FAT10 Induces cancer cell migration by stabilizing phosphorylated ABI3/NESH FAT10 Induces cancer cell migration by stabilizing phosphorylated ABI3/NESH
Um, Hyojin; Jeong, Hoim; Lee, Beomgu; Kim, Yerin; Lee, Jihyeon; Roh, Jong Seong; Lee, Seung-Geun; Park, Hae Ryoun; Robinson, William H.; Sohn, Dong Hyun
ANIMAL CELLS AND SYSTEMS 2023, VOL. 27, NO. 1, 53–60 https://doi.org/10.1080/19768354.2023.2186486 a a a a a b c,d Hyojin Um *, Hoim Jeong *, Beomgu Lee , Yerin Kim , Jihyeon Lee , Jong Seong Roh , Seung-Geun Lee , e f,g a Hae Ryoun Park , William H. Robinson and Dong Hyun Sohn a b Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan, Republic of Korea; Department of Herbal Prescription, College of Korean Medicine, Daegu Haany University, Gyeongsan, Republic of Korea; Biomedical Research Institute, Pusan National University Hospital, Busan, Republic of Korea; Division of Rheumatology, Department of Internal Medicine, Pusan National University School of Medicine, Pusan National University Hospital, Busan, Republic of Korea; Department of Oral Pathology, School of Dentistry, Pusan National University, Yangsan, Republic of Korea; Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA; VA Palo Alto Health Care System, Palo Alto, CA, USA ABSTRACT ARTICLE HISTORY Received 23 January 2023 The WAVE regulatory complex (WRC) is involved in various cellular processes by regulating actin Revised 8 February 2023 polymerization. The dysregulation of WRC components is associated with cancer development. Accepted 12 February 2023 ABI family member 3 (ABI3)/new molecule including SH3 (NESH) is one of the WRC components and it has been reported that ABI3 phosphorylation can aﬀect WRC function. Although several KEYWORDS residues of ABI3 have been reported to be possible phosphorylation sites, it is still unclear ABI family member 3 (ABI3)/ which residues are important for the function of ABI3. Furthermore, it is unclear how the new molecule including SH3 phosphorylated form of ABI3 is regulated. Here, we demonstrate that ABI3 is stabilized by its (NESH); WAVE regulatory interaction with human leukocyte antigen-F adjacent transcript 10 (FAT10). Using phospho-dead complex (WRC); human leukocyte antigen-F adjacent or phospho-mimetic mutants of ABI3, we showed that serine 213 and 216 are important transcript 10 (FAT10); phosphorylation sites of ABI3. In particular, FAT10 has a higher aﬃnity for the phosphorylated phosphorylation form of ABI3 than the non-phosphorylated form, and it stabilizes the phosphorylated form more than the non-phosphorylated form through this diﬀerential aﬃnity. The interaction between FAT10 and the phosphorylated form of ABI3 promoted cancer cell migration. Therefore, our results suggest that FAT10 stabilizes the phosphorylated form of ABI3, which may lead to WRC activation, thereby promoting cancer cell migration. Introduction translocation to the plasma membrane is signiﬁcantly reduced, and the peripheral lamellipodial structure is The WAVE regulatory complex (WRC) regulates polymer- disturbed (Sekino et al. 2015). Additionally, although ization of the actin cytoskeleton by stimulating the Arp2/ ABI1 and ABI2 were able to phosphorylate WAVE2, 3 complex. It consists of ﬁve subunits: HSPC300, ABI1 ABI3 did not (Hirao et al. 2006). (ABI2 or ABI3), NAP1/HEM2 (HEM1), SRA1/CYFIP1 It has been suggested that the phosphorylation of (PIR121/CYFIP2), and WAVE1 (WAVE2 or WAVE3) (Chen ABI3 by the PI3K/AKT pathway inhibits inactive WRC for- et al. 2014). Since WRC plays a key role in cellular pro- mation, thereby activating WRC (Moraes et al. 2017; Rana cesses, including the maintenance of cellular structure, et al. 2021). Abnormalities in the PI3K pathway are spreading, adhesion, and migration, dysregulation of common in cancers and play an important role in neo- WRC components is known to be related to cancer plastic transformation. Therefore, although ABI3 is con- development (Kurisu et al. 2005; Yamaguchi and Con- sidered a tumor suppressor, the phosphorylated form deelis 2007; Silva et al. 2009; Escobar et al. 2010; Rana of ABI3 may be associated with cancer development et al. 2021). by activating WRC. There are several putative phos- The ABI family member 3 (ABI3), also known as new phorylation sites in ABI3, including serine 213, 216, and molecule including SH3 (NESH), is one of ABL-interactor 342, which are highly conserved among humans, mice, (ABI) family proteins which include ABI1 and ABI2 (Leng rats, cows, and monkeys (Moraes et al. 2017). Although et al. 2005). It has been suggested that ABI3-containing it has been reported that these residues are likely to WRC is functionally distinct from ABI1/2-based WRC. be phosphorylation sites, it remains unclear which When ABI3 is ectopically expressed, WAVE2 CONTACT Dong Hyun Sohn email@example.com *These authors contributed equally to this work. © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 54 H. UM ET AL. residues of ABI3 are actually phosphorylated and impor- cultured in a humidiﬁed atmosphere containing 5% tant for the function of ABI3. CO₂ at 37°C. Lipofectamine 3000 transfection reagent A growing number of proteins containing domains was purchased from Thermo Fisher Scientiﬁc. GENE- with signiﬁcant homology to ubiquitin have been dis- fect was purchased from TransLab. Cycloheximide was covered over the past several years. Ubiquitin-like pro- purchased from Sigma-Aldrich. Protease and phospha- teins (UBLs), such as SUMO, NEDD8, and ISG15 have tase inhibitor cocktails were purchased from GenDEPOT. been investigated in detail, and speciﬁc E1, E2, and E3 The IP lysis buﬀer and Dynabeads protein G were pur- enzymes have been identiﬁed (Jentsch and Pyrowolakis chased from Thermo Fisher Scientiﬁc. The anti-Myc 2000). Human leukocyte antigen-F adjacent transcript (9E10)-HRP antibody was purchased from Abcam, and 10 (FAT10) is a fairly new member of the UBL family the anti-FLAG (M2)-HRP, anti-HA-HRP (3F10), anti-FLAG- that covalently modiﬁes target substrates by binding M2, anti-Myc (9E10), and β-actin-HRP antibodies were via the conserved di-glycine motif at the C-terminus purchased from Sigma-Aldrich. (Fan et al. 1996). FAT10 is encoded in the major histo- compatibility complex and is synergistically induced Plasmids by the proinﬂammatory cytokines, interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) (Liu The cDNA for ABI3 was purchased from the Korean et al. 1999; Raasi et al. 1999). Additionally, FAT10 as Human Gene Bank, and the cDNA for FAT10 was pur- well as its conjugates are rapidly degraded by the pro- chased from Origene. p3xFLAG-CMV10 or pcDNA3.1- teasome in a ubiquitin-independent manner (Hipp et al. Myc-His C was used to express FLAG-FAT10 and 2005), indicating that FAT10 is the ﬁrst ubiquitin-like ABI3-Myc, respectively. The sequences of all plasmids modiﬁer that functions as a protein signal for the were veriﬁed by sequencing (Bionics). Plasmids for rapid degradation of substrate proteins through the transfection were extracted using a Midi Prep Kit proteasome. (Qiagen). FAT10 has recently been highlighted for its associ- ation with cancer, and is overexpressed in various ABI3 site-directed mutagenesis cancers, such as gastrointestinal cancer, liver cancer, pancreatic ductal adenocarcinoma, and glioma (Yuan Information on the putative phosphorylation sites of et al. 2014). Additionally, FAT10 also suppresses the human ABI3 was obtained from the NCBI (https://www. transcriptional activity of the tumor suppressor gene ncbi.nlm.nih.gov/nuccore/NM_016428.3) and UniProt p53 and facilitates p53 degradation to accelerate (https://www.uniprot.org/uniprotkb/Q9P2A4/entry). tumorigenesis (Choi et al. 2014;Suetal. 2021;Zhang Residue numbers were deﬁned according to human et al. 2021). Furthermore, overexpression of FAT10 pro- isoform 1 (NP_057512.2), with S213, S216, and S342 as motes tumor growth (Theng et al. 2014)and malig- putative phosphorylation sites. Substitution of serine nancy, suggesting that FAT10 may play a key role in with alanine or aspartic acid was used for the production tumor development. In this study, we hypothesized of phospho-dead mutant or phospho-mimetic mutants, that FAT10 may regulate ABI3 through interaction, respectively, as previously described (Lee et al. 2011). which contributes to cancer development. We demon- Site-directed mutagenesis was performed using strated that FAT10 induced cancer cell migration by pre- primers for phospho-dead (S213A, S216A, and S342A) ferentially stabilizing phosphorylated ABI3. and phospho-mimetic (S213D, S216D, and S342D) mutations, and conﬁrmed by sequencing. Wild-type and mutant ABI3 expression plasmids were transiently Materials and methods transfected into HEK-293 T cells, and the expression of mutant proteins was evaluated by western blot analysis. Cell culture and reagents Human embryonic kidney (HEK)−293T cells were cul- Transfection and preparation of protein lysates tured in the Dulbecco’smodiﬁed eagle medium (Sigma-Aldrich) supplemented with 10% fetal bovine HEK-293T cells were plated in a 6-well plate at a density of serum (FBS), 100 units/mL of penicillin, and 100 μg/mL 0.7 × 10 cells/well a day before transfection. Transfection of streptomycin (Thermo Fisher Scientiﬁc). Human color- of plasmids was performed using lipofectamine 3000 ectal adenocarcinoma SW480 cells were cultured in the transfection reagent or GENE-fect, according to the man- Roswell Park Memorial Institute-1640 medium (Sigma- ufacturer’s instructions. For some experiments, cyclohex- Aldrich) supplemented with 10% FBS, 100 units/mL of imide was used before cell harvest. Cells were harvested penicillin, and 100 μg/mL of streptomycin. Cells were after washing with 1 × phosphate buﬀered saline (PBS) ANIMAL CELLS AND SYSTEMS 55 and lysed with IP lysis buﬀer supplemented with protease and observed using an optical microscope CKX41 and phosphatase inhibitor cocktails. The lysates were (Olympus). Migration rate was quantiﬁed using the centrifuged at 13,000 rpm for 15 min at 4°C, and the ImageJ plugin, MRI wound healing tool. supernatant was used for immunoprecipitation or western blot analysis. Total protein concentration was Statistical analysis determined using bicinchoninic acid (BCA) method (Thermo Fisher Scientiﬁc). Statistical analyses were performed using the Graph- Pad Prism software. All data are presented as mean ± standard error of the mean (SEM). One-way analysis Western blot analysis of variance (ANOVA) with Tukey’s multiple compari- For western blot analysis, cell lysates were resolved on son test was used for comparisons between multiple 8% or 10% sodium dodecyl sulfate-polyacrylamide gel groups, and p < 0.05 was considered statistically electrophoresis (SDS-PAGE) and transferred to polyviny- signiﬁcant. lidene ﬂuoride (PVDF) membranes (Merck Millipore). Subsequently, the membrane was dried at 37°C for 1 h, Results followed by blocking with 5% skim milk in 1 × Tris- buﬀered saline supplemented with 0.1% Tween-20 ABI3 interacts with FAT10 (TBS-T) for 1 h. The membrane was then probed for at It has been reported that ABI3 expression is decreased least 2 h with anti-Myc (9E10)-HRP or