Analysis of a novel DNA-binding protein kinase CKII-like enzyme of Chironomus cells.
We have previously described a Chironomus tentans nuclear 42 kDa phosphoprotein preferentially associated with transcriptionally active chromatin. In an attempt to purify and identify the kinase responsible for the phosphorylation of the 42 kDa protein, a casein-phosvitin affinity chromatography was used. Unexpectedly, in the eluted kinase fraction, a novel 42 kDa casein kinase, designated protein kinase CK42, with a kinase activity similar, but not identical, to protein kinase CKII, could be identified. In other studies, a nuclear protein that comigrates with protein kinase CK42 in electrophoresis and is capable to bind different gene promoters in single-stranded forms in a sequence-selective manner was found. The observations that both protein kinase and ssDNA-binding activities could be ascribed to a 42 kDa protein raised the possibility that the ssDNA-binding 42 kDa phosphoprotein is a protein kinase. By specific ssDNA-binding affinity chromatography, using a biotinylated oligodeoxyribonucleotide promoter probe and Streptavidine-agarose matrix, evidence that both activities arise from the same protein molecules was obtained. The similarity in the enzyme activities between protein kinase CK42 and CKII raised the question of whether the former was an alpha subunit of the latter. To provide an answer to this issue, CKII, isolated and purified from an epithelial cell line of C. tentans, was characterized and compared with protein kinase CK42 purified from the same cell system. Like other purified CKII preparations, CKII from Chironomus is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). However, the heparin and DRB sensitivities of protein kinase CKII were substantially higher than those of the protein kinase CK42. Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools of CKII could be detected. More than 80% of the nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta subunit were solubilized under the same conditions suggesting that a major portion of the nuclear CKII alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components, probably to chromatin. The biochemical and immunological data taken together strongly suggest that CK42 is a novel DNA-binding protein kinase that is not the alpha subunit of CKII.
Stigare
J
J
Karolinska Institutet, Department of Cell and Molecular Biology, Laboratory of Medical Cell Biology, Stockholm, Sweden.
Buddelmeijer
N
N
Pigon
A
A
Egyházi
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D047390:Casein Kinase II; D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D002683:Chironomidae / Q000196:embryology / Q000201:enzymology*; D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D010766:Phosphorylation; D010774:Phosvitin / Q000378:metabolism; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification
D002364:Caseins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D047428:Protein Kinase Inhibitors; D004004:Dichlororibofuranosylbenzimidazole; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D010774:Phosvitin; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; C093428:protein kinase CK42