PURIFICATION AND CHARACTERIZATION OF A UBIQUITIN CARRIER PROTEIN-KINASE FROM HELA-CELLS

Protein ubiquitination plays an important role in ATP-dependent protei n turnover, and it also may regulate other cellular events. Covalent a ttachment of ubiquitin to other proteins is catalyzed by three differe nt enzymes, E1, E2, and E3. We have previously shown that protein ubiq uitination can be regulated by phosphorylation. In the present study, we show that 20-kDa E2, an E2 molecular mass isoform, is phosphorylate d by a protein kinase from the cytosolic fraction of HeLa cells. This protein kinase was purified by a procedure involving ammonium sulfate precipitation and three column chromatographies (phenyl-Sepharose, Sup erose gel filtration, and DEAE-Sephacel). Gel-filtration chromatograph y indicated that the molecular mass of this protein kinase was about 3 00 kDa. However, SDS/PAGE showed that the purified protein kinase cons ists of three subunits with molecular masses of 120, 105, and 70 kDa, respectively. The stoichiometry of the phosphorylated 20-kDa E2 isozym e was found to be 0.45 mol of phosphate per mol of protein. The phosph orylation of 20-kDa E2 occurred only at the serine residue. The activi ty of this protein kinase required the presence of Mg2+; however, the enzyme was inhibited by a high concentration of Mg2+.