anti-FLAG (M2)- in thyroid tumors, and ectopic expression of ABI3 inhi- HRP antibodies, followed by washing three times with bits tumor formation (Latini et al. 2011). In contrast, it TBS-T for 10 min. Finally, the membranes were visualized has been suggested that phosphorylation of ABI3 may using an enhanced chemiluminescence reagent (ProNA disrupt inactive WRC formation, which may lead to ECL Ottimo, TransLab). Chemiluminescent signals were cancer development through the activation of WRC detected using an ImageQuant LAS3000 Analyzer (Fuji- (Moraes et al. 2017; Rana et al. 2021). However, it is Film). The band intensities were quantiﬁed using still unclear whether phosphorylation of ABI3 contrib- ImageJ software. The data were normalized to β-actin utes to cancer development and how the phosphory- expression (loading control) as previously described lated form of ABI3 is regulated. In various cancers, (Roh et al. 2021). FAT10 is upregulated and is known to regulate several proteins involved in cancer development, Co-immunoprecipitation assay such as MAD2, p53, and β-catenin through covalent and non-covalent interactions (Aichem and Groettrup Cells were harvested and lysed on ice for 15 min in the IP 2016; Su et al. 2021; Zhang et al. 2021). Therefore, lysis buﬀer containing protease and phosphatase inhibi- we investigated whether ABI3 is regulated by FAT10. tor cocktails, and the lysates were centrifuged at First, co-immunoprecipitation was performed to 13,000 rpm for 15 min at 4°C. For immunoprecipitation, examine the interactions between ABI3 and FAT10. 4 μg of anti-Myc (9E10) or anti-FLAG (M2) was added As shown in Figure 1, Myc-tagged ABI3 was co-immu- and incubated overnight at 4°C on a tube rotator. Dyna- noprecipitated with FLAG-tagged FAT10. Furthermore, beads protein G was added and incubated for 1 h at 4°C considering the input control, which shows weaker on a tube rotator. The immunoprecipitate was washed band intensity of the phosphorylated form compared four times with IP lysis buﬀer (0.5 mL). After the ﬁnal to the non-phosphorylated form of ABI3, the phos- washing, the beads were resuspended in 25 μL2× phorylated form interacts more strongly with FAT10 sample buﬀer. The samples were boiled for 5 min, than the non-phosphorylated form. This result shows resolved by SDS-PAGE, and western blot analysis was that the phosphorylated form of ABI3 may have a performed. higher aﬃnity for FAT10 than the non-phosphorylated form. Wound healing assay Human colorectal adenocarcinoma SW480 cells were Main phosphorylation sites on ABI3 are serine seeded in a 24-well plate and grown overnight until 213 and 216 the density reached 80%. The monolayer was scratched TM using an SPLScar Scratcher (SPL) and the ﬂoating cells Based on previous studies and database searches, S213, were removed. The migration of SW480 cells to the S216, and S342 in ABI3 are potential phosphorylation scratched areas was assayed after incubation for 24 h sites. To identify the main phosphorylation sites in ABI3, 56 H. UM ET AL. FAT10 has a higher aﬃnity for phosphorylated form of ABI3 than non-phosphorylated form Next, we tested whether the phospho-dead or phospho- mimetic mutants of ABI3 have diﬀerent binding aﬃnities for FAT10, as suggested in Figure 1. The expression plasmids for the phospho-dead mutant of ABI3 (S213A/S216A) or phospho-mimetic mutant of ABI3 (S213D/S216D) were co-transfected with the FLAG- FAT10 expression plasmid, and the interaction between ABI3 mutants and FAT10 was investigated by co-immuno- precipitation. As shown in Figure 3, the wild-type and its mutants interacted with FAT10. However, compared to the input control, the immunoprecipitate of the phospho-mimetic mutant showed a stronger signal than the phospho-dead mutant, which again conﬁrms that Figure 1. Interaction of ABI3 with FAT10. HEK-293T cells were the phosphorylated form of ABI3 has a higher binding co-transfected with Myc-tagged ABI3 (ABI3-Myc) and FLAG- aﬃnity for FAT10 than the non-phosphorylated form. tagged FAT10 (FLAG-FAT10) expression plasmids. Cell lysates (300 μg) were immunoprecipitated using anti-FLAG or anti- Myc antibodies. Western blot analysis was performed using Phosphorylation status of ABI3 is important for anti-Myc-HRP or anti-FLAG-HRP. Beta-actin was used as the the stabilization of ABI3 by FAT10 loading control. The upper band of ABI3-Myc is phosphorylated form and the lower band is non-phosphorylated form. FAT10 targets its substrates for proteasomal degradation by covalent binding in a manner similar to that of ubiqui- tin. However, recent studies have demonstrated that we introduced mutations at each potential phosphoryl- FAT10 stabilizes its interacting proteins through non- ation site. Phospho-dead mutants of ABI3 were produced covalent interactions (Yuan et al. 2014; Dong et al. by substituting serine with alanine (S213A, S216A, and 2016; Liu et al. 2016; Zhou et al. 2018; Zou et al. 2018). S342A), and the phospho-mimetic mutants of ABI3 As shown in Figures 1 and 3, ABI3 non-covalently inter- were produced by substituting serine with aspartic acid acts with FAT10. Additionally, we did not detect any (S213D, S216D, and S342D). Western blot analysis indi- covalent interactions between ABI3 and FAT10 (data cated that while ABI3 (S342A) showed almost similar not shown). Therefore, it is possible that ABI3 is also amount of phosphorylated form as the wild-type, ABI3 stabilized by FAT10, and that the phosphorylation (S213A) and ABI3 (S216A) showed relatively low status of ABI3 may be important for stabilization. To amounts of phosphorylated forms (Figure 2A). Moreover, verify this possibility, wild-type or phospho-mutants of while the phosphorylated form disappeared in ABI3 the ABI3-Myc expression plasmid were transfected with (S213A/S216A), non-phosphorylated form disappeared or without the FLAG-FAT10 expression plasmid and a in ABI3 (S213D/S216D) (Figure 2). These results suggest cycloheximide chase assay was performed. As expected, that S213 and S216 on ABI3 are important phosphoryl- ABI3 was stabilized by FAT10, and the phospho-mimetic ation sites. mutant of ABI3 showed a greater stabilization eﬀect than Figure 2. Main phosphorylation sites on ABI3 are S213 and S216. HEK-293 T cells were transfected with either phospho-dead mutants (A) or phospho-mimetic mutants of ABI3 (B), along with wild-type ABI3. The levels of the phosphorylated and non-phosphorylated forms of wild-type or mutant ABI3 were determined by western blot analysis using an anti-Myc-HRP antibody. Beta-actin was used as the loading control. ANIMAL CELLS AND SYSTEMS 57 the phospho-dead mutant (Figure 4). However, the degradation rate of each mutant was similar, even in the co-expression of FAT10, probably because of the very rapid degradation of FAT10 during the cyclohexi- mide chase assay. These results suggest that ABI3 is stabilized by FAT10, and that phosphorylation of ABI3 is important for stabilization by FAT10. Phosphorylated ABI3-induced cancer cell migration is mediated by FAT10 Considering that FAT10 is overexpressed in various cancers and stabilizes phosphorylated ABI3 (Figure 4), it is possible that the interaction between FAT10 and phosphorylated ABI3 contributes to cancer develop- ment. Therefore, we tested whether FAT10 mediates phosphorylated ABI3-induced cancer cell migration using a wound-healing assay. Overexpression of wild- type, phospho-dead mutant, or phospho-mimetic Figure 3. Phosphorylated form of ABI3 has a higher aﬃnity for mutant of ABI3 alone in SW480 colon cancer cells had FAT10. HEK-293 T cells were co-transfected with ABI3-Myc, ABI3 no eﬀect on migration (Figure 5A and B). However, (S213A/S216A)-Myc, or ABI3 (S213D/S216D)-Myc expression plasmids, along with the FLAG-FAT10 expression plasmid. Cell when FAT10 was overexpressed with wild-type ABI3 or lysates (300 μg) were immunoprecipitated using anti-FLAG or its mutants, the wild-type or phospho-mimetic mutant anti-Myc antibodies. Western blot analysis was performed of ABI3 promoted cancer cell migration, which was using anti-Myc-HRP or anti-FLAG-HRP antibodies. Beta-actin inhibited by the phospho-dead mutant (Figure 5C and was used as the loading control. D). These results suggest that the interaction between Figure 4. Phosphorylation status of ABI3 is important for the stabilization of ABI3 by FAT10. (A) HEK-293T cells were co-transfected with wild-type, phospho-dead, or phospho-mimetic mutants of ABI3-Myc expression plasmids, along with or without the FLAG-FAT10 expression plasmid. Cells were treated with cycloheximide (CHX, 100 μM) for 0, 4, 8 h. Western blot analysis was performed with anti- Myc-HRP or anti-FLAG-HRP antibodies. Beta-actin was used as the loading control. (B) Data from western blot were quantiﬁed by densitometry. Data are presented as mean ± SEM and p values were calculated by one-way ANOVA with Tukey’s multiple comparison test. **p < 0.01, ***p < 0.001. ns, not signiﬁcant. 58 H. UM ET AL. Figure 5. Phosphorylated ABI3-induced cancer cell migration is mediated by FAT10. (A and C) SW480 cells were transfected with wild- type, phospho-dead, or phospho-mimetic mutants of ABI3-Myc expression plasmids, along with or without the FLAG-FAT10 expression plasmid and the scratched areas of cells were observed at 0 and 24 h by an optical microscope (x100). (B and D) The migration rate was calculated by comparing the scratched area at 0 and 24 h. Data are presented as mean ± SEM and p values were calculated by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not signiﬁcant. FAT10 and phosphorylated ABI3 contributes to the regu- stabilizes β-catenin by blocking its ubiquitination lation of cancer cell migration. (Yuan et al. 2014). Additionally, caveolin-3, eEF1A1, survi- vin, and ZEB2 are stabilized by FAT10 (Dong et al. 2016; Liu et al. 2016; Zhou et al. 2018; Zou et al. 2018). In this Discussion study, we also showed that ABI3 is stabilized by FAT10 It has been reported that FAT10 is involved in cancer through non-covalent binding (Figures 1 and 4), but development; it is overexpressed in various cancers, the mechanism of this stabilization remains unclariﬁed. including colon and liver cancers (Lee et al. 2003). Although ABI3 plays an important role as a tumor Additionally, FAT10 promotes the survival, proliferation, suppressor, the precise mechanism by which ABI3 migration, and invasion of cancer cells (Gao et al. 2014; exerts this function is still unknown. It has been Aichem and Groettrup 2016). In particular, FAT10 sup- suggested that the phosphorylation of ABI3 by the presses the transcriptional activity of p53 and facilitates PI3K/AKT pathway may disrupt inactive WRC formation, p53 degradation to accelerate tumorigenesis (Choi et al. which may contribute to tumorigenesis. We showed that 2014; Su et al. 2021; Zhang et al. 2021). These results FAT10 mediates phosphorylated ABI3-induced cancer showed that FAT10 plays an important role in tumori- cell migration, probably through preferential interaction genesis. Although diverse UBLs, including FAT10, of FAT10 with phosphorylated ABI3. Considering the NEDD8, SUMO, and ISG15, are similar to ubiquitin, importance of phosphorylation of ABI3, we also investi- FAT10 is the only UBL that targets its substrates for pro- gated the phosphorylation sites of ABI3 and found that teasomal degradation. However, recent studies have serine 213 and 216 are important phosphorylation revealed that FAT10 can also stabilize interacting pro- sites. This is contrary to a previous report suggesting teins. Non-covalent binding of FAT10 to β-catenin that serine 342 of ABI3 is likely a phosphorylation site ANIMAL CELLS AND SYSTEMS 59 Chen B, Brinkmann K, Chen Z, Pak CW, Liao Y, Shi S, Henry L, by AKT (Moraes et al. 2017). However, their S342A Grishin NV, Bogdan S, Rosen MK. 2014. The WAVE regulatory mutant was still phosphorylated, according to their complex links diverse receptors to the actin cytoskeleton. data. In contrast, we showed that mutation of both Cell. 156:195–207. serine 213 and 216 into alanine completely abrogated Choi Y, Kim JK, Yoo JY. 2014.NFκB and STAT3 synergistically the phosphorylation of ABI3, and mutation of both activate the expression of FAT10, a gene counteracting serine 213 and 216 into aspartic acid mimics the phos- the tumor suppressor p53. Mol Oncol. 8:642–655. Epub phorylated form of ABI3. Dong D, Jiang W, Lei J, Chen L, Liu X, Ge J, Che B, Xi X, Shao J. In this study, we conﬁrmed that the phosphorylated 2016. Ubiquitin-like protein FAT10 promotes bladder cancer form and phospho-mimetic mutant of ABI3 have a progression by stabilizing survivin. Oncotarget. 7:81463– strong aﬃnity for FAT10 compared to the non-phos- phorylated form or phospho-dead mutant of ABI3. Escobar B, de Cárcer G, Fernández-Miranda G, Cascón A, Bravo- Cordero JJ, Montoya MC, Robledo M, Cañamero M, Additionally, FAT10 exhibited a stabilization eﬀect by Malumbres M. 2010. Brick1 is an essential regulator of non-covalent binding with ABI3. More importantly, the actin cytoskeleton required for embryonic development phospho-mimetic mutant of ABI3 showed a greater and cell transformation. Cancer Res. 70:9349–9359. Epub stabilization eﬀect than the phospho-dead mutant (Figure 4), conﬁrming that phosphorylation status of Fan W, Cai W, Parimoo S, Schwarz DC, Lennon GG, Weissman ABI3 is important for stabilization by FAT10. Further SM. 1996. Identiﬁcation of seven new human MHC class I region genes around the HLA-F locus. Immunogenetics. studies are required to determine the detailed stabiliz- 44:97–103. ation mechanisms of phosphorylated ABI3 by FAT10. Gao Y, Theng SS, Zhuo J, Teo WB, Ren J, Lee CG. 2014. FAT10, In conclusion, we demonstrated that FAT10 interacts an ubiquitin-like protein, confers malignant properties in with ABI3 and stabilizes it with preference for the phos- non-tumorigenic and tumorigenic cells. Carcinogenesis. phorylated form of ABI3. 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Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells. ation of data: HU, HJ, YK, SGL, HRP, WHR, and DHS; Oncogene. 24:1309–1319. Writing and editing of the manuscript: HU, HJ, BL, YK, Latini FR, Hemerly JP, Freitas BC, Oler G, Riggins GJ, Cerutti JM. JL, JSR, and DHS; Funding acquisition: DHS; Study super- 2011. ABI3 ectopic expression reduces in vitro and in vivo vision: DHS. cell growth properties while inducing senescence. BMC Cancer. 11:11. Epub 20110111. Lee CG, Ren J, Cheong IS, Ban KH, Ooi LL, Yong Tan S, Kan A, Disclosure statement Nuchprayoon I, Jin R, Lee KH, et al. 2003. Expression of the FAT10 gene is highly upregulated in hepatocellular carci- No potential conﬂict of interest was reported by the author(s). noma and other gastrointestinal and gynecological cancers. Oncogene. 22:2592–2603. Lee EJ, Shin SH, Hyun S, Chun J, Kang SS. 2011. 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