Characterization of the mUBC9-binding sites required for E2A protein degradation

Mammalian Ubc9 (mUbc9) is required for rapid degradation of the E2A protein s E12 and E47 by the ubiquitin-proteasome system. We have shown elsewhere t hat mUbc9 interacts with amino acids 477-530 of E12/E47. Here we test the h ypothesis that this region, rich in proline, glutamic acid, serine, and thr eonine (PEST) residues, serves as the E2A protein degradation domain (DD), An E2A protein lacking this region, E47 Delta(478-531), was significantly m ore stable than wild-type E47(half-life of more than 6 h versus 55 min). De letion of the E2A DD had no effect on the E-box-binding and transcriptional activity of E47, We mapped two discreet mUbc9-interacting regions within t he E2A DD: amino acids 476-494 and 505-513. E2A(505-513) interacted with mU bc9 but not with human Ubc5, MyoD, ids, or the polymyositis-scleroderma aut oantigen, Substitution of the E2A(505-513) central hydrophobic residues wit h basic residues abolished interaction with mUbc9, Also, full-length E47 la cking the second mUbc9-interacting region was significantly more stable tha n wild-type E47, Reintroduction of the E2A DD into the long-lived, naturall y occurring chimeric oncoprotein E2A-HLF (hepatic leukemic factor) destabil ized it, suggesting that this domain can transfer a degradation signal to a heterologous protein. E2A-HLF-DD chimeric protein was stabilized by the pr oteasome inhibitor LLNL, indicating the role of the ubiquitin-proteasome sy stem mediating degradation through the E2A degradation domain. Our experime nts indicate that the E2A DD mediates E2A protein interactions with the ubi quitin-proteasome system and that the E2A DD is required for metabolism of these widely expressed proteins.