In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradioldependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin-proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA͞UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor ␤ proteins.The ubiquitin-proteasome pathway is the major system in the eukaryotic cell for the selective degradation of short-lived regulatory proteins (1, 2). A common feature of proteasomemediated protein degradation is the covalent attachment of ubiquitin, a highly conserved 8.6-kDa protein, to lysine residues of proteins targeted for degradation followed by the formation of polyubiquitin chains attached covalently to the targeted protein. Ubiquitinated proteins are recognized and degraded by the multi-subunit protease complex, the 26S-proteasome (3-6). In addition to the role it plays in protein degradation, ubiquitination may serve regulatory functions such as directing the subcellular localization of proteins (3, 4). The ubiquitin-proteasome pathway also plays an important role in various cellular processes such as cell-cycle regulation, signal transduction, differentiation, antigen processing, and degradation of tumor suppressors (3, 4, 7-11).Protein ubiquitination involves three classes of enzymes, namely the E1 ubiquitin-activating enzyme (UBA), E2 ubiquitin-conjugating enzymes (UBCs), and E3 ubiquitin-protein ligases. The UBA first activates ubiquitin in an ATPdependent manner. The activated ubiquitin then forms a thioester bond between the carboxyl-terminal glycine residue of ubiquitin and a cysteine residue of the UBA. Next, ubiquitin is transferred from the E1 to one of the several E2s (UBCs), preserving the high-energy thioester bond (1, 2, 5, 11). In some cases, ubiquitin is transferred directly from the E2 to the target protein through an isopeptide bond between the -amino group of lysine residues of the target protein and the carboxy terminus of ubiquitin. In other instances, the transfer of ubiquitin from UBCs to target proteins proceeds through an E3 ubiquitin-protein ligase intermediate (12,13). It has been proposed th...

Increasing evidence indicates that transcription and pre-mRNA processing are functionally coupled to modulate gene expression. Here, we report that two members of the U2AF65 family of proteins, hCC1.3, which we call CAPERalpha, and a related protein, CAPERbeta, regulate both steroid hormone receptor-mediated transcription and alternative splicing. The CAPER proteins coactivate the progesterone receptor in luciferase transcription reporter assays and alter alternative splicing of a calcitonin/calcitonin gene-related peptide minigene in a hormone-dependent manner. The importance of CAPER coactivators in the regulation of alternative RNA splicing of an endogenous cellular gene (VEGF) was substantiated by siRNA knockdown of CAPERalpha. Mutational analysis of CAPERbeta indicates that the transcriptional and splicing functions are located in distinct and separable domains of the protein. These results indicate that steroid hormone receptor-regulated transcription and pre-mRNA splicing can be directly linked through dual function coactivator molecules such as CAPERalpha and CAPERbeta.

Approximately 40% of human breast cancers contain alterations in the tumor suppressor p53. The p53 172R-H gain-of-function mutant (equivalent to the common 175R-H human breast cancer mutant) has been shown to promote aneuploidy and tumorigenesis in the mammary gland in transgenic mice and may affect genomic stability in part by causing centrosome abnormalities. The precise mechanism of action of these gain-of-function mutants is not well understood, and has been studied primarily in fibroblast cell lines. A novel p53-null mouse mammary epithelial cell line developed from p53-null mice has been used in adenovirus-mediated transient transfection experiments to study the properties of this p53 mutant. Marked centrosome amplification and an increased frequency of aberrant mitoses were observed within 72 h of introduction of p53 172R-H. However, few cells with aberrant centrosome numbers were observed in cells stably expressing the p53 172R-H mutant. Furthermore, stable expression of this p53 mutant reduced both basal and DNA damage-induced apoptosis. This result may be mediated in part through abrogation of p73 function. The p53 172R-H mutant, therefore, appears to influence tumorigenesis at the molecular level in two distinct ways: promoting the development of aneuploidy in cells while also altering their apoptotic response after DNA damage.

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