研究者詳細 - 佐谷　秀行

論文 - 佐谷　秀行

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Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G₁ tetraploidy checkpoint function

Iida Shin Ichi, Hirota Toru, Morisaki Tetsuro, Marumoto Tomotoshi, Hara Toshihiro, Kuninaka Shinji, Honda Shinobu, Kosai Ken Ichiro, Kawasuji Michio, Pallas David C., Saya Hideyuki

Oncogene 23 （ 31 ） 5266 - 5274 2004年07月

ISSN 0950-9232

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G ₁ cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G₁ tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine-threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Mat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G₁ tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G₁ tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G₁ tetraploidy checkpoint.

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Cell-matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca²⁺ influx and PKC activation

Nagano Osamu, Murakami Daizo, Hartmann Dieter, De Strooper Bart, Saftig Paul, Iwatsubo Takeshi, Nakajima Motowo, Shinohara Masanori, Saya Hideyuki

Journal of Cell Biology 165 （ 6 ） 893 - 902 2004年06月

ISSN 0021-9525

CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and undergoes sequential proteolytic cleavages in its ectodomain and intramembranous domain. The ectodomain cleavage is triggered by extracellular Ca²⁺ influx or the activation of protein kinase C. Here we show that CD44-mediated cell-matrix adhesion is terminated by two independent ADAM family metalloproteinases, ADAM10 and ADAM17, differentially regulated in response to those stimuli. Ca²⁺ influx activates ADAM10 by regulating the association between calmodulin and ADAM10, leading to CD44 ectodomain cleavage. Depletion of ADAM10 strongly inhibits the Ca²⁺ influx-induced cell detachment from matrix. On the other hand, phorbol ester stimulation activates ADAM17 through the activation of PKC and small GTPase Rac, inducing proteolysis of CD44. Furthermore, depletion of ADAM10 or ADAM17 markedly suppressed CD44-dependent cancer cell migration on HA, but not on fibronectin. The spatio-temporal regulation of two independent signaling pathways for CD44 cleavage plays a crucial role in cell-matrix interaction and cell migration.

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Dependence of Paclitaxel Sensitivity on a Functional Spindle Assembly Checkpoint

Sudo Tamotsu, Nitta Masayuki, Saya Hideyuki, Ueno Naoto T.

Cancer Research 64 （ 7 ） 2502 - 2508 2004年04月

ISSN 0008-5472

Paclitaxel stabilizes microtubules, causing mitotic arrest and activating the spindle assembly checkpoint. We determined whether suppression of the checkpoint genes Mad2 and BubR1 affects paclitaxel resistance and whether overexpression of Mad2 protein in checkpoint-defective cells enhances paclitaxel sensitivity. Suppression of Mad2 and BubR1 in paclitaxel-treated cancer cells abolished checkpoint function, resulting in paclitaxel resistance that correlated with suppression of cyclin-dependent kinase-1 activity. In contrast, overexpression of Mad2 in cells with a checkpoint defect attributable to low Mad2 expression restored checkpoint function, resulting in enhanced paclitaxel sensitivity that correlated with enhanced cyclin-dependent kinase-1 activity. However, overexpression of Mad2 failed to enhance paclitaxel sensitivity via checkpoint activation in Mad2-independent checkpoint-defective and -intact cells. Thus, checkpoint function is required for paclitaxel sensitivity. These findings show that any molecules that could interfere with the spindle assembly checkpoint could generate paclitaxel resistance in any patient.

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Autophosphorylation of a Newly Identified Site of Aurora-B Is Indispensable for Cytokinesis

Yasui Yoshihiro, Urano Takeshi, Kawajiri Aie, Nagata Koh Ichi, Tatsuka Masaaki, Saya Hideyuki, Furukawa Koichi, Takahashi Toshitada, Izawa Ichiro, Inagaki Masaki

Journal of Biological Chemistry 279 （ 13 ） 12997 - 13003 2004年03月

ISSN 0021-9258

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.

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Activation of m-Calpain Is Required for Chromosome Alignment on the Metaphase Plate during Mitosis

Honda Shinobu, Marumoto Tomotoshi, Hirota Toru, Nitta Masayuki, Arima Yoshimi, Ogawa Michio, Saya Hideyuki

Journal of Biological Chemistry 279 （ 11 ） 10615 - 10623 2004年03月

ISSN 0021-9258

Calpains form a superfamily of Ca²⁺-dependent intracellular cysteine proteases with various isoforms. Two isoforms, μ- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca²⁺ concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not μ-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.

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Down-regulation of nuclear protein ICBP90 by p53/p21^Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition

Arima Yoshimi, Hirota Toru, Bronner Christian, Mousli Marc, Fujiwara Toshiyoshi, Niwa Shin Ichiro, Ishikawa Hiroyuki, Saya Hideyuki

Genes to Cells 9 （ 2 ） 131 - 142 2004年02月

ISSN 1356-9597

Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21^Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoIIα gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down-regulated by the p53/p21^Cip1/WAF1-dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21^Cip1/WAF1 directly led to ICBP90 reduction in p53^-/- HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21^Cip1/WAF1-dependent DNA-damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.

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PKA phosphorylation and 14-3-3 interaction regulate the function of neurofibromatosis type I tumor suppressor, neurofibromin

Feng Liping, Yunoue Shunji, Tokuo Hiroshi, Ozawa Tatsuya, Zhang Dongwei, Patrakitkomjorn Siriporn, Ichimura Toru, Saya Hideyuki, Araki Norie

FEBS Letters 557 （ 1-3 ） 275 - 282 2004年01月

ISSN 0014-5793

Neurofibromin, a neurofibromatosis type I (NF1) tumor suppressor gene product, has a domain acting as a GTPase activating protein and functions in part as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation. Therefore, regulation of the function of neurofibromin is heavily involved in cell growth and differentiation. In the present study, we identified a novel cellular neurofibromin-associating protein, 14-3-3, which belongs to a highly conserved family of proteins that regulate intracellular signal transduction events in all eukaryotic cells. The interaction of 14-3-3 is mainly directed to the C-terminal domain (CTD) of neurofibromin, and the cAMP-dependent protein kinase (PKA)-dependent phosphorylation clustered on CTD-Ser (2576, 2578, 2580, 2813) and Thr (2556) is required for the interaction. Interestingly, the increased phosphorylation and association of 14-3-3 negatively regulate the function of neurofibromin. These findings indicate that PKA phosphorylation followed by 14-3-3 protein interaction may modulate the biochemical and biological functions of neurofibromin.

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Functional analysis of APC-Cdh1.

Sudo Tamotsu, Ueno Naoto T., Saya Hideyuki

Methods in molecular biology (Clifton, N.J.) 281 189 - 198 2004年

ISSN 1064-3745

To maintain genomic integrity against various kinds of genotoxic stress, cells have multiple checkpoints in the cell cycle. When one of the cell cycle events, such as DNA synthesis, DNA repair, and chromosomal segregation, has not been successfully completed, checkpoints will delay progression until the step is correctly accomplished, and only then will they relieve the arrest to allow the cell to move to the next phase. Cells lacking functional checkpoints display genomic aberrations, resulting in the acquisition of phenotypic changes of cancer cells. Anaphase-promoting complex (APC) is activated by two regulatory proteins: Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (APC-Cdh1) activity targets mitotic cyclins from the end of mitosis to the G1 phase. Loss of Cdh1 induces unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27Kip1. Furthermore, Cdh1-deficient DT40 cells fail to maintain DNA damage-induced G2 arrest, and Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest. In light of the differences between normal and cancer cells, checkpoints can be ideal targets for developmental cancer therapeutics. In this chapter, we describe how to analyze three checkpoints (spindle assembly checkpoint, rapamycin-induced G1 checkpoint, and DNA damage-induced G2 checkpoint) in conjunction with cell synchronization.

Aurora-A Kinase Maintains the Fidelity of Early and Late Mitotic Events in HeLa Cells

Marumoto Tomotoshi, Honda Shinobu, Hara Toshihiro, Nitta Masayuki, Hirota Toru, Kohmura Eiji, Saya Hideyuki

Journal of Biological Chemistry 278 （ 51 ） 51786 - 51795 2003年12月

ISSN 0021-9258

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G₂ cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.

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CENP-A phosphorylation by Aurora-A in prophase is required for enrichment of Aurora-B at inner centromeres and for kinetochore function

Kunitoku Naoko, Sasayama Takashi, Marumoto Tomotoshi, Zhang Dongwei, Honda Shinobu, Kobayashi Osamu, Hatakeyama Katsuyoshi, Ushio Yukitaka, Saya Hideyuki, Hirota Toru

Developmental Cell 5 （ 6 ） 853 - 864 2003年12月

ISSN 1534-5807

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. We now show that Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylated CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference revealed that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. Our observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function.

