces PDL cells to form the actin cap within 10 min, flattens the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of Yes-associated protein (YAP). FSS activates p38, which plays a dual function in YAP regulation. p38 regulates the phosphorylation of Akt and cofilin, as well as induced F-actin polymerization to induce YAP activity. In addition, p38 inhibits pLATS and consecutively regulates angiomotin (AMOT) and YAP phosphorylation. AMOT competitively binds to F-actin and YAP to participate in FSS-mediated YAP nuclear translocation and cell proliferation. Taken collectively, our results provide mechanistic insights into the role of p38-AMOT-YAP in FSS-mediated PDL cells proliferation and indicate potential applications in dental regenerative medicine.

p73 is required for vessel integrity controlling endothelial junctional dynamics through Angiomotin.

Preservation of blood vessel integrity, which is critical for normal physiology and organ function, is controlled at multiple levels, including endothelial junctions. However, the mechanism that controls the adequate assembly of endothelial cell junctions is not fully defined. Here, we uncover TAp73 transcription factor as a vascular architect that orchestrates transcriptional programs involved in cell junction establishment and developmental blood vessel morphogenesis and identify Angiomotin (AMOT) as a TAp73 direct transcriptional target. Knockdown of p73 in endothelial cells not only results in decreased Angiomotin expression and localization at intercellular junctions, but also affects its downstream function regarding Yes-associated protein (YAP) cytoplasmic sequestration upon cell-cell contact. Analysis of adherens junctional morphology after p73-knockdown in human endothelial cells revealed striking alterations, particularly a sharp increase in serrated junctions and actin bundles appearing as stress fibers, both features associated with enhanced barrier permeability. In turn, stabilization of Angiomotin levels rescued those junctional defects, confirming that TAp73 controls endothelial junction dynamics, at least in part, through the regulation of Angiomotin. The observed defects in monolayer integrity were linked to hyperpermeability and reduced transendothelial electric resistance. Moreover, p73-knockout retinas showed a defective sprout morphology coupled with hemorrhages, highlighting the physiological relevance of p73 regulation in the maintenance of vessel integrity in vivo. We propose a new model in which TAp73 acts as a vascular architect integrating transcriptional programs that will impinge with Angiomotin/YAP signaling to maintain junctional dynamics and integrity, while balancing endothelial cell rearrangements in angiogenic vessels.

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release.

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1-NEDD4L WW3 interaction accounts for most of the AMOT-NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW-PPxY core interaction account for the unusually high affinity of the AMOT PPxY1-NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.

Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding.

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.

Sequestration of eIF4A by angiomotin: A novel mechanism to restrict global protein synthesis in trophoblast cells.

Enrichment of angiomotin (AMOT) in the ectoplacental cone of E7.5 murine placenta prompted our investigation on the role of AMOT in trophoblast differentiation. We show here that AMOT levels increased in mouse placenta during gestation and also upon induction of differentiation in trophoblast stem cell ex vivo. Proteomic data unravelling AMOT-interactome in trophoblast cells indicated a majority of AMOT interactors to be involved in protein translation. In-depth analysis of AMOT-interactome led to identification of eukaryotic translation initiation factor 4A (eIF4A) as the most plausible AMOT interactor. Loss of function of AMOT enhanced, whereas, gain in function resulted in decline of global protein synthesis in trophoblast cells. Bioinformatics analysis evaluating the potential energy of AMOT-eIF4A binding suggested a strong AMOT-eIF4A interaction using a distinct groove encompassing amino acid residue positions 238 to 255 of AMOT. Co-immunoprecipitation of AMOT with eIF4A reaffirmed AMOT-eIF4A association in trophoblast cells. Deletion of 238 to 255 amino acids of AMOT resulted in abrogation of AMOT-eIF4A interaction. In addition, 238 to 255 amino acid deletion of AMOT was ineffective in eliciting AMOT's function in reducing global protein synthesis. Interestingly, AMOT-dependent sequestration of eIF4A dampened its loading to the m7 -GTP cap and hindered its interaction with eIF4G. Furthermore, enhanced AMOT expression in placenta was associated with intrauterine growth restriction in both rats and humans. These results not only highlight a hitherto unknown novel function of AMOT in trophoblast cells but also have broad biological implications as AMOT might be an inbuilt switch to check protein synthesis in developmentally indispensable trophoblast cells.

Amot and Yap1 regulate neuronal dendritic tree complexity and locomotor coordination in mice.

The angiomotin (Amot)-Yes-associated protein 1 (Yap1) complex plays a major role in regulating the inhibition of cell contact, cellular polarity, and cell growth in many cell types. However, the function of Amot and the Hippo pathway transcription coactivator Yap1 in the central nervous system remains unclear. We found that Amot is a critical mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. The conditional deletion of Amot and Yap1 in neurons led to a decrease in the complexity of Purkinje cell dendritic trees, abnormal cerebellar morphology, and impairments in motor coordination. Our results indicate that the function of Amot and Yap1 in dendrite growth does not rely on interactions with TEA domain (TEAD) transcription factors or the expression of Hippo pathway-dependent genes. Instead, Amot and Yap1 regulate dendrite development by affecting the phosphorylation of S6 kinase and its target S6 ribosomal protein.

Angiomotin regulates budding and spread of Ebola virus.

The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.

Angiomotin-p130 inhibits vasculogenic mimicry formation of small cell lung cancer independently of Smad2/3 signal pathway.

Lung cancer, the most concerning malignancy worldwide and one of the leading causes of cancer-related deaths. Growing evidence indicates that Angiomotin (Amot)-p130 plays an important role in types of cancer, including breast cancer and gastric cancer. Moreover, evidence suggested that the low Amot-p130 expression correlates with the poor prognosis of lung cancer patients, however, the role and mechanism of Amot-p130 in lung cancer is still unclear. In this study, we showed that Amot-p130 expression was reduced in lung cancer tissues, compared with the adjacent para-carcinoma tissues. In addition, we observed that the reduced expression of Amot-p130 was associated with vasculogenic mimicry (VM) channels formation in lung cancer tissues. Amot-p130 expression was differently expression in lung cancer cell line H446, H1688 and H2227 compared with the normal human lung cells HFL1. To clarify the role of Amot-p130 in lung cancer, we constructed the Amot-p130 expressing H446 cells and Amot-p130 silencing H1299 cells. We confirmed that Amot-p130 overexpression inhibited the migration and invasion of lung cancer cells, whereas its silence promoted cell migration and invasion. Interestingly, we also found that Amot-p130 overexpression suppressed VM tube formation in H446 cells, while its knockdown promoted VM tube formation in H2227 cells. Further studies suggested that Amot-p130 plays roles in M tube formation of lung cancer cell V are independent on smad2/3 signaling pathway. Finally, inoculation of Amot-p130 expressing H446 cells and Amot-p130 silencing H1299 cells into nude mice suppressed tumor growth, when compared with the control group. Based on these results, Amot-p130 serves as a possible diagnostic and therapeutic target in lung cancer patients, and may be an effective mediator of VM formation in lung cancer.

De novo variant in AMOTL1 in infant with cleft lip and palate, imperforate anus and dysmorphic features.

AMOTL1 belongs to the Motin family of proteins that are involved in organogenesis and tumorigenesis through regulation of cellular migration, tube formation, and angiogenesis. While involvement of all AMOTs in development or suppression of cancers is relatively well described, little is known about the congenital phenotype of pathogenic variants in these genes in humans. Recently, a heterozygous variant in AMOTL1 was published in association with orofacial clefts and cardiac abnormalities in an affected father and his daughter. However, studies in mice did not recapitulate the human phenotype and the case was summarized as inconclusive. We present a female infant with cleft lip and palate, imperforate anus and dysmorphic features, in whom trio exome sequencing revealed a de novo variant in AMOTL1 affecting a highly conserved amino acid (c.479C>T; p.[Pro160Leu]). Bioinformatic predictions and in silico modeling supported pathogenicity. This case reinforces the conjecture regarding the disruptive effect of pathogenic variants in AMOTL1 on organ formation in humans. Studies of additional families will reveal the full phenotypic spectrum associated with this multiple malformation syndrome.

Angiomotin-like 1 plays a tumor-promoting role in glioma by enhancing the activation of YAP1 signaling.

Angiomotin-like 1 (AMOTL1) is reportedly a pivotal tumor-associated protein that is strongly associated with the tumorigenesis of multiple malignant tumors. However, the issue of whether AMOTL1 plays a role in the tumorigenesis of glioma remains unclear. The aim of this work was to explore the possible relationship between AMOTL1 and glioma progression. Results demonstrated that high levels of AMOTL1 in glioma tissues were associated with a reduced survival rate in patients with glioma. Cellular functional assays revealed that silencing of AMOTL1 in glioma cell lines substantially decreased cell proliferation and invasion and increased cell apoptosis. Further investigation revealed that silencing of AMOTL1 inhibited the activation of yes-associated protein 1 (YAP1) and decreased the expression of YAP1 target genes. Reactivation of YAP1 reversed AMOTL1-silencing-induced antitumor effects, whereas inhibition of YAP1 abolished AMOTL1-overexpression-induced tumor-promoting effects in glioma cells. Silencing of AMOTL1 also retarded the growth of glioma cell-derived xenograft tumors in vivo. In conclusion, these findings suggested that AMOTL1 may exert a tumor-promoting function in glioma by enhancing the activation of YAP1 signaling. This work suggested AMOTL1 as a potential target for the development of antiglioma therapy.

Epigenetic and transcriptomic consequences of excess X-chromosome material in 47,XXX syndrome-A comparison with Turner syndrome and 46,XX females.

47,XXX (triple X) and Turner syndrome (45,X) are sex chromosomal abnormalities with detrimental effects on health with increased mortality and morbidity. In karyotypical normal females, X-chromosome inactivation balances gene expression between sexes and upregulation of the X chromosome in both sexes maintain stoichiometry with the autosomes. In 47,XXX and Turner syndrome a gene dosage imbalance may ensue from increased or decreased expression from the genes that escape X inactivation, as well as from incomplete X chromosome inactivation in 47,XXX. We aim to study genome-wide DNA-methylation and RNA-expression changes can explain phenotypic traits in 47,XXX syndrome. We compare DNA-methylation and RNA-expression data derived from white blood cells of seven women with 47,XXX syndrome, with data from seven female controls, as well as with seven women with Turner syndrome (45,X). To address these questions, we explored genome-wide DNA-methylation and transcriptome data in blood from seven females with 47,XXX syndrome, seven females with Turner syndrome, and seven karyotypically normal females (46,XX). Based on promoter methylation, we describe a demethylation of six X-chromosomal genes (AMOT, HTR2C, IL1RAPL2, STAG2, TCEANC, ZNF673), increased methylation for GEMIN8, and four differentially methylated autosomal regions related to four genes (SPEG, MUC4, SP6, and ZNF492). We illustrate how these changes seem compensated at the transcriptome level although several genes show differential exon usage. In conclusion, our results suggest an impact of the supernumerary X chromosome in 47,XXX syndrome on the methylation status of selected genes despite an overall comparable expression profile.

Rho differentially regulates the Hippo pathway by modulating the interaction between Amot and Nf2 in the blastocyst.

The Hippo pathway modulates the transcriptional activity of Yap to regulate the differentiation of the inner cell mass (ICM) and the trophectoderm (TE) in blastocysts. Yet how Hippo signaling is differentially regulated in ICM and TE cells is poorly understood. Through an inhibitor/activator screen, we have identified Rho as a negative regulator of Hippo in TE cells, and PKA as a positive regulator of Hippo in ICM cells. We further elucidated a novel mechanism by which Rho suppresses Hippo, distinct from the prevailing view that Rho inhibits Hippo signaling through modulating cytoskeleton remodeling and/or cell polarity. Active Rho prevents the phosphorylation of Amot Ser176, thus stabilizing the interaction between Amot and F-actin, and restricting the binding between Amot and Nf2. Moreover, Rho attenuates the interaction between Amot and Nf2 by binding to the coiled-coil domain of Amot. By blocking the association of Nf2 and Amot, Rho suppresses Hippo in TE cells.

Using human sequencing to guide craniofacial research.

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

Angiomotins stimulate LATS kinase autophosphorylation and act as scaffolds that promote Hippo signaling.

The Hippo pathway controls cell proliferation, differentiation, and survival by regulating the Yes-associated protein (YAP) transcriptional coactivator in response to various stimuli, including the mechanical environment. The major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP. LATS1/2 are activated by phosphorylation on a hydrophobic motif (HM) outside of the kinase domain by MST1/2 and other kinases. Phosphorylation of the HM motif then triggers autophosphorylation of the kinase in the activation loop to fully activate the kinase, a process facilitated by MOB1. The angiomotin family of proteins (AMOT, AMOTL1, and AMOTL2) bind LATS1/2 and promote its kinase activity and YAP phosphorylation through an unknown mechanism. Here we show that angiomotins increase Hippo signaling through multiple mechanisms. We found that, by binding LATS1/2, SAV1, and YAP, angiomotins function as a scaffold that connects LATS1/2 to both its activator SAV1-MST1 and its target YAP. Deletion of all three angiomotins reduced the association of LATS1 with SAV1-MST1 and decreased MST1/2-mediated LATS1/2-HM phosphorylation. Angiomotin deletion also reduced LATS1/2's ability to associate with and phosphorylate YAP. In addition, we found that angiomotins have an unexpected function along with MOB1 to promote autophosphorylation of LATS1/2 on the activation loop motif independent of HM phosphorylation. These results indicate that angiomotins enhance Hippo signaling by stimulating LATS1/2 autophosphorylation and by connecting LATS1/2 with both its activator SAV1-MST1/2 and its substrate YAP.

Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein.

The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.