Quantitative expression analysis of the cellular specificity of HECT-domain ubiquitin E3 ligases

LILIANA E. SCARAFIA, ANDREAS WINTER and DAVID C. SWINNEY

Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California 94304


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is "homologous to E6AP C-terminus"). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20–100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.

TaqMan polymerase chain reaction; expression profiling; messenger RNA quantitation; ubiquitin E3 ligase; HECT


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UBIQUITIN-DEPENDENT PROTEOLYSIS has recently emerged as an important regulator of the half-life of many regulatory biomolecules. The biomolecules are specifically targeted for degradation by the proteasome through the posttranslational conjugation of polyubiquitin chains (for a review, see Ref. 7). Nonproteolytic ubiquitination has recently been shown to be important for endocytosis and protein trafficking (5). The process of ubiquitination is initiated by modification of the substrate protein. Modifications that target a protein for ubiquitination include phosphorylation, dephosphorylation, myristoylation, unfolding, and oxidation. The formation of polyubiquitin chains on specific molecules requires three different types of enzymes that work in concert to covalently link ubiquitin to a specific biomolecule at the appropriate time. The ubiquitin activating enzyme (E1) uses ATP to activate the ubiquitin via the formation of a high-energy thioester bond. The ubiquitin is then transferred to the second enzyme, the ubiquitin-conjugating enzyme (UBC or E2). Finally, ubiquitin is transferred to a substrate with the help of a third class of biomolecules, the E3s. There are multiple E2s and some have been shown to preferentially bind to specific E3s in cell-free systems (14). There are at least four families of E3s (7, 18); the E3{alpha} family; the large combinatorial SCF family; the ring finger family; and the HECT-domain family, which carries a 350 amino acid residue domain homologous to the COOH-terminal domain of the prototype member of the family, E6AP (hecth1). Although these E3s perform the same function, namely, the formation of polyubiquitin chains on protein substrates, they are structurally distinct. For instance, the SCF family of E3s is a multi-subunit complex, whereas the HECT domain E3s are thought to function as single proteins that both recognize the substrate and transfer ubiquitin via a thioester bond.

The HECT domain E3s have a conserved carboxy-terminal region that is involved in the formation of the thioester intermediate. Very little is known about the expression profiles of HECT E3s, their relationship to E2s, and their role in the selectivity of ubiquitination.

We undertook this study to investigate if there is differential expression of HECT domain E3s that could be associated with specific cellular functions. The results of this work show the HECT E3s to be broadly expressed at low but variable levels. We interpret this to be consistent with a requirement for the HECT E3s in rapidly processing many diverse signals. Thus the combinatorial expression of E3s and E2s allows the ubiquitin-proteasome system to be selectively and independently involved in many essential biological processes, leading us to conclude that substrate activation is necessary but not sufficient for ubiquitination selectivity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Cell lines.
Jurkat cells (T-cell leukemia), MM6 (monocytic), THP1 (peripheral blood monocyte, leukemia), Hek293 (embryonal kidney cell, epithelial morphology), HepG2 (hepatoblastoma, epithelial morphology), CCD16 (normal lung fibroblasts), CCD29 (severe emphysema lung fibroblasts), A495 (adenocarcinoma of the lung, epithelial morphology), andH292 (lung epidermoid carcinoma) are of human origin and were obtained from the American Type Culture Collection. The number of suspension cells harvested was determined by counting the cells with an hemocytometer. Jurkat cells were induced by adding human tumor necrosis factor-{alpha} (TNF-{alpha}; Calbiochem, San Diego, CA) to the cell culture medium to a final concentration of 20 ng/ml.

