Protein kinase CK2 structure-function relationship

Effects of the beta subunit on reconstitution and activity

B Boldyreff, F Meggio, L A Pinna, O G Issinger

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 1994-null
OriginalsprogEngelsk
TidsskriftCellular & molecular biology research
Vol/bind40
Udgave nummer5-6
Sider (fra-til)391-399
Antal sider8
ISSN0968-8773
StatusUdgivet - 1. jan. 1994

Fingeraftryk

Casein Kinase II
Holoenzymes
Acidic Amino Acids
Salts
Enzymes
Amino Acids
Mutagenesis
Enzyme activity
Site-Directed Mutagenesis
Alanine
Urea
Catalyst activity
Catalytic Domain
Peptide Hydrolases
Heat treatment
Mutation
Substrates

Citer dette

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title = "Protein kinase CK2 structure-function relationship: Effects of the beta subunit on reconstitution and activity",
abstract = "Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features as the native enzyme. The alpha subunit alone, although catalytically active by itself, has different biochemical and biophysical properties than the holoenzyme, e.g., it is extremely salt sensitive, already 50 mM monovalent salt can lead to a 50{\%} inhibition of the catalytic activity. Furthermore, it is readily inactivated through urea, protease, and heat treatment. In contrast, the holoenzyme, either reconstituted or native, is much more stable when similar negative insults prevail. The beta subunit has at least three functions: (a) it is necessary for maximum activity of the enzyme under physiological salt conditions, (b) it protects the alpha subunit against denaturing agents or conditions, and (c) it alters the substrate specificity of the alpha subunit. By site-directed mutagenesis, certain functions of the beta subunit could be assigned to specific amino acids or domains. Twenty one mutants of the beta subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations of certain acidic amino acids within this stretch to alanine lead to a hyperactive CK2 holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)",
keywords = "Amino Acid Sequence, Casein Kinase II, Escherichia coli, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptides, Phosphorylation, Protein Conformation, Protein Multimerization, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Sequence Deletion, Structure-Activity Relationship",
author = "B Boldyreff and F Meggio and Pinna, {L A} and Issinger, {O G}",
year = "1994",
month = "1",
day = "1",
language = "English",
volume = "40",
pages = "391--399",
journal = "Cellular & molecular biology research",
issn = "0968-8773",
publisher = "Springer",
number = "5-6",

}

Protein kinase CK2 structure-function relationship : Effects of the beta subunit on reconstitution and activity. / Boldyreff, B; Meggio, F; Pinna, L A; Issinger, O G.

I: Cellular & molecular biology research, Bind 40, Nr. 5-6, 01.01.1994, s. 391-399.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Protein kinase CK2 structure-function relationship

T2 - Effects of the beta subunit on reconstitution and activity

AU - Boldyreff, B

AU - Meggio, F

AU - Pinna, L A

AU - Issinger, O G

PY - 1994/1/1

Y1 - 1994/1/1

N2 - Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features as the native enzyme. The alpha subunit alone, although catalytically active by itself, has different biochemical and biophysical properties than the holoenzyme, e.g., it is extremely salt sensitive, already 50 mM monovalent salt can lead to a 50% inhibition of the catalytic activity. Furthermore, it is readily inactivated through urea, protease, and heat treatment. In contrast, the holoenzyme, either reconstituted or native, is much more stable when similar negative insults prevail. The beta subunit has at least three functions: (a) it is necessary for maximum activity of the enzyme under physiological salt conditions, (b) it protects the alpha subunit against denaturing agents or conditions, and (c) it alters the substrate specificity of the alpha subunit. By site-directed mutagenesis, certain functions of the beta subunit could be assigned to specific amino acids or domains. Twenty one mutants of the beta subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations of certain acidic amino acids within this stretch to alanine lead to a hyperactive CK2 holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

AB - Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features as the native enzyme. The alpha subunit alone, although catalytically active by itself, has different biochemical and biophysical properties than the holoenzyme, e.g., it is extremely salt sensitive, already 50 mM monovalent salt can lead to a 50% inhibition of the catalytic activity. Furthermore, it is readily inactivated through urea, protease, and heat treatment. In contrast, the holoenzyme, either reconstituted or native, is much more stable when similar negative insults prevail. The beta subunit has at least three functions: (a) it is necessary for maximum activity of the enzyme under physiological salt conditions, (b) it protects the alpha subunit against denaturing agents or conditions, and (c) it alters the substrate specificity of the alpha subunit. By site-directed mutagenesis, certain functions of the beta subunit could be assigned to specific amino acids or domains. Twenty one mutants of the beta subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations of certain acidic amino acids within this stretch to alanine lead to a hyperactive CK2 holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

KW - Amino Acid Sequence

KW - Casein Kinase II

KW - Escherichia coli

KW - Humans

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Peptides

KW - Phosphorylation

KW - Protein Conformation

KW - Protein Multimerization

KW - Protein Processing, Post-Translational

KW - Protein-Serine-Threonine Kinases

KW - Recombinant Fusion Proteins

KW - Sequence Deletion

KW - Structure-Activity Relationship

M3 - Journal article

VL - 40

SP - 391

EP - 399

JO - Cellular & molecular biology research

JF - Cellular & molecular biology research

SN - 0968-8773

IS - 5-6

ER -