Casein kinase-2 structure-function relationship

Creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function

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

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.
Original languageEnglish
JournalBiochemical and Biophysical Research Communications
Volume188
Issue number1
Pages (from-to)228-234
Number of pages6
ISSN0006-291X
Publication statusPublished - 15. Oct 1992

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Casein Kinase II
Holoenzymes
Catalyst activity
Phosphorylation
Sedimentation
Alanine
Sucrose
Catalytic Domain
Sediments
Substitution reactions

Keywords

  • Amino Acid Sequence
  • Casein Kinases
  • Cloning, Molecular
  • Escherichia coli
  • Humans
  • Macromolecular Substances
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Polymerase Chain Reaction
  • Protein Kinases
  • Recombinant Proteins
  • Restriction Mapping
  • Sequence Deletion

Cite this

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title = "Casein kinase-2 structure-function relationship: Creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function",
abstract = "Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.",
keywords = "Amino Acid Sequence, Casein Kinases, Cloning, Molecular, Escherichia coli, Humans, Macromolecular Substances, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Protein Kinases, Recombinant Proteins, Restriction Mapping, Sequence Deletion",
author = "B Boldyreff and F Meggio and Pinna, {L A} and Issinger, {O G}",
year = "1992",
month = "10",
day = "15",
language = "English",
volume = "188",
pages = "228--234",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Elsevier",
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}

Casein kinase-2 structure-function relationship : Creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function. / Boldyreff, B; Meggio, F; Pinna, L A; Issinger, O G.

In: Biochemical and Biophysical Research Communications, Vol. 188, No. 1, 15.10.1992, p. 228-234.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Casein kinase-2 structure-function relationship

T2 - Creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function

AU - Boldyreff, B

AU - Meggio, F

AU - Pinna, L A

AU - Issinger, O G

PY - 1992/10/15

Y1 - 1992/10/15

N2 - Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.

AB - Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.

KW - Amino Acid Sequence

KW - Casein Kinases

KW - Cloning, Molecular

KW - Escherichia coli

KW - Humans

KW - Macromolecular Substances

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Polymerase Chain Reaction

KW - Protein Kinases

KW - Recombinant Proteins

KW - Restriction Mapping

KW - Sequence Deletion

M3 - Journal article

VL - 188

SP - 228

EP - 234

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -