Degradations of a protein via protein ubiquitination involves three different enzymes and several discrete steps. Initially, ubiquitin is activated by the ubiquitin-activating enzyme, E1, which forms an E1- ubiquitin thioester. The ubiquitin carrier enzyme, E2, receives the activated ubiquitin from E1 by a thioester transacetylation process to form an E2-ubiquitin thioester and transfers the ubiquitin to its target proteins either directly or via a reaction catalyzed by a ubiquitin ligase, E3. Previously, we have purified a novel ubiquitin carrier protein kinase from HeLa cells. E220kDa, one of the E2 isozymes, is phosphorylated by this novel protein kinase. Recently, we carried out an extensive survey of this ubiquitin carrier protein kinase activity in various cytosolic fractions of normal rat tissues and found that lung contained the highest E220kDa protein kinase activity than other rat tissues. The protein kinase from rat lung shares some common properties with HeLa cell enzyme. Both enzymes bound quite tightly to phenyl- sepharose cL-4B column and could be eluted with Milli-Q H2O. Both enzymes required the presence of Mg2+; however, the enzymes were inhibited by a high concentration of Mg2+. Additionally, we also found that E220kDa can be phosphorylated by calmodulin dependent protein kinase (Cam Kinase II), with a stoichiometry of 0.15 mol of phosphate/mol of E220kDa. Phosphorylation of E220kDa caused a 45% inhibition of enzyme activity to catalyze the ubiquitination of histone H2A.
