Casein kinase-2 structure-function relationship: creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function.
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.
Boldyreff
B
B
Institut für Humangenetik, Universität des Saarlandes, Homburg, Germany.
Meggio
F
F
Pinna
L A
LA
Issinger
O G
OG
D000595:Amino Acid Sequence; D047388:Casein Kinases; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases