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The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.

Themis is a new component of the TCR signaling machinery that plays a critical role during T cell development. The positive selection of immature CD4 + CD8 + double-positive thymocytes and their commitment to the CD4 + CD8 - single-positive stage are impaired in Themis -/- mice, suggesting that Themis might be important to sustain TCR signals during these key developmental processes. However, the analysis of Themis mRNA levels revealed that Themis gene expression is rapidly extinguished during positive selection. We show in this article that Themis protein expression is increased in double-positive thymocytes undergoing positive selection and is sustained in immature single-positive thymocytes, despite the strong decrease in Themis mRNA levels in these subsets. We found that Themis stability is controlled by the ubiquitin-specific protease USP9X, which removes ubiquitin K48-linked chains on Themis following TCR engagement. Biochemical analyses indicate that USP9X binds directly to the N-terminal CABIT domain of Themis and indirectly to the adaptor protein Grb2, with the latter interaction enabling recruitment of Themis/USP9X complexes to LAT, thereby sustaining Themis expression following positive selection. Together, these data suggest that TCR-mediated signals enhance Themis stability upon T cell development and identify USP9X as a key regulator of Themis protein turnover., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth. Cancer Res; 77(18); 4921-33. ©2017 AACR ., (©2017 American Association for Cancer Research.)

USP9X (ubiquitin-specific peptidase 9, X chromosome) is the mammalian orthologue of Drosophila deubiquitinase fat facets that was previously shown to regulate the maintenance of the germ cell lineage partially through stabilizing Vasa, one of the widely conserved factors crucial for gametogenesis. Here, we demonstrate that USP9X is expressed in the gonocytes and spermatogonia in mouse testes from newborn to adult stages. By using Vasa-Cre mice, germ cell-specific conditional deletion of Usp9x from the embryonic stage showed no abnormality in the developing testes by 1 week and no appreciable defects in the undifferentiated and differentiating spermatogonia at postnatal and adult stages. Interestingly, after 2 weeks, Usp9x -null spermatogenic cells underwent apoptotic cell death at the early spermatocyte stage, and then, caused subsequent aberrant spermiogenesis, which resulted in a complete infertility of Usp9x conditional knockout male mice. These data provide the first evidence of the crucial role of the spermatogonial USP9X during transition from the mitotic to meiotic phases and/or maintenance of early meiotic phase in Usp9x conditional knockout testes., (© 2017 Society for Reproduction and Fertility.)

Within the adult mammalian brain, neurogenesis persists within two main discrete locations, the subventricular zone lining the lateral ventricles, and the hippocampal dentate gyrus. Neurogenesis within the adult dentate gyrus contributes to learning and memory, and deficiencies in neurogenesis have been linked to cognitive decline. Neural stem cells within the adult dentate gyrus reside within the subgranular zone (SGZ), and proteins intrinsic to stem cells, and factors within the niche microenvironment, are critical determinants for development and maintenance of this structure. Our understanding of the repertoire of these factors, however, remains limited. The deubiquitylating enzyme USP9X has recently emerged as a mediator of neural stem cell identity. Furthermore, mice lacking Usp9x exhibit a striking reduction in the overall size of the adult dentate gyrus. Here we reveal that the development of the postnatal SGZ is abnormal in mice lacking Usp9x. Usp9x conditional knockout mice exhibit a smaller hippocampus and shortened dentate gyrus blades from as early as P7. Moreover, the analysis of cellular populations within the dentate gyrus revealed reduced stem cell, neuroblast and neuronal numbers and abnormal neuroblast morphology. Collectively, these findings highlight the critical role played by USP9X in the normal morphological development of the postnatal dentate gyrus.

The deubiquitylating enzyme Usp9x is highly expressed in the developing mouse brain, and increased Usp9x expression enhances the self-renewal of neural progenitors in vitro. USP9X is a candidate gene for human neurodevelopmental disorders, including lissencephaly, epilepsy and X-linked intellectual disability. To determine if Usp9x is critical to mammalian brain development we conditionally deleted the gene from neural progenitors, and their subsequent progeny. Mating Usp9x(loxP/loxP) mice with mice expressing Cre recombinase from the Nestin promoter deleted Usp9x throughout the entire brain, and resulted in early postnatal lethality. Although the overall brain architecture was intact, loss of Usp9x disrupted the cellular organization of the ventricular and sub-ventricular zones, and cortical plate. Usp9x absence also led to dramatic reductions in axonal length, in vivo and in vitro, which could in part be explained by a failure in Tgf-β signaling. Deletion of Usp9x from the dorsal telencephalon only, by mating with Emx1-cre mice, was compatible with survival to adulthood but resulted in reduction or loss of the corpus callosum, a dramatic decrease in hippocampal size, and disorganization of the hippocampal CA3 region. This latter phenotypic aspect resembled that observed in Doublecortin knock-out mice, which is an Usp9x interacting protein. This study establishes that Usp9x is critical for several aspects of CNS development, and suggests that its regulation of Tgf-β signaling extends to neurons.

