Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Abstract

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase H have been isolated by immunological screening of Agtll expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinases suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.Casein kinase II is a cyclic nucleotide-independent, Ca2l-and calmodulin-insensitive protein kinase which is widely distributed among eucaryotic organisms (15). The enzyme phosphorylates a broad spectrum of both nuclear and nonnuclear substrates (1,5,11,15), suggesting that it may function in the regulation or integration of cell metabolism. Casein kinase II from most sources is composed of two dissimilar subunits, alpha (35 to 44 kilodaltons [kDa]) and beta (24 to 29 kDa), which combine to form a native 02f2 tetramer with a molecular weight of 130,000 to 150,000. The enzyme can utilize either ATP or GTP as the nucleoside triphosphate donor and phosphorylates serine or threonine residues in protein substrates. A cluster of acidic residues located immediately C-terminal to the modified residue appears to be important for substrate recognition (15, 25). The alpha polypeptide has been identified as the catalytic subunit by three independent methods (6,17,26). The beta subunit becomes phosphorylated when the enzyme is allowed to undergo autophosphorylation, but the function of this subunit and the significance of autophosphorylation are not known.In an effort to initiate a genetic analysis of casein kinase II, we previously isolated and characterized the enzyme from Drosophila melanogaster (14). The purified kinase is composed of a 37-kDa alpha and 28-kDa beta subunit which form a 130,000-dalton a232 tetramer. Like its counterpart in other species, the enzyme phosphorylates both serine and threonine residues, utilizes either ATP or GTP, exhibits autophosphorylation of the beta subunit, and is strongly inhibited by heparin, a characteristic feature of casein kinase II (15). In addition, the Drosophila enzyme displays a protein substrate specificity which is virtually indistinguishable from that exhibited by the enzyme from calf thymus (7). Peptide mapping and immunological studies demonstrate that the insect and mammalian enzymes are homologous and suggest that casein kinase II has been ...

Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IκB homologue cactus

“…The molecular mass of the catalytic a subunit of CKII is 37 kDa [20] and thus it is likely that CKII is being detected in this assay. In contrast to identical experiments where dorsal was used as substrate, no inducible protein kinase was detected.…”

“…Cactus is not phosphorylated by any kinases induced in SL2T110B cells but is modified by a protein kinase with a molecular mass of about 37 kDa which is present in all the extracts tested. The cactus sequence contains several consensus sites for CKII and the catalytic subunit of this enzyme has a mass of 37 kDa [16,20]. Thus it is likely that the phosphorylation of cactus detected by this assay is mediated by CKII.…”

Purification and characterization of echinoderm casein kinase II. Regulation by protein kinase C

“…3), as has been noted for human CKII (Pyerin et al, 1986;Litchfield et al, 1991). Analysis of the predicted primary structure of the fl-subunits of CKII from human (Jakobi et al, 1989), chicken (Maridor et al, 1991) and D. melanogaster (Saxena et al, 1987) sources reveals only two serine residues that lie within a predicted optimal phosphorylation-site consensus sequence for recognition for CKII. These are Ser-2 and Ser-3, which are found to be completely conserved where the fl-subunit amino acid sequence has been deduced.…”

Immunocytochemical localization of casein kinase II during interphase and mitosis.

“…Our results indicate that the CKII a and ß subunits are localized in the cytoplasm C ASEIN kinase II (CKII)' is a ubiquitous protein serlne/threonine kinase found in eukaryotic cells (Edelman et al . 1987 and highly conserved among eukaryotic organisms, including Drosophila, yeast, C. elegans, bovine, and human (Saxena et al ., 1987;Chen-Wu et al ., 1988;Hu and Rubin, 1990a ;Lozeman, 1990). Casein kinase II from several species share a common polypeptide subunit structure, a2ß2, with a of M 37,000-44,000 and ß of M 24,000-28,000 by electrophoresis (Edelman et al ., 1987) .…”