The protein kinase from mitotic human cells that phosphorylates Ser-209 on the casein kinase II beta-subunit is p34cdc2.
Casein kinase II is a highly conserved enzyme that is essential for viability. In cells, the casein kinase II beta-subunit is phosphorylated at an autophosphorylation site and at a site (Ser-209) that is maximally phosphorylated in mitotic cells. To identify protein kinase activities that phosphorylate Ser-209, we fractionated extracts from mitosis-arrested human Burkitt lymphoma MANCA cells. A single Ser-209 kinase activity was detected following each fractionation step. The Ser-209 kinase was purified to a specific activity of approx. 250 nmol/min per mg and efficiently phosphorylated histone H1, a synthetic peptide containing Ser-209 (Ser-209 peptide), myelin basic protein and casein. Immunoblot analysis demonstrated that all fractions containing Ser-209 kinase activity contained p34cdc2. Furthermore, depletion of the Ser-209 kinase activity with p13suc1-Sepharose and anti-p34cdc2 antiserum demonstrated conclusively that the isolated Ser-209 kinase is p34cdc2. These studies provide strong biochemical evidence that p34cdc2 is the enzyme that phosphorylates Ser-209 on the beta-subunit of CKII in mitotic cells. In addition, these results indicate that the Ser-209 peptide can be utilized as a specific reagent for the assay of p34cdc2 activity in mitotic extracts, since no other Ser-209 peptide kinase activities were detected.
Litchfield
D W
DW
Manitoba Institute of Cell Biology, Manitoba Cancer Treatment and Research Foundation, University of Manitoba, Winnipeg, Canada.
Bosc
D G
DG
Slominski
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000032:analysis* / Q000302:isolation & purification; D047390:Casein Kinase II; D002457:Cell Extracts / Q000737:chemistry; D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D012694:Serine; D011494:Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase