Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II.
Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Filhol
O
O
INSERM U244, DBMS/LBIO/BRCE, CENG, Grenoble, France.
Cochet
C
C
Wedegaertner
P
P
Gill
G N
GN
Chambaz
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D047388:Casein Kinases; D004330:Drosophila / Q000201:enzymology; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology; D011073:Polyamines / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011073:Polyamines; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases