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Adducin

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Vann Bennett – One of the best experts on this subject based on the ideXlab platform.

  • Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells
    Molecular biology of the cell, 2007
    Co-Authors: Khadar M. Abdi, Vann Bennett

    Abstract:

    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.

  • Association of protein kinase Cλ with Adducin in 3T3-L1 adipocytes
    Biochimica et Biophysica Acta, 2001
    Co-Authors: Palle G. Laustsen, William S. Lane, Vann Bennett, Gustav E. Lienhard

    Abstract:

    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.

  • Association of protein kinase C(lambda) with Adducin in 3T3-L1 adipocytes.
    Biochimica et biophysica acta, 2001
    Co-Authors: Palle G. Laustsen, William S. Lane, Vann Bennett, Gustav E. Lienhard

    Abstract:

    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.

Yoichiro Matsuoka – One of the best experts on this subject based on the ideXlab platform.

  • Rho-kinase induces association of Adducin with the cytoskeleton in platelet activation.
    Biochemical and biophysical research communications, 2005
    Co-Authors: Satoshi Tamaru, Yoichiro Matsuoka, Tetsu Fukuta, Kozo Kaibuchi, Hiroshi Shiku, Masakatsu Nishikawa

    Abstract:

    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.

  • Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
    The Journal of cell biology, 1999
    Co-Authors: Yuko Fukata, Vann Bennett, Yoichiro Matsuoka, Yoshiharu Matsuura, Noriko Oshiro, Nagatoki Kinoshita, Yoji Kawano, Kozo Kaibuchi

    Abstract:

    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.

  • 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, 1998
    Co-Authors: Yoichiro Matsuoka, Xiaolin Li, Vann Bennett

    Abstract:

    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.

Kozo Kaibuchi – One of the best experts on this subject based on the ideXlab platform.

  • Rho-kinase induces association of Adducin with the cytoskeleton in platelet activation.
    Biochemical and biophysical research communications, 2005
    Co-Authors: Satoshi Tamaru, Yoichiro Matsuoka, Tetsu Fukuta, Kozo Kaibuchi, Hiroshi Shiku, Masakatsu Nishikawa

    Abstract:

    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.

  • Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
    The Journal of cell biology, 1999
    Co-Authors: Yuko Fukata, Vann Bennett, Yoichiro Matsuoka, Yoshiharu Matsuura, Noriko Oshiro, Nagatoki Kinoshita, Yoji Kawano, Kozo Kaibuchi

    Abstract:

    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.

  • Regulation of the Association of Adducin with Actin Filaments by Rho-associated Kinase (Rho-kinase) and Myosin Phosphatase
    The Journal of biological chemistry, 1998
    Co-Authors: Kazushi Kimura, Vann Bennett, Yoichiro Matsuoka, Yuko Fukata, Yoshiharu Matsuura, Katsuya Okawa, Akihiro Iwamatsu, Kozo Kaibuchi

    Abstract:

    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.