Significance How RING-E3s, several of which are required for cell division, differentiation, or survival, achieve E2 activation at the right time and place is poorly understood. Here we used the essential RING-E3 anaphase-promoting complex (APC/C) and its E2 Ube2S to dissect the mechanism of E2 recognition and activation by RING-E3s. Our work reveals a dynamic interplay between E2 and E3 that is regulated by reversible phosphorylation; while phosphorylation of Cdc20 inhibits Ube2S binding to the APC/C, dephosphorylation by PP2A B56 allows for rapid Ube2S activation. Given that the kinetochore-bound PP2A B56 also silences the spindle checkpoint signal, this work suggests that cells coordinate Ube2S activation with other events in human cell cycle control.