Activity of recombinant alpha and beta subunits of casein kinase II from Xenopus laevis.
Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Hinrichs
M V
MV
Departamento de Bioquímica, Facultad de Medicina, Universidad de Chile, Santiago.
Jedlicki
A
A
Tellez
R
R
Pongor
S
S
Gatica
M
M
Allende
C C
CC
Allende
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047390:Casein Kinase II; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014982:Xenopus laevis
D005971:Glutamates; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D004247:DNA; D005982:Glutathione Transferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases