ANALYSIS OF A NOVEL DNA-BINDING PROTEIN-KINASE CKII-LIKE ENZYME OF CHIRONOMUS CELLS

We have previously described a Chironomus tentans nuclear 42 kDa phosp hoprotein preferentially associated with transcriptionally active chro matin. In an attempt to purify and identify the kinase responsible for the phosphorylation of the 42 kDa protein, a casein-phosvitin affinit y 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, c ould be identified. In other studies, a nuclear protein that comigrate s with protein kinase CK42 in electrophoresis and is capable to bind d ifferent gene promoters in single-stranded forms in a sequence-selecti ve manner was found. The observations that both protein kinase and ssD NA-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 bi otinylated oligodeoxyribonucleotide promoter probe and Streptavidine-a garose matrix, evidence that both activities arise from the same prote in molecules was obtained. The similarity in the enzyme activities bet ween protein kinase CK42 and CKII raised the question of whether the f ormer was an LY subunit of the latter. To provide an answer to this is sue, CKII, isolated and purified from an epithelial cell line of C. te ntans, was characterized and compared with protein kinase CK42 purifie d from the same cell system. Like other purified CKII preparations, CK II from Chironomus is able to use ATP or GTP for phosphorylation of ca sein and phosvitin, and its activity is strongly inhibited by heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylben zimidazole (DRB). However, the heparin and DRB sensitivities of protei n kinase CKII were substantially higher than those of the protein kina se CK42. Due to their differential solubilities in NaCl and (NH4),SO4 solutions, individual alpha and beta subunit pools of CKII could be de tected, More than 80% of the nuclear alpha subunit was insoluble in 0. 35 M NaCl, while all individual beta subunit were solubilized under th e 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 chromati n. The biochemical and immunological data taken together strongly sugg est that CK42 is a novel DNA-binding protein kinase that is not the al pha subunit of CKII.