Regulation of protein phosphatase‐1G from rabbit/skeletal muscle

1. Phosphorylation by cAMP‐dependent protein kinase at site 2 releases catalytic subunit from the glycogen‐bound holoenzyme

Michael J. Hubbard, Philip Cohen

    Research output: Contribution to journalArticle

    98 Citations (Scopus)

    Abstract

    The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is: (Formula Presented.) At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1G. At near physiological ionic strength, PP‐1G and glycogen concentration, site 2 was autodephosphorylated by PP‐1G with a t0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.

    Original languageEnglish
    Pages (from-to)701-709
    Number of pages9
    JournalEuropean Journal of Biochemistry
    Volume186
    Issue number3
    DOIs
    Publication statusPublished - Dec 1989

    Fingerprint

    Holoenzymes
    Phosphorylation
    Phosphoric Monoester Hydrolases
    Protein Kinases
    Muscle
    Catalytic Domain
    Skeletal Muscle
    Glycogen
    Rabbits
    Ionic strength
    Osmolar Concentration
    Proteins
    Phosphorylase Kinase
    Phosphoserine
    Glycogen Phosphorylase
    Immunoglobulin Fab Fragments
    Calcineurin
    Phosphoprotein Phosphatases
    Substrates
    Serine

    Cite this

    @article{1ff4e1d894d74d1e9662f308538f2053,
    title = "Regulation of protein phosphatase‐1G from rabbit/skeletal muscle: 1. Phosphorylation by cAMP‐dependent protein kinase at site 2 releases catalytic subunit from the glycogen‐bound holoenzyme",
    abstract = "The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is: (Formula Presented.) At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1G. At near physiological ionic strength, PP‐1G and glycogen concentration, site 2 was autodephosphorylated by PP‐1G with a t0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.",
    author = "Hubbard, {Michael J.} and Philip Cohen",
    year = "1989",
    month = "12",
    doi = "10.1111/j.1432-1033.1989.tb15263.x",
    language = "English",
    volume = "186",
    pages = "701--709",
    journal = "European Journal of Biochemistry",
    issn = "0014-2956",
    publisher = "Wiley",
    number = "3",

    }

    TY - JOUR

    T1 - Regulation of protein phosphatase‐1G from rabbit/skeletal muscle

    T2 - 1. Phosphorylation by cAMP‐dependent protein kinase at site 2 releases catalytic subunit from the glycogen‐bound holoenzyme

    AU - Hubbard, Michael J.

    AU - Cohen, Philip

    PY - 1989/12

    Y1 - 1989/12

    N2 - The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is: (Formula Presented.) At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1G. At near physiological ionic strength, PP‐1G and glycogen concentration, site 2 was autodephosphorylated by PP‐1G with a t0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.

    AB - The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is: (Formula Presented.) At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1G. At near physiological ionic strength, PP‐1G and glycogen concentration, site 2 was autodephosphorylated by PP‐1G with a t0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.

    UR - http://www.scopus.com/inward/record.url?scp=0024853130&partnerID=8YFLogxK

    U2 - 10.1111/j.1432-1033.1989.tb15263.x

    DO - 10.1111/j.1432-1033.1989.tb15263.x

    M3 - Article

    VL - 186

    SP - 701

    EP - 709

    JO - European Journal of Biochemistry

    JF - European Journal of Biochemistry

    SN - 0014-2956

    IS - 3

    ER -