The substrate-specific deubiquitylating enzyme USP9X is a putative "stemness" gene expressed in many progenitor cell populations. To test its function in embryonic stem cell-derived neural progenitor/stem cells, we expressed USP9X from a Nestin promoter. Elevated USP9X levels resulted in two phenomena. First, it produced a dramatically altered cellular architecture wherein the majority (>80%) of neural progenitors was arranged into radial clusters. These progenitors expressed markers of radial glial cells and were highly polarized with adherens junction proteins (N-cadherin, beta-catenin, and AF-6) and apical markers (Prominin1, atypical protein kinase C-zeta) as well as Notch, Numb, and USP9X itself, concentrated at the center. The cluster centers were also devoid of nuclei and so resembled the apical end-feet of radial progenitors in the neural tube. Second, USP9X overexpression caused a fivefold increase in the number of radial progenitors and neurons, in the absence of exogenous growth factors. 5-Bromo-2'-deoxyuridine labeling, as well as the examination of the brain lipid-binding protein:betaIII-tubulin ratio, indicated that nestin-USP9X enhanced the self-renewal of radial progenitors but did not block their subsequent differentiation to neurons and astrocytes. nestin-USP9X radial progenitors reformed clusters after passage as single cells, whereas control cells did not, suggesting it aids the establishment of polarity. We propose that USP9X-induced polarization of these neural progenitors results in their radial arrangement, which provides an environment conducive for self-renewal.

Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins.

Ubiquitylation is a key regulator of protein trafficking, and much about the functions of ubiquitin ligases, which add ubiquitin to substrates in this regulation, has recently come to light. However, a clear understanding of ubiquitin-dependent protein localization cannot be achieved without knowledge of the role of deubiquitylating enzymes (DUBs). DUBs, by definition, function downstream in ubiquitin pathways and, as such, have the potential to be the final editors of protein ubiquitylation status, thus determining substrate fate. This paper assimilates the current evidence concerning the substrates and activities of DUBs that regulate protein trafficking.

Doublecortin (DCX) is a microtubule-associated protein involved in neuronal migration, which causes X-linked lissencephaly and subcortical laminar heterotopia (SCLH) when mutated. Here we show that DCX interacts with the ubiquitin-specific protease Drosophila fat facets related on X chromosome (DFFRX). This interaction was confirmed by targeted mutagenesis, colocalization, and immunoprecipitation studies. DFFRX is thought to deubiquitinate specific substrates including beta-catenin, preventing their degradation by the proteasome. Interestingly, unlike beta-catenin, no ubiquitinated forms of DCX could be detected, and indeed we show that DCX interacts with a novel recognition domain in DFFRX, located outside of its catalytic site. We also show that DFFRX associates with microtubules at specific subcellular compartments, including those enriched in DCX. These results thus suggest that in addition to vesicular trafficking, DCX may play a role in the regulation of cell adhesion via its interaction with DFFRX in migrating and differentiating neurons.

Usp9x, an X-linked deubiquitylating enzyme, is stage dependently expressed in the supporting cells (i.e. Sertoli cells and granulosa cells) and germ cells during mouse gametogenesis. Af-6, a cell junction protein, has been identified as a substrate of Usp9x, suggesting a possible association between Usp9x and Af-6 in spermatogenesis and oogenesis. In this study, we examined the expression pattern of Af-6 and Usp9x and their intracellular localization in testes and ovaries of mice treated with or without pregnant mare serum gonadotropin (PMSG), an FSH-like hormone. In both testes and ovaries, Af-6 expression was predominantly observed in supporting cells, as well as in steroidogenic cells, but not in any germ cells. In Sertoli cells, Af-6 was continuously expressed throughout postnatal and adult stages, where both Af-6 and Usp9x were enriched at the sites of Sertoli-Sertoli and Sertoli-spermatid junctions especially at stages XI-VI. In the granulosa cells, Af-6, as well as Usp9x, was highly expressed in primordial and primary follicles, but its expression rapidly decreased after the late-secondary follicle stage. Interestingly, in PMSG-treated mice, the expression levels of Af-6 and Usp9x were synchronously enhanced, slightly in Sertoli cells and strongly in granulosa cells of the late-secondary and Graafian follicles. Such closely correlated expression patterns between Af-6 and Usp9x clearly suggest that Af-6 may be deubiquitylated by Usp9x in both Sertoli and granulosa cells. It further suggests that the post-translational regulation of Af-6 by Usp9x may be one potential pathway to control the cell adhesion dynamics in mammalian gametogenesis.

