Ran GTPase-activating Protein 1 Research Articles

Impact of RANGAP1 SUMOylation on Smad4 nuclear export by bioinformatic analysis and cell assays.

Small Ubiquitin-like Modifier (SUMOylation) regulates a variety of cellular activities, and its dysregulation has been associated with glioma etiology. The aim of this research was to clarify the function of SUMOylation-related genes in glioma and determine relevant prognostic markers. The Cancer Genome Atlas (TCGA) Glioma and GSE16011 datasets were analyzed through bioinformatics to identify Ran GTPase activating protein 1 (RANGAP1) as the hub gene for further study. Experimental validation consisted of quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB), and immunoprecipitation (IP) to evaluate RANGAP1 expression, function, and interaction with SUMO1. To assess the role of RANGAP1 knockdown and SUMOylation in glioma cells, various assays were conducted, including cell proliferation, migration, invasion, and apoptosis. In addition, cell cycle analysis and immunofluorescence were performed. Through bioinformatics, RANGAP1 was identified as a crucial prognostic gene for glioma. Experimental studies confirmed the downregulation of RANGAP1 in glioma cells and verified that RANGAP1 repair impedes tumor growth. When it comes to RANGAP1 silencing, it enhanced cell proliferation, invasion and migration. Additionally, SUMO1 was identified as a specific SUMO molecule coupled to RANGAP1, affecting the location of Sma and Mad related protein 4 (Smad4) in the nucleocytoplasm and the transforming growth factor (TGF)-β/Smad signaling pathway. The functional impact of RANGAP1 SUMOylation on cell proliferation and migration was further confirmed through experiments using a SUMOylation-impairing mutation (K524R). Our findings suggest that RANGAP1 may be a potential prognostic marker in gliomas and could play a role in regulating cell proliferation, migration, and invasion. SUMOylation of RANGAP1 is responsible for regulating the TGF-β/Smad signaling pathway, which is crucial for the progression of tumors. Further investigations and experiments are necessary to confirm these results.

METTL3-mediated RanGAP1 promotes colorectal cancer progression through the MAPK pathway by recruiting YTHDF1

Ran GTPase activating protein 1 (RanGAP1) has been implicated in various diseases, but its role in colorectal cancer (CRC) progression remains unclear. Using tumor tissues and public databases, we found that RanGAP1 was significantly upregulated in CRC tissues and was associated with poor prognosis of patients. N6-methyladenosine (m6A) was found to play an important role in higher expression of RanGAP1. MeRIP-seq, RIP-qPCR, Luciferase reporter assays and other related experiment elucidated the molecular mechanism underlying m6A modification of RanGAP1. Besides, cell function experiments and xenograft tumor models corroborated the function of RanGAP1 in CRC progression. By RNA-seq and related analysis, RanGAP1 was verified to influent CRC progression via the Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Therefore, N6-methyladenosine modification of RanGAP1 by METTL3/YTHDF1 plays a role in CRC progression through the MAPK pathway and could be a potential biomarker and therapeutic target for CRC.Schematic diagram showed that N6-methyladenosine modification of RanGAP1 promotes CRC progression via the MAPK signaling pathway.

Mechanism of exportin retention in the cell nucleus.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinβ1 (Kapβ1 or importinβ1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapβ1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

Abstract 2021: Identification of novel brachyury target genes in lung cancer

Abstract Lung cancer is the leading cause of cancer-related death worldwide. Previous studies have shown that the T-box transcription factor brachyury is commonly expressed in advanced lung cancer and is associated with a poor prognosis. The activation of brachyury promotes lung cancer progression, aggressiveness and resistance to conventional antitumor therapies. However, the mechanism by which brachyury drives lung cancer is unknown. Here we have sought to identify novel gene targets of brachyury to better understand the molecular pathogenesis of lung cancer. We used a bioinformatics approach and found a shortlist of putative potential brachyury target genes. We subsequently explored the modulation of candidate brachyury target genes in lung cancer. RNA interference analysis for lung cancer cell line H460 overexpressing brachyury demonstrated that fibroblast growth factor 8 (FGF8), MAS1 Proto-Oncogene G Protein-Coupled Receptor (MAS1) and Ran GTPase Activating Protein 1 (RANGAP1) played essential roles in cell growth, whereas transmembrane protein 37 (TMEM37) was involved in cell migration and invasion. ChIP-qPCR experiments confirmed the strong binding of brachyury to these functional target genes. Analysis of datasets derived from patients with lung cancer and a panel of lung cancer cell lines revealed that brachyury expression was positively correlated with FGF8, MAS1, RANGAP1 and TMEM37. In addition, the expression levels of FGF8, MAS1 and RANGAP1 were significantly associated with non-metastasis (early-stage and locally advanced lung cancer), whereas TMEM37 was associated with metastasis. Taken together, our findings suggest that brachyury may influence lung cancer progression and/or metastasis via direct regulation of FGF8, MAS1, RANGAP1 and TMEM37 expression. Identification of potential brachyury transcriptional targets provides novel insights into the molecular pathobiology of lung cancer. Citation Format: Yunping Hu, Wesley Hsu. Identification of novel brachyury target genes in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2021.

MicroRNA-1301-Mediated RanGAP1 Downregulation Induces BCR-ABL Nuclear Entrapment to Enhance Imatinib Efficacy in Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disease. Imatinib (IM), the first line treatment for CML, is excessively expensive and induces various side effects in CML patients. Therefore, it is essential to investigate a new strategy for improving CML therapy. Our immunoblot data revealed that RanGTPase activating protein 1 (RanGAP1) protein levels increased by approximately 30-fold in K562 cells compared with those in normal cells. RanGAP1 is one of the important components of RanGTPase system, which regulates the export of nuclear protein. However, whether RanGAP1 level variation influences BCR-ABL nuclear export is still unknown. In this report, using shRNA to downregulate RanGAP1 expression level augmented K562 cell apoptosis by approximately 40% after treatment with 250 nM IM. Immunofluorescence assay also indicated that three-fold of nuclear BCR-ABL was detected. These data suggest that BCR-ABL nuclear entrapment induced by RanGAP1 downregulation can be used to improve IM efficacy. Moreover, our qRT-PCR data indicated a trend of inverse correlation between the RanGAP1 and microRNA (miR)-1301 levels in CML patients. MiR-1301, targeting the RanGAP1 3′ untranslated region, decreased by approximately 100-fold in K562 cells compared with that in normal cells. RanGAP1 downregulation by miR-1301 transfection impairs BCR-ABL nuclear export to increase approximately 60% of cell death after treatment of 250 nM IM. This result was almost the same as treatment with 1000 nM IM alone. Furthermore, immunofluorescence assay demonstrated that Tyr-99 of nuclear P73 was phosphorylated accompanied with nuclear entrapment of BCR-ABL after transfection with RanGAP1 shRNA or miR-1301 in IM-treated K562 cells. Altogether, we demonstrated that RanGAP1 downregulation can mediate BCR-ABL nuclear entrapment to activate P73-dependent apoptosis pathway which is a novel strategy for improving current IM treatment for CML.

ON 01910.Na inhibits growth of diffuse large B-cell lymphoma by cytoplasmic sequestration of sumoylated C-MYB/TRAF6 complex

Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, shows either no response or development of resistance to further treatment in 30% of the patients that warrants the development of novel drugs. We have reported that ON 01910.Na (rigosertib), a multikinase inhibitor, is selectively cytotoxic for DLBCL and induces more hyperphosphorylation and sumoylation of Ran GTPase-activating protein 1 (RanGAP1) in DLBCL cells than in non-neoplastic lymphoblastoid cell line. However, the exact mechanism of rigosertib-induced cell death in DLBCL remains to be clarified. Here, we analyzed the efficacy of rigosertib against DLBCL cells invitro and invivo and its molecular effects on tumor biology. We found for the first time that rigosertib attenuated expression of unmodified and sumoylated tumor necrosis factor receptor-associated factor 6 (TRAF6) and c-Myb and inhibited nuclear entry of sumoylated RanGAP1, TRAF6, and c-Myb that was confirmed by immunofluorescence. Moreover, co-immunoprecipitation showed that rigosertib induced sequestration of c-Myb and TRAF6 in the cytoplasm by stimulating their sumoylation through the RanGAP1*SUMO1/Ubc9pathway. Specific knockdown of c-Myb and TRAF6 induced tumor cell apoptosis and cell cycle arrest at G1 phase. Xenograft mice bearing lymphoma cells also exhibited effective tumor regression on rigosertib treatment along with cytoplasmic expression of c-Myb and TRAF6. Nuclear expression of c-Myb in clinical cases of DLBCL correlated with a poor prognosis. Thus, suppression of c-Myb and TRAF6 activity may have therapeutic implication in DLBCL. These data support the clinical development of rigosertib in DLBCL.

Inhibition of RNA transportation induces glioma cell apoptosis via downregulation of RanGAP1 expression

The prognosis of glioblastoma remains poor, even treatment with surgery, radiation, or chemotherapy. Therefore, it is still important to develop a new strategy for treatment of glioblastoma. Previous reports demonstrated that rRNA is produced at abnormally high levels in tumor cells. Nuclear export of all non-coding RNAs are known to depend on RanGTPase system. Hydrolyzation of RanGTP–RNA complex by RanGTPase activating protein 1 (RanGAP1) releases RNA from nucleus to cytoplasm. Therefore, inhibition of RNA transportation would be a useful strategy to affect cancer cell fate. In this study, 5–30μM of oridonin, a natural diterpenoid compound isolated from the traditional Chinese medicine, Rabdosia rubescens, induced U87MG glioma cell apoptosis and RNA accumulation in nucleus at 12h-time point. Before U87MG cell apoptosis, the RanGAP1 protein amount decreased and RanGTP accumulated in nucleus as respectively determined by immunoprecipitation and immunofluorescence, suggesting that decrease of RanGAP1 may result in nuclear entrapment of RanGTP and RNA, and then induce U87MG cell death. In contrast, over-expression of the RanGAP1 protein reversed oridonin-induced U87MG cell apoptosis. Hence, we demonstrated that downregulation of the RanGAP1 protein level by oridonin may result in RNA accumulation in nucleus via nuclear entrapment of RanGTP which eventually led to the apoptosis of glioma cells.

SUMO1 in human sperm: new targets, role in motility and morphology and relationship with DNA damage.

In studies carried out previously, we demonstrated that small ubiquitin-like modifier 1 (SUMO1) is associated with poor sperm motility when evaluated with a protocol that reveals mostly SUMO1-ylated live sperm. Recently, with another protocol, it has been demonstrated that SUMO is expressed in most sperm and is related to poor morphology and motility, suggesting that sumoylation may have multiple roles depending on its localisation and targets. We show herein, by confocal microscopy and co-immunoprecipitation, that dynamin-related protein 1 (DRP1), Ran GTPase-activating protein 1 (RanGAP1) and Topoisomerase IIα, SUMO1 targets in somatic and/or germ cells, are SUMO1-ylated in mature human spermatozoa. DRP1 co-localises with SUMO1 in the mid-piece, whereas RanGAP1 and Topoisomerase IIα in the post-acrosomal region of the head. Both SUMO1 expression and co-localisation with the three proteins were significantly higher in morphologically abnormal sperm, suggesting that sumoylation represents a marker of defective sperm. DRP1 sumoylation at the mid-piece level was higher in the sperm of asthenospermic men. As in somatic cells, DRP1 sumoylation is associated with mitochondrial alterations, this protein may represent the link between SUMO and poor motility. As SUMO pathways are involved in responses to DNA damage, another aim of our study was to investigate the relationship between sumoylation and sperm DNA fragmentation (SDF). By flow cytometry, we demonstrated that SUMO1-ylation and SDF are correlated (r=0.4, P<0.02, n=37) and most sumoylated sperm shows DNA damage in co-localisation analysis. When SDF was induced by stressful conditions (freezing and thawing and oxidative stress), SUMO1-ylation increased. Following freezing and thawing, SUMO1-Topoisomerase IIα co-localisation and co-immunoprecipitation increased, suggesting an involvement in the formation/repair of DNA breakage.

