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Vann Bennett - One of the best experts on this subject based on the ideXlab platform.
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Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells
Molecular biology of the cell, 2007Co-Authors: Khadar M. Abdi, Vann BennettAbstract:Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that Adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with beta2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete alpha-Adducin from confluent HBE cells. Depletion of alpha-Adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of beta2-spectrin resulted in loss of Adducin from the lateral membrane. siRNA-resistant alpha-Adducin prevented loss of lateral membrane, but only if alpha-Adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phospho-mimetic versions of Adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that Adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of Adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 microm. We conclude that Adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.
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Association of protein kinase Cλ with Adducin in 3T3-L1 adipocytes
Biochimica et Biophysica Acta, 2001Co-Authors: Palle G. Laustsen, William S. Lane, Vann Bennett, Gustav E. LienhardAbstract:There is evidence that the atypical protein kinases C (PKC(lambda), PKC(zeta)) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKC(lambda/zeta) from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKC(lambda) is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of alpha- and gamma-Adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKC(lambda). Association of the Adducins with PKC(lambda) was further indicated by the finding that the Adducins coimmunoprecipitated proportionally with PKC(lambda) in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the Adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the Adducins in a whole cell lysate or on the small portion of the Adducins that coimmunoprecipitated with PKC(lambda). PKC(lambda) and the Adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKC(lambda) to Adducin may function to localize PKC(lambda) in 3T3-L1 adipocytes.
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Association of protein kinase C(lambda) with Adducin in 3T3-L1 adipocytes.
Biochimica et biophysica acta, 2001Co-Authors: Palle G. Laustsen, William S. Lane, Vann Bennett, Gustav E. LienhardAbstract:There is evidence that the atypical protein kinases C (PKC(lambda), PKC(zeta)) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKC(lambda/zeta) from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKC(lambda) is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of alpha- and gamma-Adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKC(lambda). Association of the Adducins with PKC(lambda) was further indicated by the finding that the Adducins coimmunoprecipitated proportionally with PKC(lambda) in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the Adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the Adducins in a whole cell lysate or on the small portion of the Adducins that coimmunoprecipitated with PKC(lambda). PKC(lambda) and the Adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKC(lambda) to Adducin may function to localize PKC(lambda) in 3T3-L1 adipocytes.
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Association of protein kinase Cλ with Adducin in 3T3-L1 adipocytes
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2001Co-Authors: Palle G. Laustsen, William S. Lane, Vann Bennett, Gustav E. LienhardAbstract:AbstractThere is evidence that the atypical protein kinases C (PKCλ, PKCζ) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKCλ/ζ from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKCλ is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of α- and γ-Adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKCλ. Association of the Adducins with PKCλ was further indicated by the finding that the Adducins coimmunoprecipitated proportionally with PKCλ in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the Adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the Adducins in a whole cell lysate or on the small portion of the Adducins that coimmunoprecipitated with PKCλ. PKCλ and the Adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKCλ to Adducin may function to localize PKCλ in 3T3-L1 adipocytes
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Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
The Journal of cell biology, 1999Co-Authors: Yuko Fukata, Vann Bennett, Yoichiro Matsuoka, Yoshiharu Matsuura, Noriko Oshiro, Nagatoki Kinoshita, Yoji Kawano, Kozo KaibuchiAbstract:Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-Adducin and thereby enhances the F-actin–binding activity of α-Adducin in vitro. Here we identified the sites of phosphorylation of α-Adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-Adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-Adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-Adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-AdducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-AdducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-Adducin downstream of Rho in vivo, and that the phosphorylation of Adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
Yoichiro Matsuoka - One of the best experts on this subject based on the ideXlab platform.
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Rho-kinase induces association of Adducin with the cytoskeleton in platelet activation.
Biochemical and biophysical research communications, 2005Co-Authors: Satoshi Tamaru, Yoichiro Matsuoka, Tetsu Fukuta, Kozo Kaibuchi, Hiroshi Shiku, Masakatsu NishikawaAbstract:Abstract We examined whether Adducin function is regulated through Rho-kinase after agonist stimulation in platelets. A variety of stimuli such as thrombin, STA2 (a stable analog of TXA2), Ca2+ ionophore, phorbol diester, and shear stress induced phosphorylation of α-Adducin at Thr445. Preincubation with the Rho-kinase inhibitor Y-27632 in platelets inhibited agonist-induced phosphorylation of α-Adducin. STA2 stimulation led to a redistribution of Adducin from Triton-insoluble (high speed) fraction (membrane skeleton) to Triton-insoluble (low speed) fraction (cytoskeleton) and detergent-soluble fraction. PhosphoAdducin at Thr445 was selectively isolated in the cytoskeletal fraction, whereas phosphoAdducin at Ser726 was mainly present in the Triton-soluble fraction. Y-27632 inhibition of STA2-induced α-Adducin phosphorylation at Thr445 inhibited incorporation of α-Adducin and spectrin into the platelet cytoskeleton, although Y-27632 did not affect phosphorylation of α-Adducin at Ser726. These results suggest that Rho-kinase regulates the association of α-Adducin and spectrin with the actin cytoskeleton in platelet activation.
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Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
The Journal of cell biology, 1999Co-Authors: Yuko Fukata, Vann Bennett, Yoichiro Matsuoka, Yoshiharu Matsuura, Noriko Oshiro, Nagatoki Kinoshita, Yoji Kawano, Kozo KaibuchiAbstract:Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-Adducin and thereby enhances the F-actin–binding activity of α-Adducin in vitro. Here we identified the sites of phosphorylation of α-Adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-Adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-Adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-Adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-AdducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-AdducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-Adducin downstream of Rho in vivo, and that the phosphorylation of Adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
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Adducin is an in vivo substrate for protein kinase c phosphorylation in the marcks related domain inhibits activity in promoting spectrin actin complexes and occurs in many cells including dendritic spines of neurons
Journal of Cell Biology, 1998Co-Authors: Yoichiro Matsuoka, Xiaolin Li, Vann BennettAbstract:Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in α, β, and γ Adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of Adducin involving actin and spectrin, and we demonstrate that Adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant Adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of Adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of Adducin in cells. Reactivity with phosphoAdducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of Adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant α Adducin. The mutant α Adducin was no longer concentrated at the cell membrane at sites of cell–cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant α Adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant α Adducin in a punctate pattern. Immunofluorescence with the phosphoAdducin-specific antibody revealed the RTPS-serine phosphorylation of Adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoAdducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoAdducin. These data demonstrate that Adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of Adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.
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Adducin preferentially recruits spectrin to the fast growing ends of actin filaments in a complex requiring the marcks related domain and a newly defined oligomerization domain
Journal of Biological Chemistry, 1998Co-Authors: Yoichiro Matsuoka, Vann BennettAbstract:Adducin is a protein associated with spectrin and actin in membrane skeletons of erythrocytes and possibly other cells. Adducin has activities in in vitro assays of association with the sides of actin filaments, capping the fast growing ends of actin filaments, and recruiting spectrin to actin filaments. This study presents evidence that Adducin exhibits a preference for the fast growing ends of actin filaments for recruiting spectrin to actin and for direct association with actin. beta-Adducin-(335-726) promoted recruitment of spectrin to gelsolin-sensitive sites at fast growing ends of actin filaments with half-maximal activity at 15 nM and to gelsolin-insensitive sites with half-maximal activity at 75 nM. beta-Adducin-(335-726) also exhibited a preference for actin filament ends in direct binding assays; the half-maximal concentration for binding of Adducin to gelsolin-sensitive sites at filament ends was 60 nM, and the Kd for binding to lateral sites was 1.5 microM. The concentration of beta-Adducin-(335-726) of 60 nM required for half-maximal binding to filament ends is in the same range as the concentration of 150 nM required for half-maximal actin capping activity. All interactions of Adducin with actin require the myristoylated alanine-rich protein kinase C substrate-related domain as well as a newly defined oligomerization site localized in the neck domain of Adducin. Surprisingly, the head domain of Adducin is not required for spectrin-actin interactions, although it could play a role in forming tetramers. The relative activities of Adducin imply that an important role of Adducin in cells is to form a complex with the fast growing ends of actin filaments that recruits spectrin and prevents addition or loss of actin subunits.
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Regulation of the Association of Adducin with Actin Filaments by Rho-associated Kinase (Rho-kinase) and Myosin Phosphatase
The Journal of biological chemistry, 1998Co-Authors: Kazushi Kimura, Vann Bennett, Yoichiro Matsuoka, Yuko Fukata, Yoshiharu Matsuura, Katsuya Okawa, Akihiro Iwamatsu, Kozo KaibuchiAbstract:Abstract The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as Adducin, and found that MBS specifically interacted with Adducinin vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated α-Adducin in vitro and in vivo and that the phosphorylation of α-Adducin by Rho-kinase enhanced the interaction of α-Adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward α-Adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of Adducin downstream of Rho and that the increased phosphorylation of Adducin by Rho-kinase causes the interaction of Adducin with actin filaments.
Kozo Kaibuchi - One of the best experts on this subject based on the ideXlab platform.
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Rho-kinase induces association of Adducin with the cytoskeleton in platelet activation.
Biochemical and biophysical research communications, 2005Co-Authors: Satoshi Tamaru, Yoichiro Matsuoka, Tetsu Fukuta, Kozo Kaibuchi, Hiroshi Shiku, Masakatsu NishikawaAbstract:Abstract We examined whether Adducin function is regulated through Rho-kinase after agonist stimulation in platelets. A variety of stimuli such as thrombin, STA2 (a stable analog of TXA2), Ca2+ ionophore, phorbol diester, and shear stress induced phosphorylation of α-Adducin at Thr445. Preincubation with the Rho-kinase inhibitor Y-27632 in platelets inhibited agonist-induced phosphorylation of α-Adducin. STA2 stimulation led to a redistribution of Adducin from Triton-insoluble (high speed) fraction (membrane skeleton) to Triton-insoluble (low speed) fraction (cytoskeleton) and detergent-soluble fraction. PhosphoAdducin at Thr445 was selectively isolated in the cytoskeletal fraction, whereas phosphoAdducin at Ser726 was mainly present in the Triton-soluble fraction. Y-27632 inhibition of STA2-induced α-Adducin phosphorylation at Thr445 inhibited incorporation of α-Adducin and spectrin into the platelet cytoskeleton, although Y-27632 did not affect phosphorylation of α-Adducin at Ser726. These results suggest that Rho-kinase regulates the association of α-Adducin and spectrin with the actin cytoskeleton in platelet activation.
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Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
The Journal of cell biology, 1999Co-Authors: Yuko Fukata, Vann Bennett, Yoichiro Matsuoka, Yoshiharu Matsuura, Noriko Oshiro, Nagatoki Kinoshita, Yoji Kawano, Kozo KaibuchiAbstract:Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-Adducin and thereby enhances the F-actin–binding activity of α-Adducin in vitro. Here we identified the sites of phosphorylation of α-Adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-Adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-Adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-Adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-AdducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-AdducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-Adducin downstream of Rho in vivo, and that the phosphorylation of Adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
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Regulation of the Association of Adducin with Actin Filaments by Rho-associated Kinase (Rho-kinase) and Myosin Phosphatase
The Journal of biological chemistry, 1998Co-Authors: Kazushi Kimura, Vann Bennett, Yoichiro Matsuoka, Yuko Fukata, Yoshiharu Matsuura, Katsuya Okawa, Akihiro Iwamatsu, Kozo KaibuchiAbstract:Abstract The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as Adducin, and found that MBS specifically interacted with Adducinin vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated α-Adducin in vitro and in vivo and that the phosphorylation of α-Adducin by Rho-kinase enhanced the interaction of α-Adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward α-Adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of Adducin downstream of Rho and that the increased phosphorylation of Adducin by Rho-kinase causes the interaction of Adducin with actin filaments.
Andrés F Muro - One of the best experts on this subject based on the ideXlab platform.
