The protein kinase from mitotic human cells that phosphorylates Ser-209 on the casein kinase II β-subunit is p34 cdc2

Abstract

Casein kinase II is a highly conserved enzyme that is essential for viability. In cells, the casein kinase II β-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 HI, 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 Ser-209 on the β-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.