Nuclear envelope localization of Ran-binding protein 2 and Ran-GTPase-activating protein 1 in psoriatic epidermal keratinocytes

The nuclear localization signal (NLS)-containing proteins LEDGF and STAT3 localize to the nucleus in both the spinous and basal layers of the epidermis in psoriatic skin, where they function as transcription factors or co-factors to activate epidermal keratinocytes (KCs). However, the mechanism underlying the localization of these proteins remains to be elucidated. We investigated the differential nucleocytoplasmic transport of NLS-containing proteins as a potential pathogenic mechanism for psoriasis vulgaris. Nucleoporins play an important role in the Ran-GTP-dependent nucleocytoplasmic transport of NLS-containing proteins. We showed, using immunohistochemical staining, that the nucleoporins Ran-binding protein 2 (RanBP2) and Ran-GTPase-activating protein 1 (RanGAP1) have greater expression on the nuclear envelope in psoriatic epidermal KCs than in KCs from healthy controls. We then studied the signalling pathways involved in the regulation of these proteins in HaCaT cells. The two major downstream pathways of epidermal growth factor receptor (EGFR) signalling activated in psoriatic KCs are the MAPK/Erk/1/2 and the phosphatidylinositol-3-kinase/Akt pathways. Therefore, we treated HaCaT cells with inhibitors to disrupt the MAP kinase kinase 1 (MEK1), PI3-kinase, or mTOR pathways. RanBP2 and RanGAP1 protein expression levels were significantly greater in the nuclear envelope of HaCaT cells that were not treated with inhibitors than in cells treated with a combination of PI3-kinase and MEK1 inhibitors or mTOR and MEK1 inhibitors. These results suggest that adequate nuclear envelope expression of RanBP2 and RanGAP1 could be a prerequisite for nucleocytoplasmic transport in KCs in psoriatic epidermis.

Ran GTPase-activating protein 1 is a therapeutic target in diffuse large B-cell lymphoma.

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1+, while reactive lymphoid hyperplasia (n = 12) was RanGAP1+ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin’s lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.

Abstract A132: Development of a small-molecule inhibitor targeting the Wnt signaling pathway.

Abstract Wnt signaling is a major force driving colorectal carcinogenesis, but only a small number of druggable target molecules in the Wnt pathway have been found. Our recent series of proteomic studies has revealed that various classes of nuclear proteins participate in the β-catenin and T-cell factor-4 (TCF-4) complex and modulate the activity of Wnt signalling. Those included fusion/translocated in liposarcoma (FUS/TLS) (1), poly(ADP-ribose) polymerase-1 (PARP-1) (2), Ku70/Ku80 (3), DNA topoisomerase IIα (Topo IIα) (4), splicing factor-1 (SF1) (5), Ran (ras-related nuclear protein), RanBP2 (Ran binding protein-2), and RanGAP1 (Ran GTPase-activating protein-1) (6), Traf2- and Nck-interacting kinase (TNIK) (7). Among these proteins, TNIK protein kinase attracted our current interest because various small-molecule kinase inhibitors have been applied successfully to cancer treatment. TNIK was an activating kinase for TCF-4, and colorectal cancer cells are highly dependent upon the expression and catalytic activity of TNIK for proliferation (7). High-throughput screening of a kinase-focused compound library (&amp;gt;10,000 compounds) against recombinant TNIK identified a lead candidate that inhibited the kinase activity of TNIK with an IC50 value of 8.6 nM and the transcriptional activity of TCF-4. TNIK is a feasible drug target in the Wnt signaling pathway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A132. Citation Format: Tesshi Yamada, Mari Masuda, Masaaki Sawa. Development of a small-molecule inhibitor targeting the Wnt signaling pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A132.

Abstract LB-77: Crm1-mediated nucleocytoplasmic transport inhibition perturbs multiple cellular pathways to trigger melanoma cell apoptosis

Abstract Development of multiple resistance mechanisms in melanomas has necessitated the identification of novel drug targets, which when inhibited could impact multiple cellular pathways thus circumventing potential resistance mechanisms. By performing cDNA-microarray analysis, we identified four key components of the nucleocytoplasmic transport machinery, Chromosome Region Maintenance 1 (Crm1), Ran-GTPase, Ran-GTPase Activating Protein 1 (RanGAP1) and Ran Binding Protein 1 (RanBP1) to be overexpressed in human melanoma metastases. Crm1 inhibition, employing the highly specific Crm1 inhibitor Leptomycin B (LMB) induced a dramatic depletion of pro-survival/cytoplasmic Erk1/2 and p90RSK1 and mediated persistent Erk signaling hyperactivation. Consistently, Crm1 inhibition inflicted extensive apoptosis in melanoma cells while sparing immortalized melanocytes and primary lung fibroblasts. Apoptosis required both the intrinsic and extrinsic apoptotic pathways and was associated with a nuclear entrapment and down-regulation of anti-apoptotic Survivin. Apoptosis was preceded by a G1 cell-cycle arrest and even though Crm1 inhibition mediated dramatic p53 and p21 induction exclusively in wild-type p53 melanoma cells, latter's silencing or inactivation failed to alleviate ensuing apoptosis. Notably, Crm1 inhibition elicited cell line specific, G1 to S progression-retarding changes in the expression of multiple cell-cycle regulatory proteins (Cdk2, Cdk4, p27, p16 and p18) thus potentially explaining p53 dispensability. Based on our results, we propose Crm1 as a potential therapeutic target in human melanoma, whose inhibition initiates a multitude of cell context-dependent molecular events to trigger G1-arrest followed by massive apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-77. doi:1538-7445.AM2012-LB-77

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Characterization of SENP7, a SUMO-2/3-specific isopeptidase

The modification of proteins by SUMO (small ubiquitin-related modifier) plays important roles in regulating the activity, stability and cellular localization of target proteins. Similar to ubiquitination, SUMO modification is a dynamic process that can be reversed by SENPs [SUMO-1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidases]. To date, six SENPs have been discovered in humans, although knowledge of their regulation, specificity and biological functions is limited. In the present study, we report that SENP7 has a restricted substrate specificity, being unable to process SUMO precursors and displaying paralogue-specific isopeptidase activity. The C-terminal catalytic domain of SENP7 efficiently depolymerized poly-SUMO-2 chains but had undetectable activity against poly-SUMO-1 chains. SENP7 also displayed isopeptidase activity against di-SUMO-2- and SUMO-2-modified RanGAP1 (Ran GTPase-activating protein 1) but had limited activity against SUMO-1-modified RanGAP1. in vivo, full-length SENP7 was localized to the nucleoplasm and preferentially reduced the accumulation of high-molecular-mass conjugates of SUMO-2 and SUMO-3 compared with SUMO-1. Small interfering RNA-mediated ablation of SENP7 expression led to the accumulation of high-molecular-mass SUMO-2 species and to the accumulation of promyelocytic leukaemia protein in subnuclear bodies. These findings suggest that SENP7 acts as a SUMO-2/3-specific protease that is likely to regulate the metabolism of poly-SUMO-2/3 rather than SUMO-1 conjugation in vivo.

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase ( Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.

Regulation of Wnt Signaling by the Nuclear Pore Complex

The function of beta-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, beta-catenin has no nuclear localization signal, and the mechanisms by which beta-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and beta-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and beta-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.

Chip-based analysis of SUMO (small ubiquitin-like modifier) conjugation to a target protein

A chip-based analysis of protein interactions and modifications in cell signaling pathways has been of great potential in drug discovery, diagnostics, and cell biology, because it enables rapid and high-throughput biological assays with a small amount of samples. We report a chip-based analysis of sumoylation, the post-translational modification (PTM) process that involves covalent attachment of the small ubiquitin-like modifier (SUMO) protein to a target protein through multiple enzyme reactions in eukaryotic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for sumoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip sumoylation of Ran GTPase-activating protein 1 (RanGAP1) domain resulted in highly specific fluorescence intensity compared to that of its mutant (K524A) irrelevant to SUMO conjugation. The on-chip sumoylation was also verified and quantified by using the surface plasmon resonance(SPR) spectroscopy. As the exemplary study for a parallel analysis of sumoylation, fluorescent detection of sumoylation was conducted in a microarray format on a glass slide. The chip-based analysis developed here is expected to be applicable to assay for screening of target proteins from existing protein pools and proteome arrays in a high throughput manner.

Crm1 is a mitotic effector of Ran-GTP in somatic cells

The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.

383 Preclinical antitumor activity of second generation analogs of SDX-101

Background & Aims: The function of β-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, β-catenin has no nuclear localization signal, and the mechanisms by which β-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Methods: Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. Results: TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and β-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and β-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Conclusions: Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.