Control of p53 turnover is critical to p53 function. E1A binding to p300/CBP translates into enhanced p53 stability, implying that these coactivator proteins normally operate in p53 turnover control. In this regard, the p300 C/H1 region serves as a specific in vivo binding site for both p53 and MDM2, a naturally occurring p53 destabilizer. Moreover, most of the endogenous MDM2 is bound to p300, and genetic analysis implies that specific interactions of p53 and MDM2 with p300 C/H1 are important steps in the MDM2-directed turnover of p53. A specific role for p300 in endogenous p53 degradation is underscored by the p53-stabilizing effect of overproducing the p300 C/H1 domain. Taken together, the data indicate that specific interactions between p300/CBP C/H1, p53, and MDM2 are intimately involved in the MDM2-mediated control of p53 abundance.
Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.
The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in nonsynchronized cells, its levels are regulated in a cell cycledependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.Protein ubiquitination is implicated in a variety of cellular processes, including DNA repair, cell cycle control, chromosomal organization, intracellular translocation of proteins, and apoptosis (1-3). Ubiquitin-dependent proteolysis is the best known aspect of the ubiquitin pathway. The covalent conjugation of multiple ubiquitin molecules to lysine residues of a target protein serves to signal its recognition and rapid degradation by the 26 S proteasome (3-5). Ubiquitination of protein substrates is a multi-step process that involves the concerted action of at least three classes of enzymes as follows: ubiquitinactivating enzyme (E1), 1 ubiquitin-conjugating enzymes (E2s), and ubiquitin protein ligases (E3s) (3). Although the biochemical mechanisms of ubiquitin transfer within the enzymatic components of the pathway and its subsequent conjugation to target proteins is now understood in considerable detail, it is still unclear how specific proteins are recognized by the ubiquitin system as substrates. E1 first activates ubiquitin in an ATP-dependent reaction through the formation of ubiquitin adenylate, followed by a thiol ester bond between the carboxyl terminus of ubiquitin and thiol group of a specific cysteine residue in E1. Ubiquitin is then transferred to a specific cysteine residue in one of several E2s (6). E2 enzymes, in turn, may transfer the ubiquitin either directly to a substrate or to E3 enzymes that finally catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the ⑀-amino group of lysine residues on a target protein (3,7,8). A substrate may be multiply ubiquitinated by sequential linkag...
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...
Src family tyrosine kinases are involved in modulating various signal transduction pathways leading to the induction of DNA synthesis and cytoskeletal reorganization in response to cell-cell or cell-matrix adhesion. The critical role of these kinases in regulating cellular signaling pathways requires that their activity be tightly controlled. Src family proteins are regulated through reversible phosphorylation and dephosphorylation events that alter the conformation of the kinase. We have found evidence that Src also is regulated by ubiquitination. Activated forms of Src are less stable than either wild-type or kinase-inactive Src mutants and can be stabilized by proteasome inhibitors. In addition, poly-ubiquitinated forms of active Src have been detected in vivo. Taken together, our results establish ubiquitin-mediated proteolysis as a previously unidentified mechanism for irreversibly attenuating the effects of active Src kinase.
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