Total RNA preparation.
Cells grown in suspension were pelleted by centrifugation while adherent cells were used directly after pouring off the medium. Cells were lysed by adding 1 ml Trizol Reagent (GIBCO; Life Technologies, Rockville, MD) per 1 x 107 cells, and total RNA was isolated following the manufacturer’s protocol. The exogenous standard, neomycin-enhanced green fluorescent protein (NeoEGFP) RNA, was obtained by in vitro transcription (RiboMAX Large Scale RNA Production System T7; Promega, Madison, WI) of BamHI linearized NeoEGFP plasmid (Clontech, Palo Alto, CA). The NeoEGFP RNA was added after Trizol to a determined number of cells in a ratio of 1,000 copies of RNA per cell. To eliminate genomic DNA, the RNA preparations were treated with DNase I (amplification grade, GIBCO). The purity of the RNA preparation, i.e., absence of genomic DNA, was tested by PCR assay with glyceraldehyde 3-phosphate dehydrogenase primers (human GAPDH control amplimer set; Clontech) followed by gel electrophoresis.

First strand cDNA synthesis.
The reverse transcription with oligo dT primers was done with 1 µg of total RNA using SuperScript Preamplification System for First Strand cDNA Synthesis Kit (GIBCO). After termination at 70°C for 15 min, 2 units of RNase H were added.

Primers and probes.
Primers and probes were designed to have similar amplification efficiencies. They are located when possible within the 1,500 bp of the mRNA 3' end encoding the HECT domain proteins (listed in Table 1). The probes labeled with a fluorescence dye [6-carboxyfluorescein (FAM)] on the 5' end and a quencher dye [6-carboxy-tetramethylrhodamine (TAMRA)] on the 3' end were synthesized by Perkin-Elmer Biosystems, Foster City, CA. The custom-designed primers were ordered from GIBCO (Table 2).


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Table 1. HECT sequence names and accession numbers

 

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Table 2. HECT-domain primers and probes

 
Real time quantitative RT-PCR.
Quantitative PCR was performed using real time TaqMan technology (6) and analyzed on an ABI Prism 7700 Sequence Detection System (Perkin-Elmer). Reactions (25 µl) were prepared according to the TaqMan PCR core reagent kit protocol (Perkin-Elmer) and contained 1x PCR buffer, 5 mM MgCl2, 200 µM each dNTP (except dTTP substituted with 400 µM dUTP), 0.025 U/µl AmpliTaq Gold, 0.01 U/µl AmpliEraseUNG, 300 nM forward and reverse primer, and 200 nM probe. The cDNA used as template per reaction corresponds to 4.8 ng total RNA. For better reproducibility, the template volume was increased from the recommended 1 µl (of a 1:10 cDNA dilution) to 4 µl (of a 1:40 cDNA dilution). Assays were prepared in 96-well reaction plates, capped with Optical Caps. The Ct value, corresponding to the cycle at which the fluorescent emission reaches a threshold of 10 standard deviations above the baseline emission, was measured (4). The cycling program included 2 min at 50°C for optimal AmpErase activity and 10 min at 95°C for activation of AmpliTaq Gold Polymerase, followed by 40 cycles of 95°C for 15 s alternating with 60°C for 1 min, and a final hold of 25°C. Two negative controls were added: one without reverse transcriptase, and one without cDNA template. Data were expressed relative to the expression of the housekeeping gene GAPDH. The absolute number of copies per Jurkat cell was quantitated using NeoEGFP RNA as exogenous standard.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Target sequences.
Twenty-eight HECT domain sequences were identified in the literature, public databases and Lifeseq databases (Incyte Genomics, Palo Alto, CA) using both full-length and the carboxy terminus of E6AP protein sequence (Fig. 1) as query in BLAST searches (1). The nomenclature for hecth1 to hecth20 is that of Schwarz et al. (14). Newly identified sequences were numbered hecth21 to hecth25, or letters were added to tentatively reflect the homology to other HECT sequences. Primers and probes were designed for these targets and a few other gene products associated with the ubiquitin system including a few E2s. Total RNA was isolated from nine different human cell lines and quantitated using TaqMan RT-PCR. The expression levels were determined relative to GAPDH (Table 3).



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Fig. 1. E6AP (hecth1, accession no. U84404) COOH-terminal HECT domain sequence. Conserved residues are in bold. The catalytic cysteine is marked with underscore. HECT, homologous to E6AP C-terminus.

 

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Table 3. Relative expression of HECT mRNA in Jurkat, MM6, THP1, Hek293, HepG2, A495, H292, CCD16, and CCD29 cells

 
Absolute quantitation of target sequences in Jurkat cells.
In vitro transcribed NeoEGFP RNA was added as an exogenous standard to quantitate the absolute levels in Jurkat cells. In four separate Jurkat RNA preparations, the absolute amount of GAPDH mRNA was determined to be between 16,000 and 27,000 with an average of 22,000 copies per cell, relative to the exogenous NeoEGFP standard. The levels of the HECT mRNAs were low compared with GAPDH (Fig. 2). hecth9, 24, 17b, and 18 were expressed at the highest levels; 239, 200, 185, and 122 copies per cell, respectively. Nine other HECTs were below 10 copies per cell, hecth3, 5, 7, 11, 15, 17c, 17d, 21, and 22. The mRNA levels of two E2s were also quantitated. Surprisingly, the levels of UBCH7 were ~30 times higher than for UBCH5A; 449 vs. 15 copies per cell, respectively.



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Fig. 2. HECT-domain E3s expression in Jurkat cells. The abundance of most HECT-domain mRNAs is low, between 0 and 200 copies per cell. UBCH5A and UBCH7 are included for comparison. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Expression of the target sequences in the different human cell lines.
The level of mRNA of the 28 HECT-E3s and two UBC-E2s were determined in 9 human cell lines. The PCR reactions were carried out on identical quantities of RNA isolated from the different cell lines and normalized to GAPDH. This data is shown in Table 3 and is the mean of triplicate determinations for the same experiment. Overall there were more similarities between the cell lines than differences. hecth9, 17b, and 24 had the highest expression levels. The mRNA abundance varied greater than 20-fold for two-thirds of the sequences. The least cell specific were hecth7, 8, 10, 13, 15b, 18, 19, 20, 24, and 25, whose expression levels varied less than 20-fold. There were few cases of absolute specificity; hecth3, 21, and 23 had higher levels in the three lymphocytic cell lines (Fig. 3); hecth3 in MM6 and THP1; hecth11 in HepG2 and A495 cell (Fig. 4); and hecth22 in Hek293 cells. Another difference of note was the specific lack of expression of hecth12 and 15 in fibroblast from lungs of patients with severe emphysema compared with normals (Fig. 4). A 24- and 10-fold decrease in the respective expression of these genes was observed. No differences between diseased and normal lung fibroblasts were noted in the expression levels of other HECT mRNAs.



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Fig. 3. Differential expression in cells. hecth3, 21, and 23 have higher expression in three lymphocytic cell lines.

 


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Fig. 4. Differential expression in cells. hecth11 is mainly expressed in HepG2 liver hepatoblastoma and lung A495 cell lines. hecth12 is differentially expressed in normal vs. severe emphysema lung fibroblasts.

 
The expression of the two E2s varied somewhat between cell lines; however, the levels of UBCH7 were always greater than UBCH5A. A caveat in the interpretation of these differences in expression is that four different gene loci for UBCH7 (UBE2L) have been identified: L1, L2, L3, and L4. The UBE2L3 gene is transcribed into mRNAs with four different 3' untranslated regions (10, 11). Our primers and probes have homology to and can probably hybridize to the intronless UBE2L2 (a pseudogene with an open reading frame encoding a truncated protein) or any of the transcripts from UBE2L3. This may explain the higher level of expression measured with UBCH7 compared with UBCH5A across all cell lines.

Expression of the target sequences with TNF-{alpha} treatment in Jurkat cells.
After 6-h incubation of Jurkat cells with TNF-{alpha} at a final concentration of 20 ng/ml of medium, there was no difference in the expression of HECT domain sequences compared with the no-TNF-{alpha} control. As a positive control, the transcript level of the inhibitor of NF-{kappa}B{alpha} (I{kappa}B{alpha}) increased about eightfold with TNF-{alpha} treatment (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was designed to answer the question of whether there is cellular-specific expression of any of the HECT-domain E3s that could be associated with specific functions. The results did not show any dramatic cellular specificity, although the pattern of expression was different for many of the HECT E3s. This is in contrast to the Northern analysis of Schwartz and coworkers (14) that showed a very distinct tissue-specific expression of 8 HECT E3s. Most of the 28 HECT domain genes are broadly expressed at variable levels in the tested cell lines. The level of the expression is overall low (in Jurkat cells, between 0 and 200 copies per cell).

On the whole, these results are in agreement with the relative abundance of expressed sequence tags (ESTs) corresponding to individual HECT genes that can be found in databases. As an example, we performed BLAST searches against the EMBL human EST database (release 63) with three HECT sequences: hecth9 that had the highest level of expression in Jurkat cells, hecth3 that showed low expression in lymphocytic cell lines, and hecth25 (Smurf1). The threshold E-value in these BLAST searches was set low (<1 x e-20) to retrieve only true transcripts of the genes. A total of 138 ESTs were matched with the 3'-end of hecth9 that was probed with our primers. Similarly, hecth25 and hecth3 were represented by 20 ESTs and 19 ESTs, respectively. Their rank order is the same as the transcript abundance determined in Jurkat cells (Table 3). For comparison, UBCH7 had 404 ESTs, and UBCH5 had 97. In the September 2000 version of the Lifeseq database containing 1,292 human cDNA libraries and 5,321,883 clones, there are 702, 295, and 177 sequences assigned to hecth9, hecth25, and hecth3, respectively. If the search is restricted to five Jurkat cell libraries with a total of 15,074 clones, then only hecth9 is found represented by 2 clones. UBCH7 and UBCH5 are represented with 7 and 1 entries, respectively, in the Lifeseq Jurkat cell libraries, and with 843 and 97 clone totals in all libraries. These analyses do not provide any evidence to support cell-specific, ubiquitin-dependent functions for the HECT E3s.

To put these observations in context, comprehensive surveys of genome expression in various tissues have shown that the fraction of truly tissue-specific transcripts is very small (roughly 0.1–1% of the expressed genes in any particular cell) (16). Therefore, the likelihood of finding an HECT-domain E3 expressed exclusively in a particular cell line is low. It is also likely that those displaying differential expression in this study (for instance, hecth3 and hecth21, which were only found in lymphocytic cell lines) may not be exclusively limited to those cells. On the other hand, the absence of expression of an HECT-domain E3 in certain cells may be more meaningful. What can be concluded from the results of this analysis is that the expression profile of the HECT domain E3s does not fit the criteria of housekeeping or maintenance genes.

It is also important to consider the expression levels of E2s when evaluating their role in E3-dependent ubiquitination. UBCH5A and UBCH7 were expressed in all the cells analyzed with absolute levels in Jurkat cells of 15 and 449 copies cell, respectively. Although these E2s belong to different subfamilies, they are closely related (2). Both UBCH5A and UBCH7 have been shown to participate in tumor suppressor p53 ubiquitination by E6AP (hecth1), whereas other HECT E3s preferentially interacted with either UBCH5 or UBCH7 (14). Warrington et al. (17), in analyzing the expression of 7,000 genes found UBCH5B, a close homolog of UBCH5A, expressed at uniformly, low levels in 11 diverse tissues. Because of its wide expression, UBCH5B was consequently categorized along with 535 other genes as a maintenance gene. The expression levels of UBCH7 and UBCH5A measured in this study, although less uniform, are essentially in agreement. These data suggest that the relative levels of expression of the E2s must be considered when extrapolating cell-specific outcomes from in vitro studies and are consistent with the hypothesis that ubiquitination selectivity will depend on the concentration of both E2 and E3. However, based on this limited data set and work by others, the E2s do not appear to show the cellular variability observed with the E3s.

What functional insight can be obtained from the expression profiles? The quantitation showed the transcript levels to be variable among the 28 HECT E3s. This suggests an importance for the E3s in the substrate specificity and functional outcome. A great deal of evidence points to the importance of substrate activation in the determining ubiquitination selectivity (for review see Ref. 7). The profiling reported here suggests that substrate activation is necessary but not sufficient for ubiquitination selectivity. This conclusion is consistent with structural and functional studies on the role of HECT E3s in specific outcomes. Structurally, the HECT E3s are large proteins with multiple binding domains. Some have C2 (Ca2+/lipid-binding domains) and WW domains in addition to the HECT domains (the WW domain is a 35-40 amino acid repeat containing 2 tryptophan residues). The WW domains are thought to be for substrate recognition (12). Many HECT-domain E3s have a number of different WW domains. Accordingly, it has been suggested that in vivo these proteins can interact with a number of different proteins through the WW domains (3). A regulator of chromosome condensation (RCC) domain has been found in some HECT-containing proteins. For example, p532 (hecth6 or herc1), rjs and ceb1 (hecth17b) have RCC domains that in the case of p532 have been found to process guanine nucleotide exchange activity on Ras superfamily members ARF1, Rab3A, and Rab5 (8, 9 and 13). The herc homologs (hecth6, hecth18, and hecth20) were among the most highly abundant HECT transcripts in Jurkat cells. Perhaps the best-characterized HECT domain E3 is Nedd4 (hecth5, or kiaa0093), which contains both C2 and WW domains. It was classified by Warrington et al. (17) as a fetal liver-specific transcript, but we detected it in most of the cell lines analyzed. Nedd4 has been shown to have pleotropic functions and is required for the degradation of epithelial sodium channels (15). It is postulated that the Nedd4 family of proteins are key regulators of membrane proteins, each targeting a select set of proteins for ubiquitination (3). The most recent addition to the HECT-E3 family is Smurf1 (19), which we identified as an EST and named hecth25. This transcript was present in all the cell lines tested. The expression of the Xenopus homolog of Smurf1 is developmentally regulated (expressed only from egg to swimming tadpole stages) and differentially localized in embryonic layers. Smurf was also observed to target the ubiquitination of SMADs involved in TGF-ß signaling through bone morphogenic proteins (BMPs). Smurf might serve to control both embryonic development and a wide variety of cellular responses to TGF-ß. Thus the different structural domains of the E3s interact with specific substrates of differentially expressed HECT E3s, such as Nedd4 (hecth5) and Smurf1 (hecth25), to provide specific functional outcomes.

The quantitative expression profile of 28 HECT E3s and two E2s showed overall low levels with broad range of cellular expression and some specificity. While these results give little insight to the specific functions of the HECT E3s, they are consistent with a role for the E3s in the selectivity of ubiquitination. This conclusion is consistent with studies on the structure/function relationships of specific HECT-domain E3s. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for substrate-specific ubiquitination.


    ACKNOWLEDGMENTS
 
We thank Yi-Zheng Xu, Nicole Moore, and Paula Belloni for providing some of the cell lines used in this study, and we thank Laura Garvin for the reagents for GAPDH standards.

Present address of A. Winter: Lehrstuhl für Tierzucht, Technische Universität München, Alte Akademie 12, D-85350 Freising-Weihenstephan, Germany.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. C. Swinney, m/s S3–1, Roche Bioscience, 3401 Hillview Ave., Palo Alto, CA 94304 (E-mail: david.swinney{at}roche.com).


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