Phosphorylation of the beta subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2.
To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha subunit or the beta subunit of the enzyme. When isolated from 32P-labeled cells, the beta subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the alpha subunit. In vitro, the beta subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.
Litchfield
D W
DW
Department of Pharmacology, University of Washington, Seattle 98195.
Lozeman
F J
FJ
Cicirelli
M F
MF
Harrylock
M
M
Ericsson
L H
LH
Piening
C J
CJ
Krebs
E G
EG
D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D047388:Casein Kinases; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D013388:Succinimides / Q000737:chemistry; D014407:Tumor Cells, Cultured
D010748:Phosphopeptides; D013388:Succinimides; C015752:N-chlorosuccinimide; D011494:Protein Kinases; D047388:Casein Kinases; D016203:CDC2 Protein Kinase