Protein ubiquitination plays an important role in ATP-dependent protein turnover and it may also be involved in regulating other cellular events. Covalent attachment of ubiquitin to other proteins is catalyzed by three different enzymes, E1, E2, and E3. We have previously shown that protein ubiquitination can be regulated by phosphorylation. In the present study, we show that E220kDa, an isoform of E2, is phosphorylated by a novel protein kinase from the cytosolic fraction of HeLa cells. This protein kinase was purified to almost homogeneity by a procedure involving ammonium sulfate precipitation and three column chromatographies. Gel filtration chromatography indicated that the molecular weight of this protein kinase was about 300 kDa. However, SDS- PAGE showed that the purified protein kinase consists of three subunits with molecular weights of 120 kDa, 105 kDa, and 70 kDa, respectively. The stoichiometry of the phosphorylation of E220kDa was found to be 0.45 moles of phosphate per mole of protein. The phosphorylation of E220kDa occurred only at serine residues. The activity of this protein kinase required the presence of Mg2+; however, the enzyme is inhibited by a high concentration of Mg2+. Phosphorylation of E220kDa causes about a 60% enhancement of its activity to catalyze the ubiquitination of histone H2A. Additionally, we also found that various normal rat tissues also contain E220kDa kinase activity.