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, beta-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with beta-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with beta-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with beta-catenin and E-cadherin from a higher molecular weight complex ( approximately 500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, beta-catenin or E-cadherin, which were predominantly in a larger molecular weight complex ( approximately 2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased beta-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and beta-catenin during trafficking to the plasma membrane.

During the Drosophila oogenic processes, Fat facets (Faf), an ubiquitin-specific protease essential for normal development of oocyte and eye, becomes localized at the posterior pole and is incorporated into the pole cells. This is dependent on Oskar, a key factor for pole cell determination, and suggests a role for Faf in germ cell differentiation and development. Here we show that Usp9x, an X-linked ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during mouse gonadal development in stage- and sex-dependent manners. Usp9x was first detected in PGCs at 10.5 days post coitum (dpc), and thereafter its expression both at mRNA and protein levels was enhanced in PGCs of both sexes at 11.5-13.5 dpc. In testis, Usp9x expression rapidly decreased to an undetectable level by 15.5 dpc and after birth to adult, no expression was found in any spermatogenic cells, except for weak expression in Sertoli cells. In the ovary, Usp9x expression in embryonic oocytes was also reduced at the newborn stage, its expression reappeared in oocytes at secondary follicle stage, and its products were highly accumulated in the cytoplasm of Graaffian follicles in adults. Although follicular epithelial cells also expressed Usp9x at a moderate level during postnatal development, its expression was downregulated from early secondary follicle stage. Thus, the present study is not only the first to demonstrate a conserved expression of fat facets in PGCs between mouse and fly, but also sex- and stage-dependent changes of a specific component of the deubiquitylation system during mammalian gonadal development.

Signal transduction processes can be regulated by biochemical modifications that affect protein activity or localization and by protein stability. Proteins implicated in cancer, such as beta-catenin and p53, are regulated by a combination of posttranslational modifications and protein degradation by the ubiquitin-proteasome pathway. Wood explores how ubiquitylation of these proteins may not be as unidirectional as previously thought. With the identification of substrate-specific deubiquitylating enzymes, ubiquitylation may not always lead to protein destruction, but may provide another finely tunable step for controlling protein activity.

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.

Fat facets is a Drosophila deubiquitinating enzyme required for eye development and early embryogenesis. Genetic evidence suggests that Fat facets deubiquitinates and thereby prevents the proteasomal degradation of specific substrates. The Drosophila Liquid facets protein is implicated as the critical substrate of Fat facets in the eye. A mouse homolog of Fat facets, called Fam, has been identified. The results of biochemical experiments implicate two different proteins, Af-6 and beta-catenin, as substrates for Fam. Here, the functional relationship between Fat facets and Fam is explored. We show that Fam can substitute for Fat facets in all of its essential functions in Drosophila. In addition, we tested the hypothesis that Canoe and Armadillo, the Drosophila homologs of Af-6 and beta-catenin, respectively, are important substrates for Fat facets in the Drosophila eye. We found no genetic evidence to support a role for either Canoe or Armadillo in the essential Fat facets pathways in Drosophila eye development. The significance of these results is discussed in light of the biochemical experiments that suggest that Af-6 and beta-catenin are substrates of Fam.

The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. We have shown previously that the FAM (fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6, (mammalian canoe homologue), in the MDCK epithelial cell line (Taya et al., 1998. The Ras target AF-6 is a substrate of the fam de-ubiquitinating enzyme. J. Cell Biol. 142, 1053-1062). We report here that the expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, as FAM and AF-6 also co-localize during lens fibre development as well as in sub-populations of the neural retina.

Background: In the ubiquitin-proteasome pathway, the ubiquitinated substrates either undergo degradation by the proteasome or stabilization through the action of the deubiquitinating enzyme. We have previously found that the deubiquitinating enzyme Fam is colocalized with AF-6, one of the effectors of the Ras small GTPase, at cell-cell contact sites in epithelial cells and interacts with AF-6 in vivo and in vitro. Fam has deubiquitinating activity in vitro and prevents the ubiquitination of AF-6 in intact cells. The degradation of beta-catenin, which accumulates at the cell-cell contact sites as a cadherin/catenin complex, is thought to be regulated by the ubiquitin-proteasome pathway. These observations prompted us to examine the possible Fam regulation of the stabilization of beta-catenin., Results: We found that Fam interacted with beta-catenin both in vivo and in vitro. The Fam-binding site of beta-catenin mapped to the region close to the APC or Axin-binding site of beta-catenin. Over-expression of Fam in mouse L cells resulted in an elevation of beta-catenin levels and in an elongation of the half-life of beta-catenin. In these L cells, Fam was colocalized with beta-catenin at the dot-like structures in the cytoplasm., Conclusion: These results indicate that Fam interacts with and stabilizes beta-catenin in vivo, presumably through the deubiquitination of beta-catenin.

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly. Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila.