E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation.

The E2A proteins are basic helix-loop-helix transcription factors that regulate proliferation and differentiation in many cell types. In muscle cells, the E2A proteins form heterodimers with muscle regulatory factors such as MyoD, which then bind to DNA and regulate the transcription of target genes essential for muscle differentiation. We now demonstrate that E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes, and are degraded by the ubiquitin proteasome system evidenced by stabilization following treatment with the proteasome inhibitor, MG132. During the differentiation from myoblast to myotube, the cellular abundance of E2A proteins is relatively unaltered, despite significant changes (each approximately 5-fold) in the relative rates of protein synthesis and protein degradation via the ubiquitin-proteasome system. The rate of ubiquitin-proteasome-mediated E2A protein degradation depends on the myogenic differentiation state (t 1/2 approximately 2 h in proliferating myoblasts versus t 1/2 > 10 h in differentiated myotubes), and is also associated with cell cycle in non-muscle cells. Our findings reveal an important role for both translational and post-translational regulatory mechanisms in mediating the complex program of muscle differentiation determined by the E2A proteins.

The nuclear ubiquitin-proteasome system degrades MyoD.

Many short-lived nuclear proteins are targeted for degradation by the ubiquitin-proteasome pathway. The role of the nucleus in regulating the turnover of these proteins is not well defined, although many components of the ubiquitin-proteasome system are localized in the nucleus. We have used nucleoplasm from highly purified HeLa nuclei to examine the degradation of a physiological substrate of the ubiquitin-proteasome system (MyoD). In vitro studies using inhibitors of the system demonstrate MyoD is degraded via the ubiquitin-proteasome pathway in HeLa nucleoplasm. Purified nucleoplasm in vitro also supports the generation of high molecular mass MyoD-ubiquitin adducts. In addition, in vivo studies, using leptomycin B to inhibit nuclear export, demonstrate that MyoD is degraded in HeLa cells by the nuclear ubiquitin-proteasome system.

The carboxyl-terminal domain of receptor-associated protein facilitates proper folding and trafficking of the very low density lipoprotein receptor by interaction with the three amino-terminal ligand-binding repeats of the receptor.

The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.

Identification of a region within the ubiquitin-activating enzyme required for nuclear targeting and phosphorylation.

The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 33171-33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus.

The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cell cycle-dependent manner.

The ubiquitin-activating enzyme E1 exists as two isoforms, E1a (117 kDa) and E1b (110 kDa). E1a is phosphorylated, whereas E1b is not. In the present study we have demonstrated the cell cycle dependence of E1a phosphorylation: a 2-fold increase in the specific phosphorylation of E1a in G2 compared with the basal level of phosphorylation in the other stages of the cell cycle. Two-dimensional gel electrophoresis resolved E1 into the two isoforms E1a and E1b; E1a resolved further as three phosphorylated forms and one nonphosphorylated form, while E1b resolved as one nonphosphorylated form. E1a is found predominantly in the phosphorylated forms. However, the distribution of E1a among these different phosphorylated forms was not cell cycle-dependent. We next evaluated the enzymatic activity of E1 as well as its subcellular localization throughout the cell cycle. 32P-Pyrophosphate exchange activity of E1 did not vary along the cell cycle; however, the amount of ubiquitin-protein conjugates decreased by 50% in G2. Nuclear and cytosolic fractionation of cells revealed the nuclear to cytosolic ratio of phosphorylated E1a was 3-fold greater in G2 compared with the other stages of the cell cycle. Finally, purified nuclear extracts supported E1-dependent ubiquitin conjugation of exogenous substrates as did purified cytosol. However, in nuclear extracts but not in cytosol the amount of E1 activity was rate-limiting. Thus we establish nuclear E1-dependent protein ubiquitination and propose that an increase in phosphorylation of E1a in G2 functions to increase the import and/or retention of E1a in the nucleus and may modulate nuclear protein ubiquitination.

Rescue of the complex temperature-sensitive phenotype of Chinese hamster ovary E36ts20 cells by expression of the human ubiquitin-activating enzyme cDNA.

The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubiquitin-activating enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.

Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1 antibodies can be explained by the existence of two subpopulations of E1: one cytoplasmic and partially nuclear, and one that is nuclear. Deletion of a small region at the amino terminus of the HA-1E1, including the basic sequence KKRR, transformed its immunostaining pattern to that observed with HA-2E1.

Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent.

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.