COMPONENTS OF A SYSTEM THAT LIGATES CYCLIN TO UBIQUITIN AND THEIR REGULATION BY THE PROTEIN-KINASE CDC2

Cyclin B, a positive regulatory subunit of the cdc2 protein kinase com plex, is synthesized across the cell cycle and then rapidly degraded a t the end of mitosis. Degradation of cyclin B is triggered by increase d levels of active cdc2 and is required for exit from mitosis. It was shown previously that cyclin degradation is carried out by the ubiquit in system, but the components responsible for the specificity and regu lation of cyclin-ubiquitin ligation have not been identified. The form ation of ubiquitin-protein conjugates usually requires the sequential action of three enzymes: a ubiquitin-activating enzyme (E(1)), a ubiqu itin-carrier protein (E(2)), and a ubiquitin-protein ligase (E(3)). In this work we employed a fractionation approach to identify the compon ents of a clam oocyte system responsible for specific ubiquitination o f cyclin and to determine which components are regulated by cdc2. Expe rimental conditions were established under which a fusion protein cont aining an amino-terminal fragment of cyclin B is ligated to ubiquitin only in extracts from M-phase but not from interphase cells. Fractiona tion of M-phase extracts by DEAE-cellulose and high speed centrifugati on yielded three fractions that were all required for cell cycle stage -specific cyclin-ubiquitin ligation. Only one of these fractions could be replaced by a previously known enzyme of the ubiquitin system, E(1 ). A second fraction contained a novel species of E(2), termed E(2)-C, which acts in the ligation of ubiquitin to cyclin but not to other en dogenous proteins. A third component is associated with particulate ma terial. Whereas E(2)-C from either M-phase or interphase extracts is a ctive, the particulate component is active only in M-phase. Incubation of the particulate fraction from interphase cells with the protein ki nase cdc2 activates it for cyclin-ubiquitin ligation, after a lag of a bout 30 min. These findings suggest that the particulate fraction may contain an E(3) enzyme that acts on cyclin, as well as additional fact ors activated by cdc2.