The cellular protein E6AP functions as an E3 ubiquitin protein ligase in the E6-dependent ubiquitination of p53. E6AP is a member of a family of functionally related E3 proteins that share a conserved carboxylterminal region called the Hect domain. Although several different E2 ubiquitin-conjugating enzymes have been shown to function with E6AP in the E6-dependent ubiquitination of p53 in vitro, the E2s that cooperate with E6AP in the ubiquitination of its normal substrates are presently unknown. Moreover, the basis of functional cooperativity between specific E2 and Hect E3 proteins has not yet been determined.Here we report the cloning of a new human E2, designated UbcH8, that was identified in a two-hybrid screen through specific interaction with E6AP. We demonstrate that UbcH7, an E2 closely related to UbcH8, can also bind to E6AP. The region of E6AP involved in complex formation with UbcH8 and UbcH7 was mapped to its Hect domain. Furthermore, we show that UbcH5 and UbcH6, two highly homologous E2s that were deficient for interaction with E6AP, could associate efficiently with another Hect-E3 protein, RSP5. Finally, only the E6AP-interacting E2s could function in conjunction with E6AP in the ubiquitination of an E6 independent substrate of E6AP, whereas the noninteracting E2s could not. Taken together, these studies demonstrate for the first time complex formation between specific human E2s and the Hect domain family of E3 proteins and suggest that selective physical interaction between E2 and E3 enzymes forms the basis of specificity for functionally distinct E2:E3 combinations.Ubiquitin-dependent proteolysis constitutes a major pathway in the cell for selective protein degradation (1-3). The covalent attachment of multiple ubiquitin molecules to lysine residues of a target protein serves to signal its recognition and rapid degradation by the 26 S proteasome. Ubiquitin conjugation can also result in nonproteolytic modification of target proteins (4 -7). Ubiquitination of a protein substrate requires the concerted action of three classes of enzymes; the ubiquitin activating enzyme E1 1 initially activates ubiquitin in an ATPdependent reaction through the formation of a thiol ester bond between the carboxyl terminus of ubiquitin and the thiol group of a specific cysteine residue of E1. Ubiquitin is then transferred to a specific cysteine residue on one of several ubiquitinconjugating enzymes (Ubcs or E2s). E2 enzymes in turn may transfer the ubiquitin either directly to a substrate or to the final class of enzymes known as ubiquitin protein ligases (or E3s). The E3 enzymes catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the ⑀-amino group of lysine residues on a target protein (3,8,9). A substrate may be multiply ubiquitinated through the attachment of additional ubiquitin molecules to specific lysine residues (lysine 48 or 63) of ubiquitin itself, although the processive nature of a multiubiquitination reaction is presently unclear (3, 4, 7). In order for this proce...
The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.Strong epidemiologic, virologic, and biochemical evidence indicates that the high-risk subgroup of genital human papillomaviruses (HPVs; including HPV types 16, 18, 31, 33, and 39) is the causative agent in the development of Ͼ90% of cervical cancers (reviewed in reference 64). The HPV16 E6 and E7 proteins are necessary and sufficient for immortalization of primary human keratinocytes in culture (17,20,36). HPV16 E6 also cooperates with activated ras to transform baby mouse kidney cells (51) and can transform NIH 3T3 cells (45) and immortalize human mammary epithelial cells (2) independent of E7.HPV16 E6 and E7 bind and promote the degradation of the tumor suppressor proteins p53 (44, 59) and pRB (5,8,26), respectively. HPV16 E6 induces p53 degradation by forming a complex with the cellular ubiquitin-protein ligase E6AP (22), which is then able to bind and ubiquitinate p53 (42). E6AP is a component of the ubiquitin-proteasome pathway which targets proteins for degradation by covalently linking them to multimeric chains of the 8-kDa protein ubiquitin. In this pathway, ubiquitin is first activated in an ATP-dependent reaction and then passed from the ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2). Ubiquitin is then transferred to lysine residues of the target protein with the aid of a ubiquitin-protein ligase (E3). Multiubiquitinated proteins are subsequently recognized and degraded by the 26S proteasome (18). While some classes of E3 proteins, such as the SCF complexes, function as adaptors that bring E2 enzymes into complex with their substrates (9, 47), E6AP belongs to a class o...
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