Abstract
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 carboxyl-terminal 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.
Highlights
Ubiquitin-dependent proteolysis constitutes a major pathway in the cell for selective protein degradation [1,2,3]
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
E6AP was initially identified as a 100-kDa cellular protein that in conjunction with the E6 oncoprotein of the human papillomavirus type 16 (HPV) constituted the E3 activity in the ubiquitination of p53 (8, 14 –16)
Summary
E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; HPV, human papillomavirus; 3-AT, 3-aminotriazole; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; WGE, wheat germ dependent 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. Sequence analysis of E6AP revealed a region of approximately 350 amino acids in the carboxyl terminus that was highly conserved among a number of proteins from various organisms [17] This region, termed the Hect domain (homologous to E6AP carboxyl terminus), contains a conserved cysteine residue that serves as the active site for thiol ester formation with ubiquitin [17]. It was demonstrated that inactivation of mHR6B (one of the mouse homologues of yeast Rad6/Ubc2) in mice causes male sterility associated with chromatin modification [19] This is an intriguing result in terms of functional specificity of E2 enzymes since another mouse homologue of Rad, mHR6A, which shares over 90% sequence identity with mHR6B and is expressed in all the same organs and tissues as mHR6B, was intact in these mice [19]. These studies demonstrate specific complex formation between E2 enzymes and the Hect domain family of E3 proteins, and suggest that the ability of E2s to physically associate with specific Hect E3s constitutes the basis of specificity for functionally distinct E2:E3 combinations
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