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Jean Pieters - One of the best experts on this subject based on the ideXlab platform.
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homodimerization of Coronin a through the c terminal coiled coil domain is essential for multicellular differentiation of dictyostelium discoideum
FEBS Letters, 2020Co-Authors: Thomas Fiedler, Tohnyui Ndinyanka Fabrice, Vera Studer, Adrien F. Vinet, Lenka Faltova, Richard A. Kammerer, Michel O. Steinmetz, Timothy D. Sharpe, Jean PietersAbstract:Coronin proteins are widely expressed among eukaryotic organisms. Most Coronins consist of a WD repeat domain followed by a C-terminal coiled coil. Dictyostelium discoideum expresses a single short Coronin Coronin A, which has been implicated in both actin modulation as well as multicellular differentiation. Whether Coronin A's coiled coil is important for functionality, as well as the oligomeric state of Coronin A is not known. Here, we show that the coiled-coil domain in Dictyostelium Coronin A functions in homodimerization, is dispensable for Coronin A stability and localization but essential for multicellular differentiation. These results allow a better understanding of the role for the coiled-coil domain of Coronin A in oligomerization and demonstrate that its presence is essential for multicellular differentiation.
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Homodimerization of Coronin A through the C-terminal coiled-coil domain is essential for multicellular differentiation of Dictyostelium discoideum.
FEBS letters, 2020Co-Authors: Thomas Fiedler, Tohnyui Ndinyanka Fabrice, Vera Studer, Adrien F. Vinet, Lenka Faltova, Richard A. Kammerer, Michel O. Steinmetz, Timothy D. Sharpe, Jean PietersAbstract:Coronin proteins are widely expressed among eukaryotic organisms. Most Coronins consist of a WD-repeat domain followed by a C-terminal coiled coil. Dictyostelium discoideum expresses a single short Coronin Coronin A, which has been implicated in both actin modulation and multicellular differentiation. Whether Coronin A's coiled coil is important for functionality, as well as the oligomeric state of Coronin A is not known. Here, we show that the coiled-coil domain in Dictyostelium Coronin A functions in homodimerization, is dispensable for Coronin A stability and localization but essential for multicellular differentiation. These results allow a better understanding of the role for the coiled-coil domain of Coronin A in oligomerization and demonstrate that its presence is essential for multicellular differentiation.
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Interactome and F-Actin Interaction Analysis of Dictyostelium discoideum Coronin A.
International journal of molecular sciences, 2020Co-Authors: Tohnyui Ndinyanka Fabrice, Thomas Fiedler, Vera Studer, Adrien F. Vinet, Francesco Brogna, Albrecht Schmidt, Jean PietersAbstract:Coronin proteins are evolutionary conserved WD repeat containing proteins that have been proposed to carry out different functions. In Dictyostelium, the short Coronin isoform, Coronin A, has been implicated in cytoskeletal reorganization, chemotaxis, phagocytosis and the initiation of multicellular development. Generally thought of as modulators of F-actin, Coronin A and its mammalian homologs have also been shown to mediate cellular processes in an F-actin-independent manner. Therefore, it remains unclear whether or not Coronin A carries out its functions through its capacity to interact with F-actin. Moreover, the interacting partners of Coronin A are not known. Here, we analyzed the interactome of Coronin A as well as its interaction with F-actin within cells and in vitro. Interactome analysis showed the association with a diverse set of interaction partners, including fimbrin, talin and myosin subunits, with only a transient interaction with the minor actin10 isoform, but not the major form of actin, actin8, which was consistent with the absence of a Coronin A-actin interaction as analyzed by co-sedimentation from cells and lysates. In vitro, however, purified Coronin A co-precipitated with rabbit muscle F-actin in a coiled-coil-dependent manner. Our results suggest that an in vitro interaction of Coronin A and rabbit muscle actin may not reflect the cellular interaction state of Coronin A with actin, and that Coronin A interacts with diverse proteins in a time-dependent manner.
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Coronin 1 Is Required for Integrin β2 Translocation in Platelets.
International journal of molecular sciences, 2020Co-Authors: David R. J. Riley, Jean Pieters, Jawad S. Khalil, Khalid M. Naseem, Francisco RiveroAbstract:Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant Coronins 1, 2, and 3 and the less abundant Coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of Coronin 1. Here we show that the absence of Coronin 1 is associated with impaired translocation of integrin β2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among Coronins 1, 2, and 3 in platelets, while aspects like integrin β2 translocation are specifically or predominantly dependent on Coronin 1.
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Getting in and Staying Alive: Role for Coronin 1 in the Survival of Pathogenic Mycobacteria and Naïve T Cells.
Frontiers in immunology, 2018Co-Authors: Mayumi Mori, Jean PietersAbstract:There are many different pathogenic stimuli that are able to activate the immune system, ranging from microbes that include bacteria, viruses, fungi, and parasites to host-derived triggers such as autoantigens that can induce autoimmunity as well as neoantigens involved in tumorigenesis. One of the key interactions shaping immunity toward these triggers involves the encounter of antigen-processing and -presenting cells such as macrophages and dendritic cells with T cells, resulting in immune responses that are highly selective for the antigenic trigger. Research over the past few years has implicated members of the Coronin protein family, in particular Coronin 1, in responses against several pathogenic triggers. While Coronin 1 was initially described as a host factor allowing the intracellular survival of the pathogen Mycobacterium tuberculosis, subsequent work showed it to be a crucial factor for naïve T cell homeostasis. The activity of Coronin 1 in allowing the intracellular survival of pathogenic mycobacteria is relatively well characterized, involving the activation of the Ca2+/calcineurin pathway, while Coronin 1's role in modulating naïve T cell homeostasis remains more enigmatic. In this mini review, we discuss the knowledge on the role for Coronin 1 in immune cell functioning and provide a number of potential scenarios via which Coronin 1 may be able to regulate naïve T cell homeostasis.
Tsutomu Tsuji - One of the best experts on this subject based on the ideXlab platform.
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constitutive turnover of phosphorylation at thr 412 of human p57 Coronin 1 regulates the interaction with actin
Journal of Biological Chemistry, 2012Co-Authors: Teruaki Oku, Mai Nakano, Yutaka Kaneko, Yusuke Ando, Hiroki Kenmotsu, Saotomo Itoh, Makoto Tsuiji, Yoshiyuki Seyama, Satoshi Toyoshima, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/Coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/Coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/Coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/Coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/Coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/Coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/Coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/Coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/Coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/Coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/Coronin-1 regulates its interaction with actin.
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Constitutive turnover of phosphorylation at Thr-412 of human p57/Coronin-1 regulates the interaction with actin.
The Journal of biological chemistry, 2012Co-Authors: Teruaki Oku, Mai Nakano, Yutaka Kaneko, Yusuke Ando, Hiroki Kenmotsu, Saotomo Itoh, Makoto Tsuiji, Yoshiyuki Seyama, Satoshi Toyoshima, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/Coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/Coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/Coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/Coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/Coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/Coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/Coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/Coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/Coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/Coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/Coronin-1 regulates its interaction with actin.
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phorbol ester dependent phosphorylation regulates the association of p57 Coronin 1 with the actin cytoskeleton
Journal of Biological Chemistry, 2008Co-Authors: Teruaki Oku, Yutaka Kaneko, Yoshiyuki Seyama, Satoshi Toyoshima, Koki Murofushi, Tsutomu TsujiAbstract:The p57/Coronin-1 protein is a member of the Coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/Coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/Coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/Coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/Coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/Coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/Coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/Coronin-1 antibody-conjugated Sepharose, p57/Coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/Coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/Coronin-1. These results strongly suggest that the phosphorylation of p57/Coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.
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Phorbol ester-dependent phosphorylation regulates the association of p57/Coronin-1 with the actin cytoskeleton.
The Journal of biological chemistry, 2008Co-Authors: Teruaki Oku, Yutaka Kaneko, Yoshiyuki Seyama, Satoshi Toyoshima, Koki Murofushi, Tsutomu TsujiAbstract:The p57/Coronin-1 protein is a member of the Coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/Coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/Coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/Coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/Coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/Coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/Coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/Coronin-1 antibody-conjugated Sepharose, p57/Coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/Coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/Coronin-1. These results strongly suggest that the phosphorylation of p57/Coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.
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Homotypic dimerization of the actin-binding protein p57/Coronin-1 mediated by a leucine zipper motif in the C-terminal region.
The Biochemical journal, 2005Co-Authors: Teruaki Oku, Saotomo Itoh, Satoshi Toyoshima, Rie Ishii, Kensuke Suzuki, William M Nauseef, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/Coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/Coronin-1. Recombinant p57/Coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/Coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/Coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/Coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/Coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/Coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/Coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.
Teruaki Oku - One of the best experts on this subject based on the ideXlab platform.
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constitutive turnover of phosphorylation at thr 412 of human p57 Coronin 1 regulates the interaction with actin
Journal of Biological Chemistry, 2012Co-Authors: Teruaki Oku, Mai Nakano, Yutaka Kaneko, Yusuke Ando, Hiroki Kenmotsu, Saotomo Itoh, Makoto Tsuiji, Yoshiyuki Seyama, Satoshi Toyoshima, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/Coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/Coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/Coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/Coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/Coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/Coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/Coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/Coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/Coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/Coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/Coronin-1 regulates its interaction with actin.
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Constitutive turnover of phosphorylation at Thr-412 of human p57/Coronin-1 regulates the interaction with actin.
The Journal of biological chemistry, 2012Co-Authors: Teruaki Oku, Mai Nakano, Yutaka Kaneko, Yusuke Ando, Hiroki Kenmotsu, Saotomo Itoh, Makoto Tsuiji, Yoshiyuki Seyama, Satoshi Toyoshima, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/Coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/Coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/Coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/Coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/Coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/Coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/Coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/Coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/Coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/Coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/Coronin-1 regulates its interaction with actin.
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phorbol ester dependent phosphorylation regulates the association of p57 Coronin 1 with the actin cytoskeleton
Journal of Biological Chemistry, 2008Co-Authors: Teruaki Oku, Yutaka Kaneko, Yoshiyuki Seyama, Satoshi Toyoshima, Koki Murofushi, Tsutomu TsujiAbstract:The p57/Coronin-1 protein is a member of the Coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/Coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/Coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/Coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/Coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/Coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/Coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/Coronin-1 antibody-conjugated Sepharose, p57/Coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/Coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/Coronin-1. These results strongly suggest that the phosphorylation of p57/Coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.
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Phorbol ester-dependent phosphorylation regulates the association of p57/Coronin-1 with the actin cytoskeleton.
The Journal of biological chemistry, 2008Co-Authors: Teruaki Oku, Yutaka Kaneko, Yoshiyuki Seyama, Satoshi Toyoshima, Koki Murofushi, Tsutomu TsujiAbstract:The p57/Coronin-1 protein is a member of the Coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/Coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/Coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/Coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/Coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/Coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/Coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/Coronin-1 antibody-conjugated Sepharose, p57/Coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/Coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/Coronin-1. These results strongly suggest that the phosphorylation of p57/Coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.
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Homotypic dimerization of the actin-binding protein p57/Coronin-1 mediated by a leucine zipper motif in the C-terminal region.
The Biochemical journal, 2005Co-Authors: Teruaki Oku, Saotomo Itoh, Satoshi Toyoshima, Rie Ishii, Kensuke Suzuki, William M Nauseef, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/Coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/Coronin-1. Recombinant p57/Coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/Coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/Coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/Coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/Coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/Coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/Coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.
Satoshi Toyoshima - One of the best experts on this subject based on the ideXlab platform.
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constitutive turnover of phosphorylation at thr 412 of human p57 Coronin 1 regulates the interaction with actin
Journal of Biological Chemistry, 2012Co-Authors: Teruaki Oku, Mai Nakano, Yutaka Kaneko, Yusuke Ando, Hiroki Kenmotsu, Saotomo Itoh, Makoto Tsuiji, Yoshiyuki Seyama, Satoshi Toyoshima, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/Coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/Coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/Coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/Coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/Coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/Coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/Coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/Coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/Coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/Coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/Coronin-1 regulates its interaction with actin.
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Constitutive turnover of phosphorylation at Thr-412 of human p57/Coronin-1 regulates the interaction with actin.
The Journal of biological chemistry, 2012Co-Authors: Teruaki Oku, Mai Nakano, Yutaka Kaneko, Yusuke Ando, Hiroki Kenmotsu, Saotomo Itoh, Makoto Tsuiji, Yoshiyuki Seyama, Satoshi Toyoshima, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/Coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/Coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/Coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/Coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/Coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/Coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/Coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/Coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/Coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/Coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/Coronin-1 regulates its interaction with actin.
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phorbol ester dependent phosphorylation regulates the association of p57 Coronin 1 with the actin cytoskeleton
Journal of Biological Chemistry, 2008Co-Authors: Teruaki Oku, Yutaka Kaneko, Yoshiyuki Seyama, Satoshi Toyoshima, Koki Murofushi, Tsutomu TsujiAbstract:The p57/Coronin-1 protein is a member of the Coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/Coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/Coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/Coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/Coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/Coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/Coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/Coronin-1 antibody-conjugated Sepharose, p57/Coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/Coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/Coronin-1. These results strongly suggest that the phosphorylation of p57/Coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.
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Phorbol ester-dependent phosphorylation regulates the association of p57/Coronin-1 with the actin cytoskeleton.
The Journal of biological chemistry, 2008Co-Authors: Teruaki Oku, Yutaka Kaneko, Yoshiyuki Seyama, Satoshi Toyoshima, Koki Murofushi, Tsutomu TsujiAbstract:The p57/Coronin-1 protein is a member of the Coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/Coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/Coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/Coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/Coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/Coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/Coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/Coronin-1 antibody-conjugated Sepharose, p57/Coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/Coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/Coronin-1. These results strongly suggest that the phosphorylation of p57/Coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.
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Homotypic dimerization of the actin-binding protein p57/Coronin-1 mediated by a leucine zipper motif in the C-terminal region.
The Biochemical journal, 2005Co-Authors: Teruaki Oku, Saotomo Itoh, Satoshi Toyoshima, Rie Ishii, Kensuke Suzuki, William M Nauseef, Tsutomu TsujiAbstract:The actin-binding protein p57/Coronin-1, a member of the Coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/Coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/Coronin-1. Recombinant p57/Coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/Coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/Coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/Coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/Coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/Coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/Coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.
James E. Bear - One of the best experts on this subject based on the ideXlab platform.
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Coronin 1C inhibits melanoma metastasis through regulation of MT1-MMP-containing extracellular vesicle secretion.
Scientific reports, 2020Co-Authors: Alicia C. Tagliatela, Stephanie C Hempstead, Priya S Hibshman, Max A Hockenberry, Hailey E. Brighton, Chad V. Pecot, James E. BearAbstract:Coronin 1C is overexpressed in multiple tumors, leading to the widely held view that this gene drives tumor progression, but this hypothesis has not been rigorously tested in melanoma. Here, we combined a conditional knockout of Coronin 1C with a genetically engineered mouse model of PTEN/BRAF-driven melanoma. Loss of Coronin 1C in this model increases both primary tumor growth rates and distant metastases. Coronin 1C-null cells isolated from this model are more invasive in vitro and produce more metastatic lesions in orthotopic transplants than Coronin 1C-reexpressing cells due to the shedding of extracellular vesicles (EVs) containing MT1-MMP. Interestingly, these vesicles contain melanosome markers suggesting a melanoma-specific mechanism of EV release, regulated by Coronin 1C, that contributes to the high rates of metastasis in melanoma.
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Coronin 1b regulates s1p induced human lung endothelial cell chemotaxis role of pld2 protein kinase c and rac1 signal transduction
PLOS ONE, 2013Co-Authors: Peter V Usatyuk, James E. Bear, Michael Burns, Vijay Mohan, Srikanth Pendyala, David L Ebenezer, Anantha Harijith, Long Shuang Huang, Joe G N Garcia, Viswanathan NatarajanAbstract:Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates Coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting Coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced Coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced Coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, e and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates Coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis.
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Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin
The Biochemical journal, 2012Co-Authors: Keefe T. Chan, David W. Roadcap, Nicholas Holoweckyj, James E. BearAbstract:Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg(30)) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other Coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of Coronin regulation, which has implications for how Coronins orchestrate cytoskeletal dynamics.
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Coronin 2A regulates a subset of focal-adhesion-turnover events through the cofilin pathway.
Journal of cell science, 2009Co-Authors: Thomas W Marshall, Heather L Aloor, James E. BearAbstract:Coronins are conserved F-actin-binding proteins that are important for motility and actin dynamics. Unlike type I Coronins, Coronin 2A localizes to stress fibers and some focal adhesions, and is excluded from the leading edge. Depletion of Coronin 2A in MTLn3 cells decreases cell motility and turnover of focal adhesions. Surprisingly, none of the pathways known to regulate focal-adhesion turnover are affected by depletion of Coronin 2A. Depletion of Coronin 2A does, however, increase phospho-cofilin, suggesting that misregulation of cofilin might affect adhesion dynamics. Slingshot-1L, a cofilin-activating phosphatase, localizes to focal adhesions and interacts with Coronin 2A. Depletion of Coronin 2A reduces cofilin activity at focal adhesions, as measured by barbed-end density and actin FRAP. In both fixed cells and live cells, cofilin localizes to the proximal end of some focal adhesions. Although expression of wild-type cofilin in Coronin-2A-depleted cells has no major effect on focal-adhesion dynamics, expression of an active mutant of cofilin bypasses the defects in cell motility and focal-adhesion disassembly. These results implicate both Coronin 2A and cofilin as factors that can regulate a subset of focal-adhesion-turnover events.
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The Role of Mammalian Coronins in Development and Disease
Sub-cellular biochemistry, 2008Co-Authors: David W. Roadcap, Christoph S. Clemen, James E. BearAbstract:Coronins have maintained a high degree of conservation over the roughly 800 million years of eukaryotic evolution.1,2 From its origins as a single gene in simpler eukaryotes, the mammalian Coronin gene family has expanded to include at least six members (see Chapter 4). Increasing evidence indicates that Coronins play critical roles as regulators of actin dependent processes such as cell motility and vesicle trafficking3,4 (see Chapters 6–9). Considering the importance of these processes, it is not surprising that recent findings have implicated the involvement of Coronins in multiple diseases. This review primarily focuses on Coronin 1C (HGNC symbol: CORO1C, also known as Coronin 3) which is a transcriptionally dynamic gene that is up-regulated in multiple types of clinically aggressive cancer. In addition to reviewing the molecular signals and events that lead to Coronin 1C transcription, we summarize the results of several studies describing the possible functional roles of Coronin 1C in development as well as disease progression. Here, the main focus is on brain development and on the progression of melanoma and glioma. Finally, we will also review the role of other mammalian Coronin genes in clinically relevant processes such as neural regeneration and pathogenic bacterial infections (see Chapter 10).
