SUBCELLULAR-LOCALIZATION AND UBIQUITIN-CONJUGATING ENZYME (E2) INTERACTIONS OF MAMMALIAN HECT FAMILY UBIQUITIN PROTEIN LIGASES

In most instances, the transfer of ubiquitin to target proteins is cat alyzed by the action of ubiquitin protein ligases (E3s), Full-length c DNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s, Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma viru s E6 protein, while Nedd-4 does not, Consistent with its role in p53 u biquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol, Binding studies implicate a 150 -amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C terminal portion of an E2 enzyme (UbcH5B), Ne dd-4 was determined to have a second nonoverlapping E2 binding site th at recognizes the first 67 amino acids of UbcH5B but not the more C-te rminal portion of this E2. These findings provide the first demonstrat ion of physical interactions between mammalian E2s and E3s and establi sh that these interactions occur independently of ubiquitin and an int act E3 catalytic domain, Furthermore, the presence of two E2 binding s ites within Nedd-4 suggests models for ubiquitination involving multip le E2 enzymes associated with E3s.