Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.
Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.
Heller-Harrison
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RA
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
Czech
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MP
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D047388:Casein Kinases; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000235:genetics; D005784:Gene Amplification; D005814:Genes, Viral; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D014162:Transfection
D012313:RNA; D004247:DNA; D011494:Protein Kinases; D047388:Casein Kinases