THE PROTEIN-KINASE FROM MITOTIC HUMAN-CELLS THAT PHOSPHORYLATES SER-209 ON THE CASEIN KINASE-II BETA-SUBUNIT IS P34(CDC2)

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 p34(cdc2). Furthermore, depletion of the Ser-209 kinase activity with p13(sucl)-Sepharose and anti-p34(cdc2) antiserum demonstrated conclusively that the isolated Ser-209 kinase is p34(cdc2). These studies provide strong biochemical evidence that p34(cdc2) is the enzyme that phosphorylates Set-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 p34(cdc2) activity in mitotic extracts, since no other Ser-209 peptide kinase activities were detected.