FEBS Letters 588 (2014) 2693–2703

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Review The Angiomotins – From discovery to function ⇑ Susana Moleirinho, William Guerrant, Joseph L. Kissil

Department of Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA article info abstract

Article history: Angiomotins were originally identified as angiostatin binding and implicated in the regu- Received 9 January 2014 lation of endothelial cell migration. Recent studies have shed light on the role of Angiomotins and Revised 5 February 2014 other members of the Motin family in epithelial cells and in pathways directly linked to the Accepted 6 February 2014 pathogenesis of cancer. In particular, Motins have been shown to play a role in signaling pathways Available online 15 February 2014 regulated by small G-proteins and the Hippo–YAP pathway. In this review the role of the Motin pro- Edited by Shairaz Baksh, Giovanni Blandino tein family in these signaling pathways will be described and open questions will be discussed. and Wilhelm Just Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Motin Angiomotin-like 1 Angiomotin-like 2 Hippo YAP NF2 Merlin

1. The Angiomotins – Discovery Angiomotin-like 2 (AmotL2, a.k.a. MASCOT). JEAP (junction-en- riched and -associated protein) was initially identified in a screen Sequencing of clones from human brain cDNA libraries identi- for novel tight-junction (TJ)-associated proteins by a fluorescence fied a 5061 (bp) cDNA clone, designated KIAA1071, with localization-based expression cloning method [4] and due to sim- an open reading frame (ORF) coding for a 473 amino acid protein ilarity to Angiomotin, subsequently named Angiomotin-like 1 [5]. [1]. However, it was not until 2001 through a yeast-two hybrid Human AmotL1 (hAmotL1) (NCBI Accession NM_130847) is a screen of a human placenta cDNA library, using the kringle do- 956 amino acid protein with a predicted molecular mass of mains 1–4 of angiostatin as bait that Angiomotin, the founding approximately 106 kDa. The human AmotL1 (hAmotL1) is lo- member of the motin family, was first cloned [2]. Northern blot cated at 11q21 and shares 85% analysis detected two transcripts at 6.5 kb and 7.5 kb in a broad with mouse AmotL1 (mAmotL1), an 882 amino acid protein (iso- spectrum of analyzed tissues. Given the predominant expression form 2, Uniprot database). Angiomotin-like 2 (AmotL2) (NCBI of Angiomotin in endothelial cells and its involvement in mediat- Accession NM_016201) is a 780 amino acid protein with a pre- ing the anti-migratory properties of angiostatin; it was given its dicted molecular mass of 86 kDa. The gene is mapped to chromo- name [2]. With a cytogenetic location at chromosome Xq23, it some 3q21-q22 [5], and like AmotL1 the coding sequence shares shares 85% homology with the mouse Angiomotin ortholog, and 85% homology with mouse AmotL2, a 772 amino acid protein. was given the HUGO nomenclature gene designation AMOT. Functional characterization of AmotL2 showed it colocalizes with Angiomotin is a 675 amino acid protein and with an estimated MAGI-1 at epithelial tight junctions, an interaction found to be molecular mass of 80 kDa, it was termed Amot-p80 [2,3]. dependent on the AmotL2 coiled-coil domain. The protein was Subsequent analysis of GenBank databases identified 2 additional therefore named MAGI-1-associated coiled-coil tight junction pro- polypeptides that show significant sequence homology to tein (MASCOT) [6]. In the interest of simplicity we will subse- Amot. These were Angiomotin-like 1 (AmotL1, a.k.a. JEAP) and quently refer to these as AmotL1 and AmotL2. Since these proteins exhibit such significant sequence homology, suggestive of a common evolutionary origin, this family of proteins was ⇑ Corresponding author. Address: Scripps Research Institute, 130 Scripps Way, named Motins [5]. Later studies identified an additional form of Jupiter, FL 33458, USA. Amot called p130 (Amot-p130), which arises from alternative E-mail address: [email protected] (J.L. Kissil). http://dx.doi.org/10.1016/j.febslet.2014.02.006 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2694 S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703 splicing of the Amot gene between exons 2 and 3 and carries an ex- PDZ-binding region is localized an angiostatin-binding hydropho- tended 409 amino acid N-terminus [3]. Amot-p130 (NCBI Acces- bic domain, present in Amot-p80 and Amot-p130, yet absent in sion NM_001113490) is a protein composed of 1084 amino acids AmotL1 and AmotL2 [2,5]. The consensus motif for the PDZ domain with an estimated molecular mass of 130 kDa. The apparent lack binding is highly conserved, and its presence in the Motins’ struc- of proteolytic cleavage sites suggests that Amot-p80 is not gener- ture offered the first clue for their potential involvement in signal- ated from Amot-p130 via hydrolytic catalysis, but that the Amot ing pathways. gene produces both Amot isoforms through alternative splicing [3]. In terms of protein topology, a number of models have been The Motin protein family members share several structural proposed [3,15,16]. Based on studies in mouse aortic endothelial characteristics (Fig. 1). The N-terminus, shared between Amot- (MAE) cells, in which an antibody used against the angiostatin- p130, AmotL1, and AmotL2, is composed of conserved glutamine- binding domain was effective without any prior membrane perme- rich domains, PPxY motifs (Amot: 239PPEY242 and 284PPEY287; abilization step, it was proposed that Angiomotin localizes to the AmotL1: 310PPEY313 and 367PPEY370; AmotL2: 210PPQY213 and the cell membrane as a transmembrane protein with both isoforms slightly divergent 252PPVF255), plus a recently identified unconven- forming a transmembrane loop, the angiostatin-binding domain tional LPTY motif lying upstream of the other two (Amot: oriented outwards and the N- and C-terminal domains in an intra- 106LPTY109; AmotL1: 188LPTY191; AmotL2: 104LPTY107) [7,8]. Of note, cellular orientation [16]. DNA vaccination of mice, targeting human AmotL2 differs from the other members at one of the PPxY motifs, Amot-p80, generated antibodies that bind to Amot-p80 on the as the tyrosine residue is replaced by phenylalanine. Remarkably endothelial cell surface. These studies further support a topology these motifs are highly conserved, not only among the Motin fam- where the angiostatin-binding domain is in an extracellular orien- ily members, but also across species [8]. Since Amot-p80 lacks the tation [17,18]. In contrast, in studies with bile canaliculi fractions entire N-terminal, these motifs are not present. The conserved prepared from mouse liver without the use of detergent, endoge- coiled-coil (CC) domain and the C-terminal PDZ-binding domain nous Amot-p130, Amot-p80, and AmotL2 were extracted with compose the C-terminal region. The predicted locations for the intracellular fractions, suggesting that these Motins are not coiled-coil domains are as follows: Amot-p130 (429–689 aa; trans-membrane proteins [2,15]. Interestingly, recent studies dem- 721–751 aa); AmotL1 (438–639 aa; 665–694 aa; 729–762 aa); onstrated the exclusive binding of angiostatin to glioblastoma cells AmotL2 (308–581 aa) (Uniprot database). Importantly, the N-ter- that express Angiomotin. While both proteins appear to be present minal 242 amino acids of Amot-p80 was suggested to encode for on the cell surface they do not appear to colocalize, suggesting a positively charged CC fold due to its positional conservation with Angiomotin mediates angiostatin binding indirectly. amphiphysin (bin/amphiphysin/rvs) BAR domain [9,10]. Since this The structural similarities between the Motins and the absence domain is a conserved region across the Motin family members it of an obvious angiostatin-binding domain in AmotL1 and AmotL2 was termed Amot coiled-coil homology (ACCH) domain [11]. The might suggest that this family of proteins interacts with the coiled-coil regions comprise two or more right-handed a-helices hydrophobic phospholipid membrane bilayer indirectly. Moreover, wrapped around each other with a left-handed superhelical twist protein topology prediction algorithms, such as Topcons [19] also [12]. CC domains contribute to several biological and structural paint a complex picture predicting the presence of potential hydro- functions, including oligomerization. Oligomeric regulation has phobic motifs featuring a transmembrane topology but no pre- been described for all of the members of the Motin family, either dicted extracellular domains. Thus, further experimental analysis by forming homo-oligomers through self-association, or hetero- is needed to clarify these questions. oligomers with other family members through their CC domains Although there is a high degree of similarity between the Motin [6,13,14]. Between the conserved CC domain and the C-terminal proteins, there are functional differences that are still not fully

Angiostatin PDZ LPTY PPEY PPEY CC/BAR CC binding binding Amot-p130 1084 aa *** S175 K481

Amot-p80 675 aa

LPTY PPEY PPEY AmotL1 956 aa * S262

LPTY PPQY PPVF AmotL2 780 aa S159*

Fig. 1. The Motin protein family. Angiomotin p80 (Amot-p80) is an N-terminal truncated version of Angiomotin p130 (Amot-p130) as a result of alternative splicing. Angiomotin-like proteins 1 and 2 (AmotL1 and AmotL2), share sequence identity to Amot-p130, but notably lack the angiostatin-binding domain. ⁄ depicts phosphorylation sites with available experimental confirmation. ⁄⁄ depicts ubiquitination sites with available experimental confirmation. CC/BAR – Coiled-Coil/(Bin/Amphiphysin/Rvs) domain. PDZ – Post synaptic density protein (PSD95), Drosophila disc large (Dlg1) and Zonula occludens-1 (ZO-1) domain. S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703 2695 understood. One clue to this is the finding that the Motins are dif- the stroma. Alteration of their expression pattern occurred within ferently expressed across different tissues and, if expressed in the a fairly small timeframe, specifically at 4 days post-pregnancy. The same tissue, exhibit variable spatiotemporal and expression level same expression shift was observed during the post-implantation patterns. Analysis of mRNA expression of Amot, AmotL1, and period. Intriguingly, AmotL2 expression was barely detected in AmotL2 in diverse human tissues, based on data generated by either period except on day 1 of pregnancy [21]. AmotL2 might the Illumina human BodyMap 2.0 platform, demonstrates that all therefore represent an evolutionary outlier of the Motin family, a family members are expressed in the surveyed tissues, yet at var- concept initially proposed following phylogenetic analysis [5]. iable expression levels (Fig. 2). Amot showed higher transcript lev- Finally, while several cell lines have been used in the functional els in the testis, followed by the brain and thyroid. The lowest studies of the Motin family, in many of these the expression of expression is seen in the liver and adrenal gland. AmotL1 is highly endogenous Motin family members could not be detected using expressed in skeletal muscle, representing the highest mRNA currently available antibodies and/or qPCR. In some cases, studies expression level across the entire analysis. The lowest levels are from different groups report contradictory findings in the same cell found in the blood. AmotL2 expression is highest in breast and line, suggesting experimental conditions could significantly impact lowest in the liver. This analysis is in agreement with previous expression of Motin proteins. Thus, caution should be exercised in studies examining Motin tissue expression patterns not only in hu- choosing the appropriate cell type and experimental conditions for man but also in mouse. Specifically, Amot expression was found in analysis. Table 1 summarizes expression of Motin family members mouse and rat skeletal muscle [5,20], highly expressed in mouse in cell lines based on the published literature. brain, pancreas, and salivary gland [3,5,15]. Similar to its human homologue, AmotL1 mRNA was predominantly expressed in 2. Regulation of the Angiomotins mouse skeletal muscle [5], pancreas, and salivary gland [15]. The expression pattern of the Motins is therefore tissue-type depen- A key post-transcriptional regulatory mechanism of the Motins dent, predicting functional variability within the family. appears to be by alternative splicing. The first evidence for the The expression patterns of Motin family members are also var- occurrence of coding sequencing diversity surfaced with the iable during development. Amot, AmotL1, and AmotL2 mRNAs description of two splice isoforms at the hAmotL1 , the first showed spatiotemporal-dependent expression in the mouse uterus lacking exon 2 and the second exons 2 and 3 [24]. It was predicted during pre-implantation and post-implantation periods [21–23]. that only the first isoform coded for a functional protein of 906 At a very early stage of pregnancy, Amot and AmotL1 transcripts amino acids with a predicted molecular weight of 90 kDa [24]. Uni- were initially expressed in the luminal epithelium and later on in prot protein sequence database predicts two alternative transcripts

Amot AmotL1 AmotL2

0 1 10 100 1000 0 1 10 100 1000 0 1 10 100 1000

Bone marrow Whole blood Whilte blood cells Lymph node Spleen Thymus Brain Cortex Cerebelum Retina Spinal cord Heart Smooth muscle Skeletal muscle Small intestine Colon Adipocyte Kidney Liver Lung Pancreas Thyroid Salivary gland Adrenal gland Breast Skin Ovary Uterus Placenta Prostate Testis Cervix

Fig. 2. RNA expression profiles of Motin family members in human tissue. Results are based on data generated by the Illumina human BodyMap 2.0 platform. RNA obtained from 16 normal human tissues was sequenced and mapped. Fragments Per Kilobase of exon per Million fragments mapped (FPKM) were calculated using the Cufflinks program and expression levels scaled using (100 Â FKPM)1/2 (http://cufflinks.cbcb.umd.edu/). Major tissues are colored according to 6 categories: Immune (red), Nervous (green), Muscle (yellow), Internal (blue), Secretory (violet), and Reproductive (turquoise). 2696 S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703

Table 1 Expression levels of Motin family members in different cell lines.

Cell lines Amot-p130 AmotL1 AmotL2 Analytic methodology Ref. Epithelial ACHN ÀÀUnknown WB [47] BT474 +/ÀÀ Unknown WB [46,48] BT549 À Unknown WB [48] HCC1937 + Unknown Unknown WB [46] HEK293 + +/À +/À WB/qPCR [47,48] HEK293T + – +/À WB/qPCR [46,47] HeLa ÀÀ+ WB/qPCR [47] HEpG2 ÀÀUnknown WB [48] Hs-578T +/ÀÀ Unknown WB [46,48] H2.35 À + Unknown WB [47] MCF7 +/ÀÀ Unknown WB [46,48] MCF10A – À + WB/qPCR [46–48] MDA-MB-231 +/ÀÀ Unknown WB [46,48] MDA-MB-436 + Unknown Unknown WB [46] MDA-MB-468 + Unknown Unknown WB [46] MDCK +/ÀÀ + WB/qPCR [9,46,47] SK-Br3 +/ÀÀ Unknown WB [46,48] T47D +/ÀÀ Unknown WB [46,48] ZR75 + Unknown Unknown WB [46] Endothelial BCE + À Unknown WB [13] HUVEC À + Unknown WB [13] MAE À + Unknown WB [13] MS-1 + + Unknown WB [13] Pmt-EC + + Unknown WB [13] TEC + + Unknown WB [13] Fibroblast/Fibroblast-like Cos-7 ÀÀ+ WB/qPCR [46] MEF + + Unknown WB [46] NIH-3T3 À + + WB/qPCR [46] Neural SC4 + Unknown Unknown WB [7] RT4 + Unknown Unknown WB [7] R3 + Unknown Unknown WB [7] MPNST 90-8TL + Unknown Unknown WB [7]

The endogenous expression of the Motins is based on studies that specifically screened for Motin expression across various cell lines. Motins are showed solely as present or absent in the respective cell line regardless of their expression levels. (À) Absent. (+) Present. (+/À) Inconsistent data between detection approaches. Unknown indicates that the expression of the Motin family member has not been tested in that particular cell line. WB – Western blotting. qPCR – quantitative PCR. from the hAmotL1 locus, one with 956 amino acids coding for a amino acid insertion, starting at the N-terminus, predicted to en- 106 kDa protein, and a second one generated by a 50 amino acid code a 92 kDa protein. In mice, multiple transcript variants of the deletion on AmotL1 N-terminal with a molecular mass around Motin family are predicted and we refer to the available databases 101 kDa. Regarding Amot, several studies have broadly character- for further analysis. ized the two known splice isoforms, Amot-p80 and Amot-p130. Other mechanisms involved in the regulation of the Angiomo- As previously stated, Amot-p130 carries an extended N-terminal tins have only recently begun to come to light. Previous omics-type of 409 amino acids and this is the only structural difference from approaches identified a multitude of post-translational modifica- Amot-p80 [3]. The N-terminal region alone is sufficient to generate tions of the Motin proteins, including phosphorylation, ubiquitina- temporal, spatial, and functional differences between isoforms tion, acetylation, and glycosylation. In some cases, the Motins were [3,9,14,20,25]. Even though Amot has only two transcript variants identified as substrates for specific enzymes, such as the ataxia tel- described so far, additional bands corresponding to proteins of 70 angiectasia mutase (ATM), ataxia telangiectasia and Rad3-related and 110 kDa were observed in Western blotting experiments by (ATR) or cyclin-dependent kinase 2 (CDK2) kinases [28,29]. How- different groups [14,20,26]. Whether these bands represent func- ever, these findings remain to be confirmed and have not been tional or a transient unknown Amot isoforms remains to be deter- studied in depth. There are several phosphorylation sites predicted mined. To the best of our knowledge, besides the 780 amino acid by the PhosphoSite database within the Motin family. Multiple hits protein [5], the only alternative splicing event described for the hA- were suggested and, for simplification, we will only discuss the motL2 locus is a 466 amino acid form lacking the 313 amino acids sites confirmed experimentally. Lists of potential phosphorylation located on the N-terminal of AmotL2 [24,27]. Although the molec- and ubiquitination sites (predicted by UbPred database) in human ular mass of this short isoform is not mentioned in the study [27], Motins are detailed in Table 2. commercial antibodies developed against AmotL2 detect a 51 kDa The post-translational modifications that have been studied in protein that potentially represents this isoform (Sigma–Aldrich; more detail are the ubiquitination at Lysine 481 and phosphoryla- GenWay Biotech; AvivaSystemsBiology; and others). Uniprot fur- tion of Angiomotin p130 at serine 175. In HEK293T cells, Amot- ther predicts three additional human isoforms, two of which have p130 is ubiquitinated at residue Lys481 by Atrophin-1 interacting no available experimental confirmation. Specifically, a 779 amino protein 4 (AIP4)/Itch, a member of the Nedd4 (neural-precursors- acid peptide with predicted molecular mass of 85 kDa; a 777 ami- cell-expressed developmentally down-regulated)-like ubiquitin no acid protein generated by a two residue deletion at positions E3 ligases family. AIP4 ligase activity is triggered by direct interac- 525–526 (encodes a protein with predicted molecular mass of tion between AIP4 WW1 and WW2 domains and Amot-p130 PPxY 85 kDa); and finally a 837 amino acid protein generated by a 58 motifs, which promotes either self or Amot-p130 ubiquitination S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703 2697

Table 2 Potential Phosphorylation and Ubiquitination sites in human Motin family members.

Amot-p130 AmotL1 AmotL2 Tyr109 Ser787 Tyr191 Ser724 Thr10 Tyr573 Ser175 Tyr836 Tyr218 Ser793 Tyr92 Thr596 Ser305 Tyr842 Tyr219 Ser796 Lys100 Ser608 Ser312 Tyr847 Ser225 Thr803 Tyr107 Ser609 Tyr420 Thr848 Ser241 Ser805 Ser159 Ser661 Phosphorylation sites Tyr527 Ser852 Ser262 Ser809 Ser183 Ser753 Tyr599 Ser855 Ser269 Ser828 Ser236 Ser759 Ser712 Ser1059 Ser295 Ser900 Lys408 Ser762 Ser714 Thr1061 Thr373 Thr901 Ser540 Ser718 Ser374 Ser906 Tyr661 Ser924 Ser720 Lys94 Lys520 Lys174 Lys326 Lys100 Lys156 Lys525 Lys195 Lys649 Lys111 Lys209 Lys535 Lys246 Lys881 Lys436 Ubiquitination sites Lys219 Lys545 Lys254 Lys929 Lys721 Lys231 Lys553 Lys302 Lys943 Lys255 Lys619 Lys481

The phosphorylation and ubiquitination sites displayed are based on predictions made by Phosphosite and UbPred databases.

[8,30]. This regulation is mediated by the large tumor suppressor by Amot-p130 is still not fully elucidated and will be discussed (LATS)1/2, a core component of the Hippo signaling pathway that further below. also binds AIP4 [30]. Amot-p130 is thus a physiological substrate for the E3 ligase, a role extended to AmotL1 but not to AmotL2 3. Functional redundancy in the Motin family or Amot-p80 [30]. Other members of the E3 ubiquitin ligase family, Nedd4 and Nedd4.2, also induced Amot-p130 and AmotL1 poly- The extent of functional and genetic redundancy between the ubiquitination and proteasome-dependent degradation. However, members of the Motin family is unknown. As discussed above, Mo- phosphorylation of Nedd4.2 by the non-receptor tyrosine kinase tin expression patterns suggest both distinct and overlapping roles, c-Abl was shown to inhibit AmotL1 degradation [31]. Recruitment however limited functional evidence exists so far. In the mouse of c-Abl is promoted by yes-associate protein (YAP), a transcrip- embryo (zygote), simultaneous silencing of Amot/AmotL1/AmotL2 tional co-factor involved in Hpo–YAP signaling. It was found that resulted in a remarkable increase in cells expressing the differenti- YAP promotes Nedd4.2 phosphorylation by c-Abl, thus ensuring ation gene Cdx2+ compared to depletion of Amot alone. Thus genet- Nedd4.2 does not target AmotL1 for 26S proteasomal degradation ic redundancy between the Motin family appears to occur in [31]. Furthermore, through its WW domains, Nedd4 binds to promotion of embryonic inner cell mass (ICM) differentiation Amot-p130 first and second L/PPXY motifs, akin to the YAP- [22]. Furthermore, simultaneous knockdown of the Motin proteins Amot-p130 association. This suggests that YAP might compete in HEK293T cells caused a striking up-regulation in the activity of a with Nedd4 for Amot-p130 binding [8]. Whether YAP, by binding Wnt reporter when compared to knocking down each member to Amot-p130, prevents its Nedd4 ubiquitin-dependent degrada- alone, which resulted in the down-regulation of the Wnt signaling tion is unknown and further studies are needed to address this [27]. Together with other studies [8], these findings present initial question. Importantly, both direct (BT474, HEK293T, and MCF7 cell evidence for the occurrence of both genetic and functional redun- lines) and indirect (in MDA-MB-468 cells) correlations between dancy within the Motin family. AIP4 activity and Amot-p130 steady state levels were found to be cell-context dependent [8,30]. 4. Function: role in endothelial cell biology and angiogenesis Experimental confirmation of Motin family member phosphor- ylation was recently reported by multiple groups who identified Given that the Angiomotins were originally discovered as medi- LATS1/2-dependent phosphorylation of Amot-p130(Ser175), ators of endothelial cell migration, early studies into the role of the AmotL1(Ser262), and AmotL2(Ser159) at conserved HVRSLS mo- Motin family during development focused on endothelial cells (EC) tifs [23,30,32,33]. LATS2 binds AmotL2 on its N-terminal 307 biology and blood vessel formation. Given the focus of this review, amino acids [34], and Amot-p130. The direct binding between we will only cover these studies briefly. LATS2 and Amot was enhanced by Amot Ser175 phosphorylation Studies to examine the role of Motin family members in vivo and is dependent on LATS kinase activity [23,33]. The phosphor- have suggested these proteins have critical functions during devel- ylation of Motins by LATS has been shown to occur both in vitro opment. Amot knockout 129/SvEv background mice displayed and in vivo [23,30,32,33]. Finally, a model was proposed where early lethality and most mice died soon after gastrulation due to both phosphorylation of Amot-p130 at serine 175 and ubiquitina- defects in cell migration [35]. The severity of this phenotype is tion at Lysine 481 act together as a major regulatory mechanism dependent on genetic background, as crossing the Amot+/À 129/ to mediate YAP activity and Hpo–YAP pathway signaling [30]. SvEv mice to C57/B6 to generate a mixed 129/B6 background re- Under serum starvation conditions, activation of the Hippo path- sulted in a delayed lethality of the AmotÀ/À mice (75% die between way triggers Amot-p130 phosphorylation by LATS1/2 at Ser175. E11 and E15). Careful analysis of these mice indicated that Amot AIP4 binding to phosphorylated Amot-p130 impedes the AIP4– plays a critical role in blood vessel formation through regulation LATS1/2 interaction and subsequent LATS degradation. Once asso- of endothelial cell migration and polarity. Similarly, the knockout ciated, Amot-p130 is ubiquitinated by AIP4 at Lys481 increasing of Amot in Zebrafish embryo also impaired endothelial cell migra- its steady state levels. The increased levels of LATS and Amot- tion and blood vessel formation [36]. Efforts to manipulate the p130 promote YAP ubiquitination [30]. The regulation of YAP expression of other members of the Motin family have also been 2698 S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703 reported. The knockdown of AmotL1 in Zebrafish embryos yields a phenotype similar to Amot knockdown with a migratory defect of Apical Surface Crb3 endothelial cells and vessel formation, although the mechanisms Pals1 might differ [13]. Concerning AmotL2, knockdown in Zebrafish em- bryos resulted in cell migration and proliferation defects [37,38]. Overall, the effect of knocking down either one or several members Rac1/Cdc42 Patj of the Motin family leads to defective phenotypes such as impaired TJ EC migration, polarization, and proliferation. GTP GDP The regulation of endothelial cell migration and proliferation by Angiomotin PDZ Angiomotins requires their C-terminal PDZ-binding domains. Dele- tion of those amino acids in AmotL2/Amot-p80 knockdown Zebra- Coiled-coil fish embryos was sufficient to restrict migration [38,39].In BAR AJ mouse embryos, the deletion of Amot PDZ-binding domain under Coiled-coil the EC-specific Tie promoter inhibited the response of MAE cells CT GAP Rich1 Merlin to chemotactic cues resulting in lethality by day E9.5 [25]. Mecha- FERM nistic studies show that Amot, by binding the polarity complex (PxxP)n Patj/Mupp1:Pals1, indirectly associates with Syx, a RhoA GTPase exchange factor (RhoGEF) binding-domain protein, forming a ter- nary complex [39,40]. Specifically, Amot directly binds to the Fig. 3. Angiomotin, Rich1, and Merlin regulate Rac1/Cdc42 activity. A model PDZ3 domain of Patj/Mupp1, whilst Syx mostly interacts with showing mutually-exclusive interactions between Angiomotin with Merlin or Rich1 Patj/Mupp1 PDZ10 domain. AmotL1 and AmotL2 also associate that modulate Rac1/Cdc42 activity. Merlin, by binding Angiomotin through their mutual CC domains, releases Rich1 from the inhibitory Amot–Rich1 complex. Rich1 with Patj/Mupp1 via its PDZ1 and PDZ2 domains, respectively is then able to inactivate Rac1/Cdc42 by converting Rac1/Cdc42-GTP to Rac1/ [15,39]. Importantly, Amot was shown to strongly associate with Cdc42-GDP. monophosphorylated phosphatidylinositols PI(4)P and PI(3)P [11]. Thus, it is suggested that Amot, by interacting through its PDZ-binding domain with Patj/Mupp1:Syx polarity complex, medi- like MCF7, SKBR3 or MDA-MB-468 [46]. Conversely, knockdown ates its trafficking through endocytic vesicles from EC junctions to of AmotL2, but not AmotL1, in MCF10A cells activated both AKT the leading edge of endothelial migrating EC resulting in and ERK/MAPK pathways, suggestive of a negative, rather than po- recruitment of Syx activity to the leading edge. The targeting of sitive, mediation of these pro-proliferation pathways [37]. The dif- the endocytic vesicles is likely mediated by Amot ACCH domain ferences in consequences of AmotL2 knockdown could be (lipid-binding domain) [39,41]. attributed to cell-type specificity. As AmotL2 mRNA and protein levels are barely detectable in MCF10A cells [47,48] (Table 1), fur- 5. Function: role in small G-protein signaling ther studies are needed to gain a clear understanding of AmotL2 function in these cells. Since Motins have pivotal functions during development, EC migration and proliferation, additional studies probed whether 6. Function: Angiomotin regulation of Hpo/YAP signaling epithelial cell polarity is regulated by Amot. Interestingly, all mem- bers of the Motin family have been shown to associate with tight The Hpo/YAP pathway has been described in a number of recent junctions through binding to the tight junction-associated proteins reviews [49–51]. Briefly, this pathway was initially characterized Patj and Pals1 [9,15,39]. Patj and Pals1 are cytoplasmic scaffolding in flies and is highly conserved in mammalian cells. It plays a cen- proteins that together with the transmembrane protein CRB3 form tral role in various cellular behaviors including proliferation, sur- the apical CRB complex [42]. Work conducted by us and by others vival, and cell contact inhibition [52–54]. The pathway is showed that in mammalian epithelial cells, Amot interacts with composed of a core kinase cascade, in which the Mst1/2 kinases Rich1, a Cdc42/Rac1 GTPase Activating Protein (GAP), via their mu- (Hippo in flies), in complex with scaffold protein WW45 (Salvador tual BAR/CC domains [9,43]. This association targets Amot to the in flies), phosphorylate LATS1/2 (Warts in flies) and Mob1 (Mats in TJs, wherein by binding Patj, Amot forms a complex composed flies). Phosphorylated LATS1/2 binds to and further activates the by Amot:Patj:Pals1:Rich1 localized at the apical membrane. Impor- Mob1 kinase, which in turn, phosphorylates YAP (Yorkie in flies), tantly, Amot negatively regulates Rich1 GAP activity, which has a transcriptional co-activator. The phosphorylation of YAP prevents been suggested to impair TJ integrity [9]. In addition, Amot directly it from entering into the nucleus, where it can form transcription- binds to Merlin, a negative mediator of Rac1-signaling, also ally active complexes with TEAD (Scalloped in flies) and other tran- through their mutual BAR/CC domains [43]. Thus a model emerges scription factors to drive the expression of pro-proliferative or anti- in which Amot plays a role in mediating Rac1 signaling at junc- apoptotic . A series of studies have demonstrated that akin to tional structures as Merlin and Rich1 compete for Amot binding. what has been observed in flies, the mammalian Hpo–YAP path- Merlin, by releasing Rich1 from the Amot inhibitory complex, way can also regulate organ size. For example, inducible overex- facilitates Rich1 GAP activity of Rac1 and subsequent signal trans- pression of YAP in adult mouse liver results in rapid increase in duction through direct downstream effectors such as the p21-acti- liver size [55,56]. Comparable hepatomegaly phenotypes were ob- vated kinases and MAPK pathway (Fig. 3) [43]. This coordination served when Mst1, Mst2, and WW45 were ablated specifically in was also observed in the pathological setting of neurofibromatosis the liver [57–59]. Finally, increased YAP activity appears to be a type 2 (NF2), a condition caused by the loss of the NF2 gene, which common occurrence in human hepatocellular carcinoma [36]. codes the Merlin protein [44,45]. Amot knockdown in Nf2À/À Schw- Recently there has been a flurry of reports implicating Motin ann cells inhibits proliferation in vitro and leads to impaired tumor family members in the regulation of the Hpo/YAP signaling in formation in vivo [43]. Other studies have also reported positive mammalian cells. The Drosophila Hippo signaling cascade is highly regulation of the ERK/MAPK pathway by Amot and AmotL2 in dif- conserved throughout evolution, and functional conservation was ferent cell contexts such as Zebrafish embryos, angiogenic endo- confirmed in many cases by the ability of mammalian orthologs thelial cells (HUVEC) [37], or epithelial breast cancer cell lines to rescue the corresponding Drosophila mutants in vivo [60,61], S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703 2699 although this is not observed in all cases [62]. Remarkably, the A number of recent studies have described the negative regula- Motin family members do not appear to have Drosophila orthologs tion of YAP by members of the Motin family [33,34,37,47,48,72] [63]. The protein Expanded (Ex), which shares significant sequence (Fig. 4). Regarding YAP/TAZ subcellular localization, it has been homology to both Amot-p130 and to FRMD6/Willin, was proposed suggested that upon ectopic expression of Angiomotins in HeLa to represent a functional equivalent in Drosophila [62,64,65].Ex and MCF7 cells, YAP and TAZ translocate from the nucleus to the PPXY motifs directly interact with Yorkie (Yki) WW domains form- cytoplasm and in MCF10A cells, Amot-p130 co-localized with the ing a complex localized at the apical junctions of cells in the eye actin cytoskeleton [37,47,48]. Interestingly, the expression of exog- imaginal disc [66–68]. This mechanism of interaction, as discussed enous Angiomotins in some of these systems resulted in the local- below, resembles the physical association between Amot and ization to cytoplasmic puncta instead of targeting to TJs [47]. AmotL1 with YAP and TAZ. However, FRMD6/Willin alone was Furthermore, in the tight junction-forming MDCK cells, not able to rescue Ex loss in Drosophila [62], thus raising the possi- Amot-p130 expression induced partial translocation of YAP to bility that Ex function has been split during evolution into the the cytoplasm and co-localization with the TJ protein ZO-1 [47]. cooperative actions of Angiomotins and FRMD6/Willin. Loss-of-function analysis in cell lines expressing at least one of Elegant work conducted by Sowa et al. (2009) first described the three members of the family added further insights into Motins Amot as a YAP binding partner. This finding was part of a global involvement in the negative regulation of YAP function. Consistent proteomic survey analyzing de-ubiquitinating enzymes and their with findings from overexpression studies in MDCK or MCF10A interacting protein complexes in a mammalian cell system [69]. cells, the knockdown of either Amot-p130 or AmotL2 not only Several studies have subsequently demonstrated that Amot-p130 caused loss of TJs with augmented localization of YAP and TAZ in and AmotL1 directly interact with YAP via Motins LPTY and the nucleus, but also promotion of Epithelial–Mesenchymal Transi- first PPEY motif (Amot-p130: 106LPTY109 239PPEY242; AmotL1: tion (EMT) and up-regulation of YAP downstream target genes 188LPTY191 310PPEY313), and mostly the first WW domain of YAP CTGF and Cyr61 [34,37,47,48]. Noteworthy, attenuation of AmotL1 (localized between 171 and 208 residues of YAP [70]. This binding expression was refractory to these phenotypic and transcriptional is independent of their second PPEY motif and PDZ-binding do- changes, pointing out the distinct behaviors between Motin family main as mutations in these regions showed only mild effects members. Interestingly, in H441 human lung cancer cells, AmotL2 [7,31,37,47,48,71]. Yet considerable decrease in the Amot–YAP induced a robust shuttling of TAZ from the nucleus to the cyto- interaction was observed with Amot coiled-coil (CC) domain mu- plasm [72]. This highlights cell-context dependency, suggesting tants [47]. Regarding AmotL2, its PPXY motif (210PPEY213) was the tissue or organs wherein they are expressed determines Motin shown to bind YAP mainly through the first WW domain, and inde- functions (Fig. 2). pendently of the PDZ-binding motif [34,37]. Noteworthy, the asso- Models for the negative regulation of YAP/TAZ activity by the ciation of Amot-p130, AmotL1, and AmotL2 to TAZ (YAP1 paralog) Angiomotins suggest a number of possibilities leading to localiza- occurs exclusively via the Motin PPXY motif and TAZ WW domain tion of YAP/TAZ to the cytoplasm/cell junctions. As discussed pre- [48,72]. Consistently, neither YAP nor TAZ show interaction with viously, a major mechanism of YAP/TAZ regulation is through the short isoform Amot-p80. Therefore, at least under the condi- exclusion from the nucleus. This is regulated to a significant extent tions used in the above-mentioned analyses, the Motin family through YAP and TAZ phosphorylation sites Ser127 and Ser89 res- members represent a strong binding partner of YAP. Interestingly, idues, respectively, leading to cytoplasmic sequestration through several other proteins including LATS1/2 also bind to the WW do- 14-3-3 binding [54,86]. Both YAP and TAZ are kinase substrates mains of YAP through their PPXY motifs [73–78]. of LATS1/2 [76,54]. Once activated, LATS1/2 phosphorylates YAP It should be noted that some potential discrepancies exist in on five HxRxxS consensus sites – Ser61, Ser109, S127, S164, and the literature as to which isoform of YAP interacts with the Mo- Ser381 [87], and TAZ on four HxRxxS motifs – Ser66, Ser89, S117, tins. Some studies have shown that the Motin family members and Ser311 [88]. Phosphorylation of YAP Ser127 and Ser381 resi- bind YAP isoform 2, which has two WW domains, but not to iso- dues by LATS1/2 are key events for its inhibition [87] whilst on form 1, with one WW domain [30,71,79]. The difference between TAZ, phosphorylation of Ser89 and Ser311 sites were shown to in- the two isoforms consists of an extra 38 amino acids that com- hibit its transcriptional activity [88]. pose the second WW domain. Intriguingly, a number of other Accordingly, it has been suggested that Motins serve as a scaf- studies have identified an interaction between Motins and the fold for Hippo pathway kinases and YAP at TJs, leading to activation YAP1 isoform. These differences could result from multiple rea- of LATS2 and phosphorylation of YAP2 (Fig. 4) [34]. Yet other stud- sons such as different experimental systems or protein expression ies show only mild up-regulation of phosphorylated YAP in beyond physiological levels. In any case, caution is advised when HEK293 cells upon exogenous expression of AmotL2 [47]. Other determining which YAP isoform is being used in a given experi- models suggest that Motin-mediated localization of YAP/TAZ to mental setup. Indeed, at least two out of the eight YAP isoforms the cytoplasm/cell junctions is independent of YAP/TAZ phosphor- appear to differently regulate the Hpo/YAP signaling pathway ylation by LATS1/2 [47]. This is supported by studies showing that [71,74]. this localization it is still observed with YAPS127A and TAZS89A mu- Besides YAP, the Motins also associate with other effectors of tants (Fig. 4) [37,48]. the Hpo/YAP signaling pathway. Specifically, Merlin interacts with In contrast to the findings ascribing a YAP-inhibitory role for Amot-p130 through their mutual CC domains [43]; LATS2 associ- Angiomotins, we have recently found that Amot-p130 is required ates with Amot-p130 CC domain, and with the N-terminal 307 for YAP function both in vivo and in vitro. By using both gain- amino acids of AmotL2 through its kinase domain, including the and loss-of-function approaches, we find that in multiple cell types adjacent Mob-1 binding region [23,34]. AmotL2 also binds MST2, Amot-p130 directly interacts with YAP and functions as a positive yet the domains involved in this interaction are currently unknown regulator by antagonizing YAP interaction with LATS1, promoting [34]. KIBRA, an upstream component of the pathway, binds to the YAP dephosphorylation and translocation to the nucleus (Fig. 4) second 284PPEY287 motif of Amot-p130 through its N-terminus [7]. The differences between the observations described above [23]. Whether KIBRA mediates Motin function is not known, yet might reflect use of different cell lines or experimental setups. this protein is involved not only in regulation of the Hpo–YAP For example, a potential caveat is the expression of exogenous pathway [80–82] but also in the regulation of epithelial cell polar- Motin family proteins tagged at their C-terminal PDZ binding ity [83–85]. Future analyses will help to clarify the significance of domain, which might mask interactions with binding partners this interaction. such as Patj/Mupp1. Yet another possibility is that previously 2700 S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703

Cytoplasm TJ TJ TJ TJ TJ TJ AMOT Merlin Cytoplasm MST1/2 AMOT Merlin LATS1/2 AMOT P YAP P YAP ?? 3 AMOT 1 P Tao1 P P ?? YAP P MST1/2 Sav1 Actin Mob1 4

LATS1/2 P Amot Cytoplasmic Retention Degradation by ubiquitination 1 p130 2 P YAP 14-3-3 P P YAP P P 1 YAP AMOT 2 (??) YAP

P P ?? ? Alternative Amot transcriptional programs Nucleus p130 YAP YAP TEAD 1-4 TEAD 1-4

Fig. 4. Proposed models for YAP regulation by the Motins. Amot-p130 as a positive regulator of YAP: (1) Amot-p130 interaction with YAP prevents its association with LATS1/ 2 in the cytoplasm and LATS-mediated YAP phosphorylation. Subsequently Amot-p130:YAP complex translocates to the nucleus to promote the transcription of TEAD-target genes. Amot as a negative regulator of YAP: (2) LATS-mediated phosphorylation of Amot promotes Amot-YAP association and subsequent inhibition of YAP activity. Whether Amot phosphorylation regulates YAP sequestration to the cytoplasm, and/or localization at the TJs is currently unknown. (3) Amot acts as a scaffold protein at the TJ, bringing together MST and LATS, promoting LATS activation and subsequent LATS-mediated YAP phosphorylation. (4) Amot physically interacts with YAP leading to localization at the TJ or to the actin cytoskeleton, independent of LATS-mediated phosphorylation events.

unappreciated post-translational modifications play a role in regu- 7. Function: Angiomotin involvement in the pathological lating the function of Motin family members. Interestingly, recent setting studies suggest that Motin-YAP association might also be regulated by phosphorylation of the Motins by LATS1/2 (Fig. 4). These studies The relationship of Angiomotins to the pathogenesis and pro- show that phosphorylation regulates Motin subcellular localization gression of human diseases is still poorly understood. The majority and activities related to cell proliferation and migration of investigations have centered upon the expression of Amot in a [23,30,32,33]. Whether this phosphorylation induces translocation variety of cancers and its role in modulating angiogenesis, an of YAP/TAZ from the nucleus to the cytoplasm and subsequent essential process for cancer progression and survival. A mouse localization to TJs remains to be determined. model of hemangioendothelioma that overexpresses Amot-p80 re- Finally, in addition to mechanisms involving the sequestration sulted in fast-growing invasive tumors that were larger than con- of Yap out of the nucleus, we have recently identified a nuclear trols [90]. In contrast, an Amot mutant-expressing cell line in function for Amot-p130 in regulating YAP activity [7]. Amot- which the C-terminus PDZ-binding motif was deleted produced p130 was found in the nucleus where it forms a complex with smaller, dormant tumors that displayed a high level of apoptosis. YAP and the DNA-binding transcriptional factor TEAD1. Gene The Amot mutant tumors remained dormant for more than 21 days expression profiling further supported these findings and demon- and did not invade surrounding tissues. Both Amot and deletion strates a strong concordance between Amot-p130 and YAP tran- mutant tumors were vascularized, but the deletion of the C-termi- scriptional profiles, suggesting functional cooperation. In fact, nal amino acids resulted in inhibited cell migration, suggested as microarray analysis and gene set enrichment analysis (GSEA) indi- an explanation for the lack of invasiveness [90]. cated that 99.5% of the genes co-regulated by Amot-p130 and YAP Subsequent screening of human breast tumors has revealed sig- are regulated in the same direction, with significant correlation be- nificantly increased expression of Amot in tumors relative to nor- tween the top-ranked Amot-p130 and YAP-regulated genes. Lucif- mal tissue [91]. In contrast, AmotL1 and AmotL2 have shown no erase assays and Chromatin immunoprecipitation (ChIP) coupled significant correlation with tumor burden. Furthermore, Amot to RT-PCR show that in HEK293 cells, knockdown of Amot-p130 overexpression correlated with tumor grade, decreased survival, expression inhibited the binding of a YAPS112A mutant to the pro- and metastatic disease. Taken together, these findings imply an moter of ApoE (which encodes Apolipoprotein E). Surprisingly, important role for Amot in the context of tumor progression and binding of YAPS112A to the CTGF promoter was not affected by underscore the suitability of Amot as a potential biomarker and a Amot-p130 knockdown. Sequential ChIP analysis further corrobo- target for anticancer therapeutics. rated that Amot-p130 functions as a co-factor in a transcriptionally Efforts thus far to design Amot-based therapies have centered active YAP-TEAD1 complex, involved in recruiting the complex to on immunological-based strategies. A DNA-based vaccine against promoters of a subset of YAP-regulated genes, which does not in- human Amot-p80 produced in mice has shown promising results clude CTGF [7,89]. in a mouse model of breast carcinoma [18]. Implantation of mouse S. Moleirinho et al. / FEBS Letters 588 (2014) 2693–2703 2701

TUBO (Turin–Bologna) mammary carcinoma cells produced rapidly choroidal plaques by 73% [17]. Finally, recent research has corre- growing tumors in mice vaccinated with empty vector. By contrast, lated Amot expression to microRNA-induced tumor dormancy vaccination with Amot-p80 inhibited tumor growth completely in [97]. In this disease state, proliferation is balanced by increased the majority of mice. In the few examples where tumors did devel- apoptosis and an impairment of angiogenesis. Dormant glioblas- op the tumors grew much slower compared to control mice. The toma multiforme tumors that expressed dormancy-associated significant suppression of TUBO tumor growth in response to miRs also showed elevated levels of Amot with enriched angio- Amot-p80 vaccination was partially abrogated in CD4-depleted statin, suggesting a potential antiangiogenic mechanism for tumor mice and completely nullified in B cell knockout mice, suggesting dormancy. vaccination effects are antibody-dependent. Amot-p80 vaccination Overall, the identified interactions of Amot within disease path- was also tested using a more aggressive transgenic breast cancer ways present a complex picture, with angiogenic signaling and model, based on Her-2/neu oncogene under the control of the Hpo–YAP pathway interactions both driving the pathology of a mouse mammary tumor virus promoter (MMTV). Vaccination with variety of tumor types. It is clear from the genetic and immuno- Amot-p80 slightly delayed tumor progression, but when combined logic studies that Amot is a promising therapeutic target and future with vaccination against Her-2 extracellular and transmembrane work will undoubtedly reveal a more thorough and cohesive pic- domains, a synergistic effect was observed and 80% of vaccinated ture of Amot dysregulation and interactions within a wide range mice survived for more than 70 weeks, compared to the complete of tumor and angiogenic disease types. lethality at 20 and 25 weeks for control and Amot-alone vaccina- tion, respectively [18,92]. Significantly, the expression of Amot 8. Open questions was observed to increase with the progression of neoplastic lesions to invasive carcinomas [92]. Vaccination with Amot-p80 inhibited There are major gaps in our understanding of the Motin family angiogenesis in implanted tumors, and isolated Amot antibodies of proteins as detailed throughout this review. One of the major inhibited the migration of mouse aortic endothelial cells, replicat- open questions relates to functional redundancy between mem- ing the effects of angiostatin. Finally, Amot-p80 vaccination re- bers of the family. While there are clear areas of overlap it is also sulted in increased permeability of TUBO tumors, which clear that there are non-redundant roles that are likely to be cell- increased the efficacy of chemotherapy [92]. type specific. Another critical question is which are the relevant Interestingly, Amot expression is found to be significantly pathways through which the Motins exert their functions. The dis- upregulated in schwannomas from NF2 patients, which displayed covery that the Motins orchestrate the function of multiple signal- a primarily localization of Amot to the nucleus [7]. Further analysis ing pathways, including pathways regulated by small G-proteins of a role for Amot in this tumor type found that Amot is essential and the Hpo/YAP pathway, draws a complex picture. Interestingly, for the development of schwannomas by Nf2-null Schwann cells the majority of studies examining Amot in animal tumor models [43]. Using an orthotopic model of NF2-associated schwannoma, and human tumor samples suggest Amot expression is upregulated luciferase-expressing Nf2-null schwann cells (SC4) that stably ex- and, in some cases, required for disease progression. This raises press Amot shRNA were implanted intraneurally into the sciatic questions regarding which Amot-related pathways are involved nerve and tumor progression was monitored via bioluminescent and how these pathways modulate Amot functions within the con- imaging. In contrast to control SC4 cells, which formed substantial text of the disease. Again, this is also likely cell-type and context- tumors one week after implantation, Amot knockdown resulted in dependent. Further complexity has been recently introduced with a significant impairment in the growth of schwannomas. Western the identification of post-translational modifications that likely blotting revealed sustained suppression of Amot expression and regulate the function of the Motin themselves. The role of these MAPK signaling, which explains the slower tumor growth and is modifications, as well as other transcriptional and post-transcrip- consistent with the proposed mechanism. Together, these results tional mechanisms, are only beginning to emerge and ongoing demonstrate the requirement of Amot for development of schwan- research by multiple groups will surely lead to a better under- noma in response to loss of Nf2. standing of these mechanisms in short order. 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