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Technetium-99m Sestamibi Single Photon Emission Computed Tomography Findings Correlated with P-glycoprotein Expression, Encoded by the Multidrug Resistance Gene-1 Messenger Ribonucleic Acid, in Intracranial Meningiomas

Saya Hideyuki, Yamada Kazuo

Neurologia Medico-Chirurgica 43 （ 12 ） 581 2003年12月

ISSN 0470-8105

Prognostic Value of Epidermal Growth Factor Receptor in Patients with Glioblastoma Multiforme

Shinojima Naoki, Tada Kenji, Shiraishi Shoji, Kamiryo Takanori, Kochi Masato, Nakamura Hideo, Makino Keishi, Saya Hideyuki, Hirano Hirofumi, Kuratsu Jun Ichi, Oka Koji, Ishimaru Yasuji, Ushio Yukitaka

Cancer Research 63 （ 20 ） 6962 - 6970 2003年10月

ISSN 0008-5472

Glioblastoma multiforme (GBM) frequently involves amplification and alteration of the epidermal growth factor receptor (EGFR) gene, resulting in overexpression of varied mutations, including the most common mutation, EGFRvIII, as well as wild-type EGFR (EGFRwt). To test the prognostic value of EGFR, we retrospectively analyzed the relationship between treatment outcomes and the EGFR gene in 87 newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. The EGFR gene status was assessed by Southern blots and EGFR expression by immunohistochemistry using three monoclonal antibodies (EGFR.25 for EGFR, EGFR.113 for EGFRwt, and DH8.3 for EGFRvIII). EGFR amplification was detected in 40 (46%) of the 87 GBM patients; in 39 (97.5%) of these, EGFR was overexpressed. On the other hand, in 46 of 47 patients without EGFR amplification (97.9%), no EGFR overexpression was present. There was a close correlation between EGFR amplification and EGFR overexpression (P < 0.0001). EGFRwt was overexpressed in 27 of the 40 (67.5%) patients with, and in none without, EGFR amplification (P < 0.0001). Similarly, EGFRvIII was overexpressed in 18 (45.0%) of 40 patients with and in 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). The finding that 8 (20%) of the patients with EGFR amplification/EGFR overexpression manifested overexpression of neither EGFRwt nor EGFRvIII indicates that they overexpressed other types of EGFR. Multivariate analysis demonstrated that EGFR amplification was an independent, significant, unfavorable predictor for overall survival (OS) in all patients (P = 0.038, HR = 1.67). With respect to the relationship of age to EGFR prognostication, the EGFR gene status was a more significant prognosticator in younger patients, particularly in those <60 years (P = 0.0003, HR = 3.15), whereas not so in older patients. EGFRvIII overexpression, on the other hand, was not predictive for OS. However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71). This finding indicates that EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis. In GBM patients, EGFR is of significant prognostic value for predicting survival, and the overexpression of EGFRvIII with amplification plays an important role in enhanced tumorigenicity.

Preliminary observations on genetic alterations in pilocytic astrocytomas associated with neurofibromatosis 1

Tada Kenji, Kochi Masato, Saya Hideyuki, Kuratsu Jun Ichi, Shiraishi Shoji, Kamiryo Takanori, Shinojima Naoki, Ushio Yukitaka

Neuro-Oncology 5 （ 4 ） 228 - 234 2003年10月

ISSN 1522-8517

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder that predisposes sufferers to various forms of neoplasia. Among affected individuals, 15%-20% develop astrocytomas, especially pilocytic astrocytomas (PA), which are benign and classified as grade I by the World Health Organization. They are generally well circumscribed, and their progression is slow. NF1-associated PAs (NF1-PAs) occasionally behave as aggressive tumors. To elucidate underlying genetic events in clinically progressive NF1-PAs, we performed molecular genetic analysis on 12 PAs, including 3 NF1-PAs, for p53, p16, and epidermal growth factor receptor genes, as well as loss of heterozygosity (LOH) on chromosome 1p, 10, 17, and 19q. None of the obvious genetic alterations typically seen in higher grade astrocytomas were found in 9 sporadic PAs. However, in 2 of 3 NF1-PAs, microsatellite analysis showed LOH10, including the PTEN (phosphatase and tensin homolog deleted on chromosome 10) gene locus, despite the diagnosis of pilocytic astrocytoma; one of these also manifested homozygous deletion of the p16 gene. The other NF1-PA harbored only LOH of the NF1 gene locus (17q). Our preliminary results support the hypothesis that some NF1-PAs differ genetically from sporadic PAs.

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Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells

Hirota Toru, Kunitoku Naoko, Sasayama Takashi, Marumoto Tomotoshi, Zhang Dongwei, Nitta Masayuki, Hatakeyama Katsuyoshi, Saya Hideyuki

Cell 114 （ 5 ） 585 - 598 2003年09月

ISSN 0092-8674

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.

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Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

Sugahara Kazuki N., Murai Toshiyuki, Nishinakamura Hitomi, Kawashima Hiroto, Saya Hideyuki, Miyasaka Masayuki

Journal of Biological Chemistry 278 （ 34 ） 32259 - 32265 2003年08月

ISSN 0021-9258

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.

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Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras

Yunoue Shunji, Tokuo Hiroshi, Fukunaga Kohji, Feng Liping, Ozawa Tatsuya, Nishi Toru, Kikuchi Akira, Hattori Seisuke, Kuratsu Junichi, Saya Hideyuki, Araki Norie

Journal of Biological Chemistry 278 （ 29 ） 26958 - 26969 2003年07月

ISSN 0021-9258

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance.

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Serum response factor is modulated by the SUMO-1 conjugation system

Matsuzaki Kazuhito, Minami Takeshi, Tojo Masahide, Honda Yoshiomi, Uchimura Yasuhiro, Saitoh Hisato, Yasuda Hideyo, Nagahiro Shinji, Saya Hideyuki, Nakao Mitsuyoshi

Biochemical and Biophysical Research Communications 306 （ 1 ） 32 - 38 2003年06月

ISSN 0006-291X

Serum stimulation leads to activation of the serum response factor (SRF)-mediated transcription of immediate-early genes such as c-fos via various signal transduction pathways. We have previously reported that promyelocytic leukemia protein (PML) is involved in the transcriptional regulation by SRF. PML is one of the well-known substrates for modification by small ubiquitin-related modifier-1 (SUMO-1) and several SUMO-1-modified proteins associate with PML. Here, we report that SRF is modified by SUMO-1 chiefly at lysine¹⁴⁷ within the DNA-binding domain. Substitution of this target lysine for alanine did not affect the translocation of SRF to PML-nuclear bodies. The SRF mutant augmented the transcriptional activity under Rho A-stimulated condition but not under serum-starved condition, suggesting that activated SRF is suppressed by its sumoylation. These data support the transcriptional role of SUMO-1 conjugating system in cellular serum response.

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MCAF mediates MBD1-dependent transcriptional repression

Fujita Naoyuki, Watanabe Sugiko, Ichimura Takaya, Ohkuma Yoshiaki, Chiba Tsutomu, Saya Hideyuki, Nakao Mitsuyoshi

Molecular and Cellular Biology 23 （ 8 ） 2834 - 2843 2003年04月

ISSN 0270-7306

DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

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Presenilin-dependent γ-secretase activity mediates the intramembranous cleavage of CD44

Murakami Daizo, Okamoto Isamu, Nagano Osamu, Kawano Yoshiaki, Tomita Taisuke, Iwatsubo Takeshi, De Strooper Bart, Yumoto Eiji, Saya Hideyuki

Oncogene 22 （ 10 ） 1511 - 1516 2003年03月

ISSN 0950-9232

CD44 is the major adhesion molecule for the extracellular matrix components and is implicated in a wide variety of physiological and pathological processes including the regulation of tumor cell growth and metastasis. Our previous studies have shown that CD44 undergoes sequential proteolytic cleavages in the extracellular and transmembrane domains and the cleavage product derived from CD44 intramembranous cleavage acts as a signal transduction molecule. However, the underlying mechanism of the intramembranous cleavage of CD44 remains to be elucidated. In the present study, we report for the first time that CD44 is a substrate of the presenilin (PS)-dependent γ-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by γ-secretase inhibitors in U251MG cells and that this cleavage is also inhibited in PS-deficient mouse embryonic fibroblasts. Furthermore, we showed that PSI is redistributed to ruffling areas of the plasma membrane similarly to CD44 after TPA treatment, supporting our biochemical observation that PS1 is involved in the intramembranous cleavage of CD44. Our present findings suggest important implications for understanding CD44-dependent signal transduction and a potential role of PS/γ-secretase activity in the functional regulation of adhesion molecules.

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PML-nuclear bodies are involved in cellular serum response

Matsuzaki Kazuhito, Minami Takeshi, Tojo Masahide, Honda Yoshiomi, Saitoh Noriko, Nagahiro Shinji, Saya Hideyuki, Nakao Mitsuyoshi

Genes to Cells 8 （ 3 ） 275 - 286 2003年03月

ISSN 1356-9597

Background: Serum stimulation leads to the activation of various signal transduction pathways in cells, and the resultant signals are integrated into the serum response factor (SRF)-dependent transcription of immediate-early genes such as c-fos. Results: To further characterize this response, we investigated the mechanism which controls serum response transcription in cultured human cells. Frequency of PML (promyelocytic leukaemia)-nuclear bodies (NBs) formation increases shortly after serum stimulation, probably facilitating the interaction of SRF and CBP acetyltransferase at the NBs. PML modulates SRF-mediated c-fos promoter activities upon addition of serum to cells or expression of constitutively active Rho family GTPases. We mapped the region in the SRF that interacts with PML to the C-terminal transactivation domain. An SRF mutant deleted of the transactivation domain neither co-localizes with CBP in NBs nor fulfills its transcriptional role. Under conditions of serum stimulation, the formation of NBs coincides with the immediate-early expression of the endogenous c-fos gene in fibroblasts and in all-trans retinoic acid-treated acute promyelocytic leukaemia NB4 cells. Conclusion: These data provide an insight into the involvement of NBs in modulating the transcription of serum-induced immediate-early genes.

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