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William A. Catterall - One of the best experts on this subject based on the ideXlab platform.
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Identification of the sites of selective Phosphorylation and dePhosphorylation of the rat brain Na+ channel alpha subunit by cAMP-dependent protein kinase and phosphoprotein phosphatases.
The Journal of biological chemistry, 1993Co-Authors: Brian J. Murphy, Sandra Rossie, K S De Jongh, William A. CatterallAbstract:Abstract Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent Phosphorylation results from selective Phosphorylation and dePhosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.
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identification of the sites of selective Phosphorylation and dePhosphorylation of the rat brain na channel alpha subunit by camp dependent protein kinase and phosphoprotein phosphatases
Journal of Biological Chemistry, 1993Co-Authors: Brian J. Murphy, Sandra Rossie, K S De Jongh, William A. CatterallAbstract:Abstract Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent Phosphorylation results from selective Phosphorylation and dePhosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.
Toshinori Kinoshita - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of in vitro Phosphorylation and dePhosphorylation of the plasma membrane h atpase
Plant and Cell Physiology, 2010Co-Authors: Yuki Hayashi, Suguru Nakamura, Atsushi Takemiya, Yohei Takahashi, Ken-ichiro Shimazaki, Toshinori KinoshitaAbstract:Stomatal opening, which is mediated by blue light receptor phototropins, is driven by activation of the plasma membrane H + -ATPase via Phosphorylation of the penultimate threonine in the C-terminus and subsequent binding of a 14-3-3 protein. However, the biochemical properties of the protein kinase and protein phosphatase for H + -ATPase are largely unknown. We therefore investigated in vitro Phosphorylation and dePhosphorylation of H + -ATPase. H + -ATPase was phosphorylated in vitro on the penultimate threonine in the C-terminus in isolated microsomes from guard cell protoplasts of Vicia faba. Phosphorylated H + -ATPase was dephosphorylated in vitro, and the dePhosphorylation was inhibited by EDTA, a divalent cation chelator, but not by calyculin A, an inhibitor of type 1 and 2A protein phosphatases. Essentially the same results were obtained in purified plasma membranes from etiolated Arabidopsis seedlings, indicating that a similar protein kinase and phosphatase are involved in plant cells. Further analyses revealed that Phosphorylation of the H + -ATPase is insensitive to K-252a, a potent inhibitor of protein kinase, and is hypersensitive to Triton X-100, a non-ionic detergent. Moreover, dePhosphorylation required Mg 2+ but not Ca 2+ , and protein phosphatase was localized in the 1% Triton X-100-insoluble fraction. These results demonstrate that a protein kinase-phosphatase pair, K-252a-insensitive protein kinase and Mg 2+ -dependent type 2C protein phosphatase, co-localizes at least in part with the H + -ATPase in the plasma membrane and regulates the Phosphorylation status of the penultimate threonine of the H + -ATPase.
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Biochemical Characterization of In Vitro Phosphorylation and DePhosphorylation of the Plasma Membrane H⁺-ATPase
Plant and Cell Physiology, 2010Co-Authors: Yuki Hayashi, Suguru Nakamura, Atsushi Takemiya, Yohei Takahashi, Ken-ichiro Shimazaki, Toshinori KinoshitaAbstract:Stomatal opening, which is mediated by blue light receptor phototropins, is driven by activation of the plasma membrane H + -ATPase via Phosphorylation of the penultimate threonine in the C-terminus and subsequent binding of a 14-3-3 protein. However, the biochemical properties of the protein kinase and protein phosphatase for H + -ATPase are largely unknown. We therefore investigated in vitro Phosphorylation and dePhosphorylation of H + -ATPase. H + -ATPase was phosphorylated in vitro on the penultimate threonine in the C-terminus in isolated microsomes from guard cell protoplasts of Vicia faba. Phosphorylated H + -ATPase was dephosphorylated in vitro, and the dePhosphorylation was inhibited by EDTA, a divalent cation chelator, but not by calyculin A, an inhibitor of type 1 and 2A protein phosphatases. Essentially the same results were obtained in purified plasma membranes from etiolated Arabidopsis seedlings, indicating that a similar protein kinase and phosphatase are involved in plant cells. Further analyses revealed that Phosphorylation of the H + -ATPase is insensitive to K-252a, a potent inhibitor of protein kinase, and is hypersensitive to Triton X-100, a non-ionic detergent. Moreover, dePhosphorylation required Mg 2+ but not Ca 2+ , and protein phosphatase was localized in the 1% Triton X-100-insoluble fraction. These results demonstrate that a protein kinase-phosphatase pair, K-252a-insensitive protein kinase and Mg 2+ -dependent type 2C protein phosphatase, co-localizes at least in part with the H + -ATPase in the plasma membrane and regulates the Phosphorylation status of the penultimate threonine of the H + -ATPase.
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Phosphorylation and DePhosphorylation of Guard-Cell Proteins from Vicia faba L. in Response to Light and Dark.
Plant Physiology, 1993Co-Authors: Toshinori Kinoshita, Ken-ichiro Shimazaki, Mitsuo NishimuraAbstract:Phosphorylation and dePhosphorylation of proteins were investigated in guard-cell protoplasts from Vicia faba L. When guard-cell protoplasts were incubated with 32Pi in the dark for 80 min, several proteins, with molecular masses of 42, 40, 34, 32, 26, and 19 kD, were phosphorylated. Illumination of the dark-adapted protoplasts with red light caused dePhosphorylation of the 26-kD protein, but there was no detectable change in levels of Phosphorylation in other proteins. In the dePhosphorylation of the 26-kD protein, far-red light of 730 nm was most effective, but when the light was turned off, the protein was phosphorylated to the original level within 10 min. Subcellular fractionation of guard-cell protoplasts indicated that the 26-kD protein was located in the chloroplast. The migration pattern of the 26-kD protein was exactly the same as the light-harvesting Chl a/b protein complex of photosystem II (LHCPII) from Vicia mesophyll cells on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dephosphorylated 26-kD protein was phosphorylated by adding sodium hydrosulfite, a strong reducing agent, under the far-red illumination of guard-cell protoplasts. The magnitude of dePhosphorylation by red light (660 nm) was increased by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an electron transfer inhibitor of photosystem II (PSII). Light-induced dePhosphorylation was inhibited by 1 nM okadaic acid, an inhibitor of serine/threonine protein phosphatase. From these results, it is concluded that the 26-kD protein is LHCPII and that LHCPII is present mostly in the phosphorylated form in the dark and is dephosphorylated by type 2A protein phosphatase under the light absorbed by photosystem I in Vicia guard-cell protoplasts.
Brian J. Murphy - One of the best experts on this subject based on the ideXlab platform.
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Identification of the sites of selective Phosphorylation and dePhosphorylation of the rat brain Na+ channel alpha subunit by cAMP-dependent protein kinase and phosphoprotein phosphatases.
The Journal of biological chemistry, 1993Co-Authors: Brian J. Murphy, Sandra Rossie, K S De Jongh, William A. CatterallAbstract:Abstract Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent Phosphorylation results from selective Phosphorylation and dePhosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.
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identification of the sites of selective Phosphorylation and dePhosphorylation of the rat brain na channel alpha subunit by camp dependent protein kinase and phosphoprotein phosphatases
Journal of Biological Chemistry, 1993Co-Authors: Brian J. Murphy, Sandra Rossie, K S De Jongh, William A. CatterallAbstract:Abstract Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent Phosphorylation results from selective Phosphorylation and dePhosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.
Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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rhodopsin Phosphorylation and dePhosphorylation in vivo
Journal of Biological Chemistry, 1995Co-Authors: Hiroshi Ohguro, Preston J Van Hooser, Ann H Milam, Krzysztof PalczewskiAbstract:Abstract Rhodopsin is an important member of the superfamily of G protein-coupled receptors. In vitro studies have suggested that multiPhosphorylation of rhodopsin is a pivotal step in phototransduction. Because the in vitro biochemical experiments were conducted under non-physiological conditions, we investigated the Phosphorylation of mouse rhodopsin in vivo and determined the sites of Phosphorylation and the time course of dePhosphorylation. We found that a single phosphate group is incorporated into the rhodopsin molecule in a light-dependent manner, primarily at Ser after flashes and at Ser after continuous illumination. DePhosphorylation of these sites had different kinetics and spatial distribution in rod outer segments. DePhosphorylation of Ser was complete within 30 min, while Ser was dephosphorylated much slower (requiring up to 60 min), correlating with the regeneration of rhodopsin. These results suggest that Phosphorylation of Ser and Ser plays different roles in phototransduction.
Sandra Rossie - One of the best experts on this subject based on the ideXlab platform.
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Identification of the sites of selective Phosphorylation and dePhosphorylation of the rat brain Na+ channel alpha subunit by cAMP-dependent protein kinase and phosphoprotein phosphatases.
The Journal of biological chemistry, 1993Co-Authors: Brian J. Murphy, Sandra Rossie, K S De Jongh, William A. CatterallAbstract:Abstract Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent Phosphorylation results from selective Phosphorylation and dePhosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.
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identification of the sites of selective Phosphorylation and dePhosphorylation of the rat brain na channel alpha subunit by camp dependent protein kinase and phosphoprotein phosphatases
Journal of Biological Chemistry, 1993Co-Authors: Brian J. Murphy, Sandra Rossie, K S De Jongh, William A. CatterallAbstract:Abstract Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent Phosphorylation results from selective Phosphorylation and dePhosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.