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β-Adducin (Add2) KO mice show synaptic plasticity, motor coordination and behavioral deficits accompanied by changes in the expression and phosphorylation levels of the α- and γ-Adducin subunits
Genes brain and behavior, 2009Co-Authors: Fabiola Porro, Luisa Costessi, M. Rosato-siri, E. Leone, Alessandra Iaconcig, E. Tongiorgi, Andrés F MuroAbstract:Adducins are a family of proteins found in cytoskeleton junctional complexes, which bind and regulate actin filaments and actin-spectrin complexes. In brain, Adducin is expressed at high levels and is identified as a constituent of synaptic structures, such as dendritic spines and growth cones of neurons. Adducin-induced changes in dendritic spines are involved in activity-dependent synaptic plasticity processes associated with learning and memory, but the mechanisms underlying these functions remain to be elucidated. Here, β-Adducin knockout (KO) mice were used to obtain a deeper insight into the role of Adducin in these processes. We showed that β-Adducin KO mice showed behavioral, motor coordination and learning deficits together with an altered expression and/or phosphorylation levels of α-Adducin and γ-Adducin. We found that β-Adducin KO mice exhibited deficits in learning and motor performances associated with an impairment of long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. These effects were accompanied by a decrease in phosphorylation of Adducin, a reduction in α-Adducin expression levels and upregulation of γ-Adducin in hippocampus, cerebellum and neocortex of mutant mice. In addition, we found that the mRNA encoding β-Adducin is also located in dendrites, where it may participate in the fine modulation of LTP and LTD. These results strongly suggest coordinated expression and phosphorylation of Adducin subunits as a key mechanism underlying synaptic plasticity, motor coordination performance and learning behaviors.
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The erythrocyte skeletons of beta-Adducin deficient mice have altered levels of tropomyosin, tropomodulin and EcapZ.
FEBS Letters, 2004Co-Authors: Fabiola Porro, Luisa Costessi, Martín L Marro, Francisco E Baralle, Andrés F MuroAbstract:The erythrocyte membrane cytoskeleton is organized as a polygonal spectrin network linked to short actin filaments that are capped by Adducin at the barbed ends. We have constructed a mouse strain deficient in beta-Adducin having abnormal erythrocytes. We show here that the levels of several skeletal proteins from beta-Adducin mutant erythrocytes are altered. In fact, CapZ, the main muscle actin-capping protein of the barbed ends that in the erythrocytes is cytoplasmic, is 9-fold upregulated in mutant skeletons of erythrocytes suggesting a compensatory mechanism. We also detected upregulation of tropomodulin and downregulation of alpha-tropomyosin and actin. In addition, purified Adducin can be re-incorporated into Adducin-deficient ghosts.
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The erythrocyte skeletons of β-Adducin deficient mice have altered levels of tropomyosin, tropomodulin and EcapZ
FEBS Letters, 2004Co-Authors: Fabiola Porro, Luisa Costessi, Martín L Marro, Francisco E Baralle, Andrés F MuroAbstract:The erythrocyte membrane cytoskeleton is organized as a polygonal spectrin network linked to short actin filaments that are capped by Adducin at the barbed ends. We have constructed a mouse strain deficient in β-Adducin having abnormal erythrocytes. We show here that the levels of several skeletal proteins from β-Adducin mutant erythrocytes are altered. In fact, CapZ, the main muscle actin-capping protein of the barbed ends that in the erythrocytes is cytoplasmic, is 9-fold upregulated in mutant skeletons of erythrocytes suggesting a compensatory mechanism. We also detected upregulation of tropomodulin and downregulation of α-tropomyosin and actin. In addition, purified Adducin can be re-incorporated into Adducin-deficient ghosts.
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Hypertension-Linked Decrease in the Expression of Brain γ-Adducin
Circulation research, 2002Co-Authors: Hong Yang, Andrés F Muro, Sharon C. Francis, Mia Debarros, Chengwen Sun, Colin Sumners, Carlos M. Ferrario, Michael J. Katovich, Kathleen W. Sellers, Mohan K. RaizadaAbstract:Gene profiling data coupled with Adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which Adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of Adducins in neurotransmission at the cellular level. All three Adducin subunits (α, β, and γ) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the γ-Adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in γ-Adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize γ-Adducin gene expression. A hypertension-linked decrease of γ-Adducin was confirmed by demonstrating a decrease in γ-Adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a γ-Adducin–specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous γ-Adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in γ-Adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters.
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Mild spherocytic hereditary elliptocytosis and altered levels of α- and γ-Adducins in β-Adducin-deficient mice
Blood, 2000Co-Authors: Andrés F Muro, Fabiola Porro, Martín L Marro, Srećko Gajović, Lucio Luzzatto, Francisco E BaralleAbstract:The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of β-Adducin produces an 80% decrease of -Adducin and a fourfold up-regulation of γ-Adducin in erythrocytes. β-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of −/− erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of Adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of −/− mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of Adducin remaining in the mutant animals (presumably γ Adducin) could be functional and might account for the mild phenotype.
Giuseppe Bianchi - One of the best experts on this subject based on the ideXlab platform.
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Rostafuroxin Protects from Podocyte Injury and Proteinuria Induced by Adducin Genetic Variants and Ouabain
The Journal of pharmacology and experimental therapeutics, 2014Co-Authors: Mara Ferrandi, Giuseppe Bianchi, Isabella Molinari, Maria Pia Rastaldi, Patrizia Ferrari, Paolo ManuntaAbstract:Glomerulopathies are important causes of morbidity and mortality. Selective therapies that address the underlying mechanisms are still lacking. Recently, two mechanisms, mutant β-Adducin and ouabain, have been found to be involved in glomerular podocytopathies and proteinuria through nephrin downregulation. The main purpose of the present study was to investigate whether rostafuroxin, a novel antihypertensive agent developed as a selective inhibitor of Src-SH2 interaction with mutant Adducin- and ouabain-activated Na,K-ATPase, may protect podocytes from Adducin- and ouabain-induced effects, thus representing a novel pharmacologic approach for the therapy of podocytopathies and proteinuria caused by the aforementioned mechanisms. To study the effect of rostafuroxin on podocyte protein changes and proteinuria, mice carrying mutant β-Adducin and ouabain hypertensive rats were orally treated with 100 μg/kg per day rostafuroxin. Primary podocytes from congenic rats carrying mutant α-Adducin or β-Adducin (NB) from Milan hypertensive rats and normal rat podocytes incubated with 10(-9) M ouabain were cultured with 10(-9) M rostafuroxin. The results indicated that mutant β-Adducin and ouabain caused podocyte nephrin loss and proteinuria in animal models. These alterations were reproduced in primary podocytes from NB rats and normal rats incubated with ouabain. Treatment of animals, or incubation of cultured podocytes with rostafuroxin, reverted mutant β-Adducin- and ouabain-induced effects on nephrin protein expression and proteinuria. We conclude that rostafuroxin prevented podocyte lesions and proteinuria due to mutant β-Adducin and ouabain in animal models. This suggests a potential therapeutic effect of rostafuroxin in patients with glomerular disease progression associated with these two mechanisms.
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Role of rat α Adducin in angiogenesis: Null effect of the F316Y polymorphism
Cardiovascular research, 2007Co-Authors: Claudia Cappuzzello, Giuseppe Bianchi, Grazia Tripodi, Lucia Torielli, Patrizia Ferrari, Roberta Melchionna, Antonella Mangoni, Diego Arcelli, Mauro Helmer-citterich, Maurizio C. CapogrossiAbstract:Objective Rat α Adducin point mutation (F316Y) has been associated with primary systemic arterial hypertension. As microcirculatory abnormalities are present in most forms of hypertension, the aim of the present study was to investigate whether rat α Adducin may regulate endothelial cell (EC) functions in vitro and in vivo . Methods and results The overexpression of rat wild type α Adducin (WT-Add1) in ECs induced capillary-like structure development in Matrigel in vitro and enhanced capillary formation in Matrigel implants in vivo in CD1 mice. In contrast, the overexpression of the mutated form (MUT-Add1) of rat α Adducin had a Null effect in vitro and lacked any significant activity in vivo . Further, adenovirus-mediated rat WT-Add1 but not MUT-Add1 gene transfer to murine ischemic hindlimb enhanced capillary formation in skeletal muscles. Gene profiling of human umbilical vein endothelial cells overexpressing α Adducin was performed in order to identify putative effector molecules of α Adducin-mediated activities on ECs. Interestingly, among a number of genes involved in angiogenesis regulation, retinoic acid-induced protein (RAI17) was found to be upregulated in WT-Add1 vs MUT-Add1 overexpressing cells, possibly representing a key molecule/axis for the functional Add1-induced effect. Conclusions Rat WT α Adducin enhanced EC functions both in vitro and in vivo. The expression of the F316Y variant, associated with the hypertensive phenotype, had a Null effect and might contribute to endothelial rarefaction/dysfunction in hypertension. RAI17 was found to be a putative effector molecule differentially regulated by the overexpression of the two forms of Add1 in endothelial cells.
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Pharmacogenomics and Pharmacogenetics of Hypertension: Update and Perspectives—The Adducin Paradigm
Journal of the American Society of Nephrology : JASN, 2006Co-Authors: Paolo Manunta, Giuseppe BianchiAbstract:There is a growing literature on the potential prospective use of genome information to enhance success in finding new medicines. An example of a prospective efficacy of pharmacogenetic and pharmacogenomics is the detection and impact of Adducin polymorphism on hypertension. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2, and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered Adducin function might cause hypertension through an enhanced constitutive tubular sodium reabsorption. In particular, six linkage studies, 18 of 20 association studies, and four of five follow-up studies that measured organ damage in hypertensive patients support the clinical impact of Adducing polymorphism. As many modulatory genes and environment affect the Adducin activity, the context must be taken into account to measure the clinical effect size of Adducins. Pharmacogenomics is giving an important contribution to this end. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar BP reduction in carriers of the mutated Adducin may support new strategies that aim to optimize the use of antihypertensive agents for the prevention of hypertension-associated organ damage.
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Genetics of hypertension: the Adducin paradigm.
Annals of the New York Academy of Sciences, 2003Co-Authors: Giuseppe Bianchi, Grazia TripodiAbstract:: The following were investigated: (1) how we became interested in studying Adducin genes and what we know about Adducin; (2) studies in animals and humans supporting the role of Adducin polymorphisms in hypertension, including some methodological problems related to the dissection of the role of a given genetic molecular mechanism in a complex multifactorial polygenic disease like hypertension; (3) biochemical mechanisms underlying the effect of Adducin and its interaction with the Na-K pump; and (4) future directions.
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Association between hypertension and variation in the α- and β-Adducin genes in a white population
Kidney international, 2002Co-Authors: Ji-guang Wang, Cristina Barlassina, Jan A. Staessen, Lorena Citterio, Tatiana Kuznetsova, Robert Fagard, Harry A.j. Struijker-boudier, Laura Zagato, Elisabetta Messaggio, Giuseppe BianchiAbstract:Association between hypertension and variation in the α- and β-Adducin genes in a white population. Background The substitution of tryptophan for glycine at amino acid 460 (Gly460Trp polymorphism) of the α-subunit of the heterodimeric cytoskeleton protein Adducin increases renal sodium reabsorption and may be involved in the pathophysiology of essential hypertension. In the present study, we investigated in multivariate analyses whether the risk of hypertension was associated with the C1797T polymorphism of the β-Adducin gene. Methods A total of 1848 subjects randomly selected from a white population were genotyped. Study nurses measured blood pressure at the participants' homes. Results The frequencies of the α-Adducin Trp and β-Adducin T alleles were 0.23 and 0.11, respectively. In men ( N = 904), the β-Adducin T allele was not associated with hypertension [adjusted relative risk (RR) vs. CC homozygotes 0.94, P = 0.77], but T allele carriers had lower plasma renin activity (PRA) and 24-hour urinary aldosterone excretion ( P N = 944), β-Adducin T allele carriers had a higher risk of hypertension than CC homozygotes (RR 1.81, CI 1.18–2.77, P = 0.007), but similar PRA and 24-hour urinary aldosterone excretion ( P > 0.29). In 345 post-menopausal women and 190 users of oral contraceptives, the RRs of hypertension were 2.47 (CI 1.34–4.64, P = 0.003) and 2.56 (CI 0.83–7.86, P = 0.10), respectively. For systolic pressure in women, there was a significant interaction ( P = 0.02) between the α- and β-Adducin polymorphisms. Only in female carriers of the mutated α-Adducin Trp allele was the systolic pressure significantly higher in β-Adducin T allele carriers compared with CC homozygotes (+3.8mm Hg, P = 0.02). Furthermore, in the presence of the mutated α-Adducin Trp allele, the RRs associated with the β-Adducin T allele were 2.35 ( P = 0.01) in all women, 2.92 ( P = 0.03) in post-menopausal subjects, and 3.79 ( P = 0.09) in users of oral contraceptives. Conclusions The 1797T allele of the β-Adducin gene is associated with increased risk of hypertension in post-menopausal women and in users of oral contraceptives, particularly in the presence of the mutated α-Adducin Trp allele. We hypothesize that inhibition of the renin-aldosterone system in men and absence of such a compensatory mechanism in women may explain, at least to some extent, the sexual dimorphism of the blood pressure phenotype in relation to the C1797T β-Adducin polymorphism.
