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Toshio Hakoshima - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of cargo recognition by the myosin-X MyTH4-FERM Domain
The EMBO journal, 2011Co-Authors: Yoshinori Hirano, Taiki Hatano, Aya Takahashi, Michinori Toriyama, Naoyuki Inagaki, Toshio HakoshimaAbstract:Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail Domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM Domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 Domain is folded into a helical VHS-like structure and is associated with the FERM Domain. We found an unexpected binding mode of the DCC peptide to the subDomain C groove of the FERM Domain, which is distinct from previously reported β–β associations found in radixin–adhesion molecule complexes. We also revealed direct interactions between the MyTH4–FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 Domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.
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Crystallographic characterization of the radixin FERM Domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2008Co-Authors: Shin-ichi Terawaki, Ken Kitano, Miki Aoyama, Toshio HakoshimaAbstract:ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM Domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM Domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM Domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6122, with unit-cell parameters a = b = 122.7, c = 128.3 A, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 A.
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Crystallographic characterization of the radixin FERM Domain bound to the cytoplasmic tail of adhesion molecule CD44.
Acta crystallographica. Section F Structural biology and crystallization communications, 2007Co-Authors: Tomoyuki Mori, Shin-ichi Terawaki, Ken Kitano, Ryoko Maesaki, Toshio HakoshimaAbstract:CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell-cell and cell-matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane-cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM Domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM Domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 A.
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Crystallographic characterization of the radixin FERM Domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Yumiko Takai, Ken Kitano, Shin-ichi Terawaki, Ryoko Maesaki, Toshio HakoshimaAbstract:Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM Domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM Domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM Domain bound to CD43 belong to space group P4322, with unit-cell parameters a = b = 68.72, c = 201.39 A, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM Domain bound to PSGL-1 belong to space group P212121, with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 A, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 A for the FERM–CD43 complex and 2.8 A for the FERM–PSGL-1 complex.
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Crystallographic characterization of the radixin FERM Domain bound to the C-terminal region of the human Na+/H+-exchanger regulatory factor (NHERF).
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Shin-ichi Terawaki, Ryoko Maesaki, Kengo Okada, Toshio HakoshimaAbstract:Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The N-terminal FERM Domains of ERM proteins interact with Na+/H+-exchanger regulatory factors (NHERFs), which are PDZ-containing adaptor proteins, to modulate the ion-channel activity. Here, crystals of complexes between the radixin FERM Domain and the C-terminal regions of NHERF and NHERF2 have been obtained. The crystals of the FERM–NHERF complex were found to belong to space group P212121, with unit-cell parameters a = 69.38 (2), b = 146.27 (4), c = 177.76 (7) A. The crystal contains four complexes in the asymmetric unit. An intensity data set was collected to a resolution of 2.50 A.
Yoichi Furukawa - One of the best experts on this subject based on the ideXlab platform.
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Abstract 318: Identification of FERM Domain-containing protein 5 (FRMD5) as a novel target of β-catenin/TCF7L2 complex
Molecular and Cellular Biology Genetics, 2017Co-Authors: Yoichi Furukawa, Chi Zhu, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Kiyoshi YamaguchiAbstract:Deregulated Wnt signaling is one of the earliest steps in colorectal tumorigenesis. The impairment results most frequently from APC mutations, which leads to the accumulation of β-catenin and subsequent activation of TCF7L2. Although previous studies have identified a number of target genes of the activated β-catenin/TCF7L2 transcriptional complex such as cMYC and CCND1, the comprehensive effect of the activation on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for novel genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with β-catenin siRNAs, and combined these data with public microarray data of LS174 cells treated with a dominant-negative form of TCF7L2. As a result, we identified a total of 134 genes that were regulated by both β-catenin and TCF7L2. Subsequent ChIP-sequence with TCF7L2 antibody selected 11 genes from the 134 genes as candidates of direct target genes. In this study, we focused on FERM Domain-containing protein 5 (FRMD5) among the 11 candidates. Quantitative PCR confirmed that its expression was reduced by β-catenin siRNA in HCT116 and SW480 cells. ChIP-quantitative PCR analysis with ant-TCF7L2 antibody also corroborated the interaction of TCF7L2 with a region (hg19; chr15:44,449,680-44,450,487) within intron 1 of FRMD5. Reporter assay with plasmids containing this region revealed that the reporter activity was down-regulated by the knockdown of β-catenin. These data suggested that FRMD5 is a direct target of β-catenin/TCF7L2 complex, and that the region is involved in the transcriptional activation through an interaction with the complex. Consistently, its expression was elevated in colorectal tumors compared to normal colonic mucosa in public microarray data. To uncover the role of FRMD5 in colorectal carcinogenesis, we analyzed expression profile of HCT116 cells treated with FRMD5 siRNA, and combined the data with profile with β-catenin siRNA. Gene set analysis with 53 commonly up-regulated and 36 commonly down-regulated genes by both FRMD5 and β-catenin disclosed that the gene sets of DNA replication, cell cycle, and extracellular matrix (ECM) were altered by FRMD5. These data may be useful for the future studies of colorectal carcinogenesis. Citation Format: Yoichi Furukawa, Chi Zhu, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Kiyoshi Yamaguchi. Identification of FERM Domain-containing protein 5 (FRMD5) as a novel target of β-catenin/TCF7L2 complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2017-318
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abstract 318 identification of FERM Domain containing protein 5 frmd5 as a novel target of β catenin tcf7l2 complex
Cancer Research, 2017Co-Authors: Yoichi Furukawa, Chi Zhu, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Kiyoshi YamaguchiAbstract:Deregulated Wnt signaling is one of the earliest steps in colorectal tumorigenesis. The impairment results most frequently from APC mutations, which leads to the accumulation of β-catenin and subsequent activation of TCF7L2. Although previous studies have identified a number of target genes of the activated β-catenin/TCF7L2 transcriptional complex such as cMYC and CCND1, the comprehensive effect of the activation on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for novel genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with β-catenin siRNAs, and combined these data with public microarray data of LS174 cells treated with a dominant-negative form of TCF7L2. As a result, we identified a total of 134 genes that were regulated by both β-catenin and TCF7L2. Subsequent ChIP-sequence with TCF7L2 antibody selected 11 genes from the 134 genes as candidates of direct target genes. In this study, we focused on FERM Domain-containing protein 5 (FRMD5) among the 11 candidates. Quantitative PCR confirmed that its expression was reduced by β-catenin siRNA in HCT116 and SW480 cells. ChIP-quantitative PCR analysis with ant-TCF7L2 antibody also corroborated the interaction of TCF7L2 with a region (hg19; chr15:44,449,680-44,450,487) within intron 1 of FRMD5. Reporter assay with plasmids containing this region revealed that the reporter activity was down-regulated by the knockdown of β-catenin. These data suggested that FRMD5 is a direct target of β-catenin/TCF7L2 complex, and that the region is involved in the transcriptional activation through an interaction with the complex. Consistently, its expression was elevated in colorectal tumors compared to normal colonic mucosa in public microarray data. To uncover the role of FRMD5 in colorectal carcinogenesis, we analyzed expression profile of HCT116 cells treated with FRMD5 siRNA, and combined the data with profile with β-catenin siRNA. Gene set analysis with 53 commonly up-regulated and 36 commonly down-regulated genes by both FRMD5 and β-catenin disclosed that the gene sets of DNA replication, cell cycle, and extracellular matrix (ECM) were altered by FRMD5. These data may be useful for the future studies of colorectal carcinogenesis. Citation Format: Yoichi Furukawa, Chi Zhu, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Kiyoshi Yamaguchi. Identification of FERM Domain-containing protein 5 (FRMD5) as a novel target of β-catenin/TCF7L2 complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2017-318
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Identification of FERM Domain-containing protein 5 as a novel target of β-catenin/TCF7L2 complex.
Cancer science, 2017Co-Authors: Chi Zhu, Kiyoshi Yamaguchi, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Yoichi FurukawaAbstract:Deregulation of the canonical Wnt signaling pathway plays an important role in human tumorigenesis through the accumulation of β-catenin and subsequent transactivation of TCF7L2. Although some of the consequences associated with the accumulated β-catenin have been clarified, the comprehensive effect of activated β-catenin/TCF7L2 transcriptional complex on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with β-catenin siRNAs, and ChIP-sequencing using anti-TCF7L2 antibody. Combination of these data with public microarray data of LS174 cells with a dominant-negative form of TCF7L2 identified a total of 11 candidate genes. In this paper, we focused on FERM Domain-containing protein 5 (FRMD5), and confirmed that it is regulated by both β-catenin and TCF7L2. An additional reporter assay disclosed that a region in intron1 transcriptionally regulated the expression of FRMD5. ChIP assay also corroborated that TCF7L2 associates with this region. These data suggested that FRMD5 is a novel direct target of the β-catenin/TCF7L2 complex.
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identification of FERM Domain containing protein 5 as a novel target of β catenin tcf7l2 complex
Cancer Science, 2017Co-Authors: Chi Zhu, Kiyoshi Yamaguchi, Tomoyuki Ohsugi, Yumi Terakado, Rei Noguchi, Tsuneo Ikenoue, Yoichi FurukawaAbstract:Deregulation of the canonical Wnt signaling pathway plays an important role in human tumorigenesis through the accumulation of β-catenin and subsequent transactivation of TCF7L2. Although some of the consequences associated with the accumulated β-catenin have been clarified, the comprehensive effect of activated β-catenin/TCF7L2 transcriptional complex on tumorigenesis remains to be elucidated. To understand the precise molecular mechanisms underlying colorectal cancer, we searched for genes regulated by the complex in colorectal tumors. We performed expression profile analysis of HCT116 and SW480 colon cancer cells treated with β-catenin siRNAs, and ChIP-sequencing using anti-TCF7L2 antibody. Combination of these data with public microarray data of LS174 cells with a dominant-negative form of TCF7L2 identified a total of 11 candidate genes. In this paper, we focused on FERM Domain-containing protein 5 (FRMD5), and confirmed that it is regulated by both β-catenin and TCF7L2. An additional reporter assay disclosed that a region in intron1 transcriptionally regulated the expression of FRMD5. ChIP assay also corroborated that TCF7L2 associates with this region. These data suggested that FRMD5 is a novel direct target of the β-catenin/TCF7L2 complex.
Baorong Zhang - One of the best experts on this subject based on the ideXlab platform.
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stable cell lines of human sh sy5y uniformly expressing wild type or mutant type FERM Domain containing 7 gene
Experimental and Therapeutic Medicine, 2017Co-Authors: Yanfang Mao, Tingting Zheng, Baorong ZhangAbstract:It has been reported that FERM Domain containing 7 (FRMD7) may cause X-linked idiopathic congenital nystagmus (ICN). A total of >40 mutations of the FRMD7 gene have been identified, however their pathogenic role remains unclear. In the present study, enhanced green fluorescent protein-tagged wild-type (WT) and mutant (MT) FRMD7 (c. C781>G) were expressed in stably expressing human neuroblastoma SH-SY5Y cells following viral transfection and antibiotic selection. Uniform expression of the FRMD7 fusion proteins was confirmed via fluorescence microscopy and western blotting. The expression profiles of neuron-specific proteins and Rho guanine triphosphatases (GTPases) differed significantly between the wild-type and mutant cell lines. Levels of Mtap2, NF-M, nestin, GAP43 and Rac1 mRNA were significantly increased in MT-FRMD7 cells compared with controls (P<0.01). However, the expression of Rac1 protein did not differ significantly among the two cell lines. Taken together, the results of the current study suggest that MT-FRMD7 influences the expression of neuron-specific genes and Rho GTPases, which may be involved in the pathogenesis of ICN. The FRMD7 stable expression cell line may facilitate future studies investigating the role of this protein in neuronal development.
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Stable cell lines of human SH-SY5Y uniformly expressing wild-type or mutant-type FERM Domain containing 7 gene.
Experimental and therapeutic medicine, 2017Co-Authors: Yanfang Mao, Tingting Zheng, Baorong ZhangAbstract:It has been reported that FERM Domain containing 7 (FRMD7) may cause X-linked idiopathic congenital nystagmus (ICN). A total of >40 mutations of the FRMD7 gene have been identified, however their pathogenic role remains unclear. In the present study, enhanced green fluorescent protein-tagged wild-type (WT) and mutant (MT) FRMD7 (c. C781>G) were expressed in stably expressing human neuroblastoma SH-SY5Y cells following viral transfection and antibiotic selection. Uniform expression of the FRMD7 fusion proteins was confirmed via fluorescence microscopy and western blotting. The expression profiles of neuron-specific proteins and Rho guanine triphosphatases (GTPases) differed significantly between the wild-type and mutant cell lines. Levels of Mtap2, NF-M, nestin, GAP43 and Rac1 mRNA were significantly increased in MT-FRMD7 cells compared with controls (P
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FERM Domain containing protein 7 interacts with the Rho GDP dissociation inhibitor and specifically activates Rac1 signaling.
PloS one, 2013Co-Authors: Yanfang Mao, Guohua Zhao, Yaping Yan, Jun Tian, Xiaoguang Lei, Xinzhen Yin, Baorong ZhangAbstract:The FERM Domain containing protein 7 gene (FRMD7) associated with the X-linked disorder idiopathic congenital nystagmus (ICN) is involved in the regulation of neurite elongation during neuronal development. Members of the Rho family of small G-proteins (Rho GTPases) are key regulators of the actin cytoskeleton and are implicated in the control of neuronal morphology. The Rho GDP dissociation inhibitor alpha, RhoGDIα, the main regulator of Rho GTPases, can form a complex with the GDP-bound form of Rho GTPases and inhibit their activation. Here, we demonstrate that the full length of the mouse FRMD7, rather than the N-terminus or the C-terminus alone, directly interacts with RhoGDIα and specifically initiates Rac1 signaling in mouse neuroblastoma cell line (neuro-2a). Moreover, we show that wild-type human FRMD7 protein is able to activate Rac1 signaling by interacting with RhoGDIα and releasing Rac1 from Rac1-RhoGDIα complex. However, two missense mutations (c.781C>G and c.886G>C) of human FRMD7 proteins weaken the ability to interact with RhoGDIα and release less Rac1, that induce the activation of Rac1 to a lesser degree; while an additional mutant, c.1003C>T, which results in a C-terminal truncated protein, almost fails to interact with RhoGDIα and to activate Rac1 signaling. Collectively, these results suggest that FRMD7 interacts with one of the Rho GTPase regulators, RhoGDIα, and activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth. We predict that human mutant FRMD7 thus influences Rac1 signaling activation, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.
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A novel missense mutation in the FERM Domain containing 7 (FRMD7) gene causing X-linked idiopathic congenital nystagmus in a Chinese family.
Molecular vision, 2013Co-Authors: Zhirong Liu, Baorong Zhang, Shanying Mao, Yao Ding, Meiping DingAbstract:Purpose: Idiopathic congenital nystagmus (ICN) is a genetically heterogeneous disease. Thus far, the disease gene has been identified as the FERM Domain containing 7 (FRMD7) gene. The purpose of this study was to elucidate the clinical and genetic characteristics of a four- generation Chinese family with ICN. Methods: The clinical data and the genomic DNA of a Chinese ICN family were collected following the provision of informed consent. All coding exons of the FRMD7 gene were amplified by PCR and then sequenced. Affinity GST-p21 activated kinase 2 (PAK2) precipitation was used to investigate whether this novel FRMD7 mutant influenced Rac1 signaling activation in the human embryonic kidney 293 T cells (HEK 293T) cells transiently cotransfected with wild-type or mutant FRMD7 and Rac1. Results: A novel missense mutation (c.635T>C) was identified in all affected members. Obligate female carriers were heterozygous in these mutations and the affected males were homozygous, consistent with X-linked inheritance. This mutation is a substitution of proline for leucine. Function analysis showed that this novel mutant influences Rac1 signaling in human HEK 293T cells. Conclusions: This study widens the mutation spectrum of the FRMD7 gene. This mutant was shown to activate GTPase Rac1 signaling in vitro; however, the quantity of activated Rac1 was obviously decreased compared with the wild type (p
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a novel missense mutation in the FERM Domain containing 7 frmd7 gene causing x linked idiopathic congenital nystagmus in a chinese family
Molecular Vision, 2013Co-Authors: Zhirong Liu, Baorong Zhang, Shanying Mao, Yao Ding, Meiping DingAbstract:Purpose: Idiopathic congenital nystagmus (ICN) is a genetically heterogeneous disease. Thus far, the disease gene has been identified as the FERM Domain containing 7 (FRMD7) gene. The purpose of this study was to elucidate the clinical and genetic characteristics of a four- generation Chinese family with ICN. Methods: The clinical data and the genomic DNA of a Chinese ICN family were collected following the provision of informed consent. All coding exons of the FRMD7 gene were amplified by PCR and then sequenced. Affinity GST-p21 activated kinase 2 (PAK2) precipitation was used to investigate whether this novel FRMD7 mutant influenced Rac1 signaling activation in the human embryonic kidney 293 T cells (HEK 293T) cells transiently cotransfected with wild-type or mutant FRMD7 and Rac1. Results: A novel missense mutation (c.635T>C) was identified in all affected members. Obligate female carriers were heterozygous in these mutations and the affected males were homozygous, consistent with X-linked inheritance. This mutation is a substitution of proline for leucine. Function analysis showed that this novel mutant influences Rac1 signaling in human HEK 293T cells. Conclusions: This study widens the mutation spectrum of the FRMD7 gene. This mutant was shown to activate GTPase Rac1 signaling in vitro; however, the quantity of activated Rac1 was obviously decreased compared with the wild type (p<0.05). Taken together, our data strongly support the hypothesis that the identified FRMD7 mutant influences GTPase Rac1 signaling, which regulates neurite development. This mutation may be related to the pathogenesis of X-linked ICN.
Shoichiro Tsukita - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for neurofibromatosis type 2. Crystal structure of the merlin FERM Domain.
The Journal of biological chemistry, 2001Co-Authors: Toshiyuki Shimizu, Keisuke Hamada, Shoichiro Tsukita, Azusa Seto, Nobuo Maita, Toshio HakoshimaAbstract:Neurofibromatosis type 2 (NF2) is a dominantly inherited disease associated with the central nervous system. The NF2 gene product merlin is a tumor suppressor, and its mutation or inactivation causes this disease. We report here the crystal structure of the merlin FERM Domain containing a 22-residue α-helical segment. The structure reveals that the merlin FERM Domain consists of three subDomains displaying notable features of the electrostatic surface potentials, although the overall surface potentials similar to those of ezrin/radixin/moesin (ERM) proteins indicate electrostatic membrane association. The structure also is consistent with inactivation mechanisms caused by the pathogenic mutations associated with NF2.
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Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM Domain.
Acta Crystallographica Section D Biological Crystallography, 2001Co-Authors: Keisuke Hamada, Takeshi Matsui, Toshiyuki Shimizu, Shoichiro Tsukita, Azusa Seto, Yoshimi Takai, Toshio HakoshimaAbstract:The Rho guanine nucleotide-dissociation inhibitor (RhoGDI) is a general regulator that forms a complex with the GDP-bound form of Rho-family GTPases and suppresses their activation. The FERM Domains of ERM (ezrin/radixin/moesin) proteins bind to RhoGDI and dissociate Rho from RhoGDI. The formation of a complex between RhoGDI and the FERM Domain is an important step in the regulatory cycle of Rho activation. In this study, crystals of RhoGDI complexed with the FERM Domain of radixin were obtained. The crystals of the binary complex belong to the space group P21212, with unit-cell parameters a = 130.9 (2), b = 151.2 (2), c = 71.2 (1) A, and contain two protein complexes in the crystallographic asymmetric unit. A 2.9 A resolution data set was collected using synchrotron radiation at SPring-8.
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Crystallographic characterization of the radixin FERM Domain bound to the cytoplasmic tail of the adhesion protein ICAM-2.
Acta crystallographica. Section D Biological crystallography, 2001Co-Authors: K Hamada, Shoichiro Tsukita, T Shimizu, T Matsui, Toshio HakoshimaAbstract:Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The FERM Domains located at the N-terminal regions of ERM proteins are responsible for membrane association through direct interactions with the cytoplasmic Domains of integral membrane proteins. Here, crystals of the complex between the radixin FERM Domain and the full-length cytoplasmic tail (28-residue peptide) of intercellular adhesion molecule 2, ICAM-2, have been obtained. The crystals were found to belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 100.44 (9), c = 99.49 (6) A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.60 A.
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structural basis of the membrane targeting and unmasking mechanisms of the radixin FERM Domain
The EMBO Journal, 2000Co-Authors: Keisuke Hamada, Takeshi Matsui, Toshiyuki Shimizu, Shoichiro Tsukita, Toshio HakoshimaAbstract:Radixin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which play a role in the formation of the membrane-associated cytoskeleton by linking actin filaments and adhesion proteins. This cross-linking activity is regulated by phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP2) in the downstream of the small G protein Rho. The X-ray crystal structures of the radixin FERM Domain, which is responsible for membrane binding, and its complex with inositol-(1,4,5)-trisphosphate (IP3) have been determined. The Domain consists of three subDomains featuring a ubiquitin-like fold, a four-helix bundle and a phosphotyrosine-binding-like Domain, respectively. These subDomains are organized by intimate interDomain interactions to form characteristic grooves and clefts. One such groove is negatively charged and so is thought to interact with basic juxta-membrane regions of adhesion proteins. IP3 binds a basic cleft that is distinct from those of pleckstrin homology Domains and is located on a positively charged flat molecular surface, suggesting an electrostatic mechanism of plasma membrane targeting. Based on the structural changes associated with IP3 binding, a possible unmasking mechanism of ERM proteins by PIP2 is proposed.
Michael J. Eck - One of the best experts on this subject based on the ideXlab platform.
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the FERM Domain organizing the structure and function of fak
Nature Reviews Molecular Cell Biology, 2010Co-Authors: Margaret C Frame, Hitesh Patel, Bryan Serrels, Daniel Lietha, Michael J. EckAbstract:Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) Domain. Recent structural work reveals how regulatory proteins activate FAK by binding to its FERM Domain, enabling it to coordinate diverse cellular responses.
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structural basis for the autoinhibition of focal adhesion kinase
Cell, 2007Co-Authors: Daniel Lietha, Derek F. Ceccarelli, Michael D. Schaller, Xinming Cai, Michael J. EckAbstract:Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM Domain directly binds the kinase Domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM Domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase Domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM Domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.
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crystal structure of the FERM Domain of focal adhesion kinase
Journal of Biological Chemistry, 2006Co-Authors: Derek F. Ceccarelli, Michael D. Schaller, Hyun Kyu Song, Florence Poy, Michael J. EckAbstract:Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM Domain. FERM Domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM Domain and a portion of the regulatory linker that connects the FERM and kinase Domains. The tertiary folds of the three subDomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subDomain alters the nature of the interDomain interface, and the phosphoinositide binding site found in ERM family FERM Domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM Domain can be regulated by binding of Src kinases to the linker segment.
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FERM Domain interaction promotes FAK signaling.
Molecular and cellular biology, 2004Co-Authors: Jill M. Dunty, Veronica Gabarra-niecko, Michelle L. King, Derek F. Ceccarelli, Michael J. Eck, Michael D. SchallerAbstract:From the results of deletion analyses, the FERM Domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM Domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM Domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM Domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM Domain.