Transcriptional Regulation of N-Acetylglucosaminyltransferase V by the src Oncogene*

(Received for publication, April 3, 1997)

Phillip Buckhaults Dagger §, Lin Chen Dagger , Nevis Fregien and Michael Pierce Dagger par

From the Dagger  Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30602 and the  Department of Cell Biology and Anatomy, University of Miami Medical School, Miami, Florida 33101

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Transformation of baby hamster kidney fibroblasts by the Rous sarcoma virus causes a significant increase in the GlcNAcbeta (1,6)Man-branched oligosaccharides by elevating the activity and mRNA transcript levels encoding N-acetylglucosaminyltransferase V (GlcNAc-T V). Elevated activity and mRNA levels could be inhibited by blocking cell proliferation with herbimycin A, demonstrating that Src kinase activity can regulate GlcNAc-T V expression. 5' RACE analysis was used to identify a 3-kilobase 5'-untranslated region from GlcNAc-T V mRNA and locate a transcriptional start site in a 25-kilobase pair GlcNAc-T V human genomic clone. A 6-kilobase pair fragment of the 5' region of the gene contained AP-1 and PEA3/Ets binding elements and, when co-transfected with a src expression plasmid into HepG2 cells, conferred src-stimulated transcriptional enhancement upon a luciferase reporter gene. This stimulation by src could be antagonized by co-transfection with a dominant-negative mutant of the Raf kinase, suggesting the involvement of Ets transcription factors in the regulation of GlcNAc-T V gene expression. The src-responsive element was localized by 5' deletion analysis to a 250-base pair region containing two overlapping Ets sites. src stimulation of transcription from this region was inhibited by co-transfection with a dominant-negative mutant of Ets-2, demonstrating that the effects of the src kinase on GlcNAc-T V expression are dependent on Ets.


INTRODUCTION

The glycosylation of cell surface glycoproteins is a dynamic process that can be regulated by agents that cause differentiation, such as retinoic acid (1) or transforming growth factor-beta (2), or by those that induce cellular proliferation, for example, interleukin-1 or tumor necrosis factor-alpha (3). In many instances, alterations of the oligosaccharides on cell surface glycoproteins cause significant changes in the adhesive or migratory behavior of a cell (4, 5). An induced alteration in the glycosylation of cell surface glycoproteins that has been documented for many years concerns the significant increase in oligosaccharide size caused by oncogenic transformation using a variety of agents (6-14). This increase in size was found to result mainly from an increase in the levels of asparagine-linked oligosaccharides containing N-acetylglucosamine linked beta 1,6 to the alpha (1,6)-linked mannose in the trimannosyl core, (GlcNAcbeta (1,6)Man), and in many cases these oligosaccharides express polylactosamine that can be sialylated (15-18). The (GlcNAcbeta (1,6)Man) branch is synthesized by N-acetylglucosaminyltransferase V (GlcNAc-T V),1 the enzyme whose activity is significantly and selectively increased after transformation by tumor viruses or isolated oncogenes (16, 19-22). Moreover, decreased expression of the GlcNAcbeta (1,6)Man branch has been correlated with decreased metastatic potential (23, 24), whereas the increased expression of this branch appears in some instances to correlate with the progression of invasive malignancies (25).

The transformation of baby hamster kidney (BHK) fibroblasts by the src oncogene causes an increase in N-linked oligosaccharide (GlcNAcbeta (1,6)Man) branching, and the mechanism by which this increase occurs has been under investigation in our laboratories. To elucidate this mechanism, we examined GlcNAc-T V enzyme activity and mRNA levels in BHK cells and their Rous sarcoma virus-transformed counterparts (RSV-BHK) in the presence of the Src kinase inhibitor, herbimycin A. The results from these experiments led us to examine the 5' region of the human gene encoding GlcNAc-T V and its increased expression caused by Src activity. Our results indicate that the N-acetylglucosaminyltransferase V gene can be transcriptionally activated by Src tyrosine kinase activity, and this control is dependent on both the Raf-1 kinase and an Ets family transcriptional activator.


EXPERIMENTAL PROCEDURES

Glycosyltransferase Activity Assays

Cells were grown to confluency and harvested in 50 mM MES 6.5, 150 mM NaCl, and lysed by addition of Trition X-100 to 1%. Lysates were assayed according to the method of Palcic et al. (22). Briefly, 106 cpm of UDP-[3H]GlcNAc (25 cpm/pmol) and 10 nmol of synthetic trisaccharide acceptor for GlcNAc-T V (octyl 6-O-[2-O-(2-acetamido-2-deoxy-beta -D-glucosyl-pyranosyl)-alpha -D-mannopyranosyl]-beta -D-glucopyraoside) were dried under vacuum in a 1.5-ml microcentrifuge tube. Extracts of various protein concentrations were prepared, and 10 µl were added to the assay tube. Assays were incubated at 37 °C for 4 h and quenched by the addition of 500 µl of water. Radiolabeled product was isolated on a C18 Sep-Pak (Waters) column, eluted in 2 ml of methanol, and counted in a scintillation counter. Assays were performed in duplicate or triplicate, at two or three protein concentrations, and specific activity was calculated by linear least squares regression analysis of the data.

Northern Analysis

20 µg of total RNA was electrophoresed on a 1% formaldehyde-agarose gel and blotted to nylon. A 1-kb fragment of a partial rat GlcNAc-T V cDNA clone was random prime-labeled (26), and the blot was probed according to the method of Church and Gilbert (27). Data were collected and quantitated with a PhosphorImager.

Phosphotyrosine Quantitation

Cells were harvested by the addition of 1 ml of preheated SDS-polyacrylamide gel electrophoresis sample buffer to a 10-cm plate and shearing through a 20 gauge needle. Protein concentrations were determined on trichloroacetic acid precipitates using the BCA reagents (Pierce). 20 µg of protein were electrophoresed on a 4-20% gradient acrylamide gel and transferred to nitrocellulose (Bio-Rad). Blots were probed with an anti-phosphotyrosine antibody (a kind gift from Dr. Bart Sefton) followed by a goat anti-mouse horseradish peroxidase conjugate. Bands were detected using the ECL reagents (Amersham Corp.) and quantitated by scanning densitometry.

5' RACE Analysis

Marathon Race Ready cDNA from human whole brain (CLONTECH) was used as a template in a 5' RACE PCR according to the manufacturer's instructions. A 3' PCR primer (303, CCTGGACCTCAGCAAAAGGTACATCAAGGC) designed near the 5' end of the published human cDNA sequence was used along with the 5' anchor primer in a primary round of RACE PCR. Products were separated on a 1% Tris-acetic acid-EDTA-agarose gel and blotted to nitrocellulose. A nested PCR product was generated using the primers 501 (GAATGGAAGTGAGGGAAGGC) and 305 (GGAAGTTGTCCTCTCAGAAGCTGGGCTTT) and a genomic clone template. This product was random prime-labeled (26) and used to probe the membrane to which the RACE PCR product was transferred (see Fig. 6). To improve yields of authentic products, a secondary round of RACE PCR was then performed. A nested GlcNAc-T V 3' primer (305) and nested 5' anchor primer were used in the secondary round of RACE PCR using as templates the products from the primary RACE PCR. The products from this round of RACE PCR were directly subcloned into the TA cloning vector (Invitrogen), and clones were sequenced.


Fig. 6. RACE PCR Southern blot analysis. A RACE-anchored human brain cDNA library was used for PCR using a 3' GlcNAc-T V primer (GCCTTGATGTACCTTTTGCCGAGGTCCAGG) and a 5' anchor primer, and the products were separated and subjected to Southern blot analysis. The blot was probed with a biotinylated GlcNAc-T-V PCR product to detect authentic GlcNAc-T V products, as described under "Experimental Procedures." The largest band visualized by this analysis (left lane) was calculated to be 2.8 kb in length. Size markers are shown at right.
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Genomic Clone Isolation

The human GlcNAc-T V promoter sequences were isolated from a human genomic library cloned in the lambda -FIX II vector (Stratagene, Inc.). A 687-bp EcoRI fragment containing the 5'-untranslated region of the rat cDNA (28) was used as a probe to screen this library using standard plaque hybridization procedures. Screening 5 × 106 phage plaques yielded two overlapping genomic clones that span a region of approximately 30 kb.

Nucleotide Sequencing

Sequencing of RACE PCR clones and human genomic clones was performed using the Applied Biosystems, Inc. reagents by the UGA Molecular Genetics Instrumentation Facility.

Luciferase Reporter Construction

For pGL2-TV1, a 6-kb XhoI-SacI fragment of the genomic clone was band-purified (Sephaglass, Pharmacia Biotech Inc.) and subcloned into XhoI-SacI sites of the pGL2-Basic vector (Promega). For pGL2-TV2 through pGL2-TV4, PCR products were generated using the genomic clone as a template and subcloned into the TA vector. TA clones of the correct orientation were cut with XhoI-SacI, and inserts were cloned into XhoI-SacI sites of pGL2-Basic.

Promoter Activity Determinations

SV40-beta -galactosidase (2 µg) and reporter constructs (2 µg) ± effector plasmids (2 µg) were transfected by the calcium-phosphate precipitation method (29) into 50% confluent cultures of HepG2 cells grown in 6-well culture plates. 40 h post-transfection, cell lysates were prepared and assayed for beta -galactosidase and luciferase (Promega). Luciferase activity was normalized to vector-dependent beta -galactosidase activity.

Plasmids

The plasmids encoding the Raf-1 kinase and its dominant negative form (30) were kind gifts from Dr. Ulf Rapp. Plasmids encoding Ets-2 and its dominant negative form (31) were kinds gifts from Dr. K. E. Boulukos. The v-src expression plasmid was a kind gift from Dr. Tony Hunter.


RESULTS

Earlier experiments utilizing BHK and RSV-BHK cells metabolically radiolabeled with [2-3H]mannose indicated at least a 2-fold increase in the total amount of (2,6)-substituted mannose in the RSV-BHK cells, normalized to total mannose-labeled glycopeptides. Although the specific activity of GlcNAc-T V was increased over 6-fold in the RSV-transformed cells, no significant differences in the kinetic properties of GlcNAc-T V in the transformed cells could be detected. These results suggested that the increases in GlcNAcbeta (1,6)Man levels after transformation were most likely not due to post-translational effects on the enzyme (22). The specific activity of GlcNAc-T V and its mRNA levels were measured, therefore, to determine if the increase in GlcNAc-T V activity in the transformed cells could result from differences in mRNA levels. GlcNAc-T V activity was assayed under optimal conditions in BHK and RSV-BHK cells using a synthetic trisaccharide acceptor. The transformed BHK cells demonstrated a GlcNAc-T V enzyme specific activity 6-fold higher than the untransformed BHK cells. By contrast, no difference was seen in the specific activity of another N-acetylglucosaminyltransferase that functions in the synthesis of N-linked oligosaccharides, GlcNAc-T I (Fig. 1), indicating the specificity of Rous sarcoma virus transformation on GlcNAc-T V activity. To investigate the possibility that the difference in GlcNAc-T V specific activity is associated with a difference in steady-state mRNA levels, Northern blots were performed using a fragment of a cDNA encoding GlcNAc-T V. Compared with BHK cells, RSV-transformed cells were found to have a 6-fold increase in the expression levels of both 8.7 and 9.3 kb GlcNAc-T V transcripts, but no change in either GAPDH or GlcNAc-T I transcripts (Fig. 2). Although not apparent in Fig. 2, PhosphorImager quantitation demonstrated an equivalent increase in both GlcNAc-T V mRNA transcripts. These results demonstrated that the elevation of enzyme activity in the RSV-transformed cells was a result of either transcriptional activation or increased mRNA stability and argue against postranslational modifications of the enzyme causing a significant increase in its catalytic activity.


Fig. 1. BHK and RSV-BHK cell GlcNAc-T V and GlcNAc-T I specific activities. The specific activities of both enzymes were measured in cell lysates under optimal conditions. The specific activity of each enzyme in BHK cells was set at 100%.
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Fig. 2. BHK and RSV-BHK cell GlcNAc-T V mRNA levels. Upper panel, Northern blot of total RNA from BHK (lane 1) and RSV-BHK cells (lane 2) probed with a 1-kb radiolabeled fragment from the open reading frame of murine GlcNAc-T V and visualized using a PhosphorImager. Lower panel, PhosphorImager quantitation of the band intensities from the analysis shown in the upper panel. The blot was stripped and hybridized with a rat GAPDH radiolabeled cDNA to demonstrate loading equivalence.
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To obtain convincing evidence that the differences in GlcNAc-T V expression result from src tyrosine kinase activity, we made use of a src-selective tyrosine kinase inhibitor, herbimycin A, a metabolite produced by Streptomyces sp. MH237-CF8. This inhibitor was first identified for its ability to reverse the transformed morphology of Rous sarcoma virus-infected rat kidney cells (32), and this reversion of morphology was associated with a reduction in total cellular phosphotyrosine levels (33). Herbimycin A was unable, however, to reverse the transformed morphologies induced by the ras, raf, or myc oncogenes, demonstrating its specificity for the src family of tyrosine kinase oncogenes (34). Herbimycin A is also able to reverse src-stimulated expression of the glucose transporter gene (35) and to cause a reversible Go contact arrest in src-transformed normal rat kidney cells (36). We utilized herbimycin A, therefore, to test the hypothesis that the expression of GlcNAc-T V is positively regulated by the src tyrosine kinase.

First, to monitor the effects of src kinase and demonstrate its inhibition by herbimycin A, we measured total cellular phosphotyrosine levels by performing Western blots with an alpha -phosphotyrosine antibody on extracts made from cells treated for 24 h with various concentrations of the drug. These results demonstrate that, as expected, herbimycin A caused a dose-dependent decrease in cellular phosphotyrosine levels (Fig. 3). At a concentration of 1 µg/ml, herbimycin A caused a reversal of the RSV-transformed morphology and a complete inhibition of cell division (data not shown). Consistent with the drug's effect of inhibiting the src kinase (33) and blocking cell division in Go (36), herbimycin A caused a dose-dependent decrease in GlcNAc-T V enzyme specific activity in RSV-transformed BHK cells (Fig. 4). Interestingly, although the drug blocked cell division and caused a modest decrease in phosphotyrosine levels in the untransformed BHK cells (data not shown), it had little effect on the expression level of GlcNAc-T V enzyme activity in confluent cultures of untransformed cells (Fig. 4). This result suggests that regulation of GlcNAc-T V expression is complex, with both src-dependent and src-independent factors. Herbimycin A had no effect on the specific activity of GlcNAc-T I (data not shown), arguing against nonspecific toxic effects on the transformed cells and confirming that GlcNAc-T I is not regulated by src. To determine if the inhibition of expression of GlcNAc-T V enzyme activity by herbimycin A was a result of inhibiting the expression of the mRNA encoding the enzyme, Northern blots were performed on RNA samples prepared from RSV-transformed cells treated with various concentrations of the drug. Similar to its effects on GlcNAc-T V enzyme specific activity, herbimycin A caused a decrease in GlcNAc-T V message levels in the RSV-BHK cells in a dose-dependent manner (Fig. 5). Taken together, these results indicate that expression of the GlcNAc-T V mRNA in the src-transformed cells is under the control of the src tyrosine kinase.


Fig. 3. BHK and RSV-BHK cell phosphotyrosine levels. Total cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and subjected to Western blotting using as a probe an anti-phosphotyrosine antibody. Bound antibody was visualized using luminescent reagents and quantitated by densitometer scanning.
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Fig. 4. BHK and RSV-BHK GlcNAc-T V specific activities after treatment with herbimycin A. GlcNAc-T V enzyme specific activity was measured in cell lysates that had been incubated for 24 h with various concentrations of herbimycin A.
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Fig. 5. BHK and RSV GlcNAc-T V mRNA levels after treatment with herbimycin A. Total RNA was extracted from cells treated for 24 h with herbimycin A and subjected to Northern blot analysis, and the results were quantitated using a PhosphorImager. The level of GlcNAc-T V mRNA in untreated cells was set at 100%.
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To elucidate the mechanism by which src induces the expression of GlcNAc-T V, we isolated the 5'-flanking region of the gene and analyzed this region for promoter activity. The GlcNAc-T V message is approximately 9 kb in most rodent and human tissues, with brain having high expression levels. To locate a promoter for GlcNAc-T V, 5' RACE PCR techniques were used to isolate and sequence the 5' end of the message from human brain. RACE PCR products were first generated from the genomic clone using the 303 primer (designed against the 5' end of the human GlcNAc-T V cDNA sequence) and then analyzed by Southern blotting. GlcNAc-T V-specific sequences were detected using a nested 501-305 PCR product as the hybridization probe. Multiple bands were detected, the longest of which was 2.9 kb (Fig. 6). PCR products were ligated into the TA cloning vector, and clones corresponding to the 600- and 1200-bp products were obtained and found to overlap and differ only in the length of their 5' ends. To isolate a product that encompassed all of the 5'-untranslated region of the GlcNAc-T V message, a second round of RACE PCR was then performed using two nested primers designed near the 5' end of the 0.6 kb clone, and the resulting products were subcloned and sequenced. Clones corresponding to the 1.8- and 2.9-kb bands were isolated from this second round of RACE PCR and sequenced. As before, these clones were found to differ only in the length of their 5' ends. A third round of RACE PCR using a 3' primer designed to the sequence near the 5' end of the 2.9-kb clone produced a single product of the expected size (70 bp). The assembled sequence of the RACE clones was co-linear with the sequence of the genomic clone in this region, indicating that no splicing events occurred in the 5'-UTR of the message from human brain. These results demonstrate, therefore, that the location of the 5'-most transcriptional start site utilized in brain is located approximately 2.9 kb upstream of the ATG, corresponding to the band of this size shown in Fig. 6.

To examine the 5' region flanking the 5'-most transcriptional start site, a 6-kb SacI-XhoI genomic fragment containing 848 bp of the brain 5'-UTR and 5.5 kb of 5'-flanking sequence (depicted in Fig. 7), designated pGL2-TV1, was cloned into the luciferase expression vector pGL2-basic. The activity of this region as a promoter and its responsiveness to src were then examined in transiently transfected HepG2 cells. pGL2-TV1 was found to act as a weak promoter, shown in Fig. 8, consistent with the low levels of GlcNAc-T V transcript observed in HepG2 cells and most tissues. Moreover, this DNA fragment conferred transcriptional responsiveness to src when co-transfected with a src-containing expression plasmid (Fig. 8). The similarity between the increases in GlcNAc-T V expression in the RSV-transformed cells and src stimulation of transcription from the GlcNAc-T V promoter in HepG2 cells suggests that transcriptional control is likely the most important regulatory influence of src on GlcNAc-T V activity.


Fig. 7. Schematic representation of the 5' region of the GlcNAc-T V gene. The fine line represents 8390 bp of genomic sequence whose 5' boundary is a SacI site, which is also the 5' boundary of the pGL2-TV1 construct and can be found in the GenBankTM accession number: AF004882. The XhoI site marks the 3' boundary of the pGL2-TV1 construct. TV2, TV3, and TV4 mark the 5' boundaries of the pGL2-TV2, pGL2-TV3, and pGL2-TV4 constructs, respectively. The 3' boundaries of these three constructs are each 70 bp 3' of the transcriptional start site. The PEA-3 (ets-2 binding sites) and AP-1 sites necessary for full src- responsiveness are denoted above the line. The bold line denotes the GlcNAc-T V open reading frame.
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Fig. 8. The TV1 fragment promoter activity is responsive to src and is dependent on raf. The TV1 fragment depicted in Fig. 7, representing a 6240-bp SacI-XhoI fragment of the 5'-flanking region, was cloned into the pGL2-Basic luciferase reporter vector and used to transfect Hep G-2 cells in the presence and the absence of a co-transfected src-expression plasmid. A dominant negative raf-1 expression plasmid, Raf C4B, was also utilized in some transfections, as well as an expression plasmid containing an inactive point mutant of the same, Raf C4B pm17.
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Transformation and some transcriptional activation by src occurs via the MAPK pathway in a Raf-1-dependent manner. For example, a dominant-negative Raf-1 mutant suppresses src transformation of BALB/c mouse fibroblasts (37) and is able to block src-stimulated transcriptional activation of the EGR gene (38). Furthermore, this mutant is able to block serum or Ras stimulation of the transcription of an AP-1/Ets-driven gene (30). If transcriptional activation of GlcNAc-T V by src occurs at least in part via the MAPK pathway, we reasoned that the activation should be inhibited by the dominant-negative Raf-1 mutant. Consistent with this hypothesis, the transcriptional stimulation of the GlcNAc-T V promoter by src was significantly inhibited when co-transfected with a plasmid encoding a dominant-negative mutant Raf, RafC4B-DN (Fig. 8). Because proliferation often occurs in a Raf-dependent manner, this result is consistent with correlations noted between GlcNAc-T V expression and cellular proliferation in nontransformed cells (39, 40) and may predict in certain cell types a general association between GlcNAc-T V enzyme activity and cell proliferation.

To map more closely the region of the GlcNAc-T V promoter responsible for transcriptional activation by src, a series of 5' deletions containing 70 bp of the brain 5'-UTR and different amounts of the 5'-flanking region were constructed by PCR amplification from the genomic clone, the boundaries of which are depicted in Fig. 7. Promoter fragments were cloned into the pGL2-basic vector and tested for basal promoter activity and src responsiveness. Based on the results from several sets of experiments, both pGL2-TV2, which contained about 1.2 kb, and pGL2-TV3, which contained 739 bp, were both found to be weakly active as promoters and transcriptionally responsive to src (Fig. 9). The pGL2-TV4 construct containing 339 bp, however, was found to be inactive as a basic promoter and completely unresponsive to src. These results suggest a requirement for the two overlapping PEA-3 sites located near the transcriptional start site, contained in pGL2-TV3, in the src-mediated transcriptional activation of the GlcNAc-T V gene (Figs. 7 and 10).


Fig. 9. Mapping the Src-responsive region of the GlcNAc-T V genomic fragments. Fragments TV1 through TV4 were separately subcloned into the pGL2-Basic luciferase reporter vector and tested for src responsiveness. The TV3 fragment contained the shortest sequence that showed responsiveness and includes two overlapping PEA-3 sites upstream from a single AP-1 site.
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Fig. 10. Nucleotide sequence of the GlcNAc-T V genomic fragment TV3. This sequence corresponds to the shortest fragment tested that conferred src responsiveness to a GlcNAc-T V promoter-driven luciferase gene. The fragment was generated as described under "Experimental Procedures" and included 70 bp 3' of the transcriptional start site. PEA-3 (Ets-2 binding sites) and AP-1 binding sites are denoted by solid underlining and dotted underlining, respectively. The 5' and 3' ends correspond to positions -659 and +70, respectively, as depicted in Fig. 7.
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PEA-3 sites are bound by the Ets family of transcriptional activators and mediate transcriptional activation in response to mitogenic signals from plasma membrane-associated tyrosine kinase oncogenes (41). To obtain more direct evidence for the involvement of an Ets family transcription factor in src-stimulated transcription of the GlcNAc-T V gene, we exploited the availability of an ets-2 expression plasmid along with an expression plasmid that encoded a truncated ets-2 with a dominant-negative activity, ets-2Delta 1-328 (31). Co-transfection of pGL2-TV3 with ets-2 resulted in stimulation of transcription, whereas co-transfection with ets-2Delta 1-328 was able to block src-transcriptional stimulation (Fig. 11). This result demonstrates that this region is capable of being transcriptionally stimulated in response to ets-2 expression and suggests that src transcriptionally activates this region through a factor that binds to the PEA-3 site(s).


Fig. 11. Src responsiveness to the TV3 fragment can be inhibited by a dominant negative ets-2. pGL2-TV3 was transfected into HepG2 cells alone, with src, ets-2, and/or a dominant negative mutant of ets-2, Delta 1-328.
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DISCUSSION

A major regulatory mechanism for controlling the expression of glycan structures is alteration in the activities of the enzymes involved in their synthesis. Changes in glycosyltransferase activity may occur by several different mechanisms, including post-translational interactions with specifier proteins (42) and translational control of glycosyltransferase transcripts. For example, the beta (1,4)-galactosyltransferase is expressed at low to moderate levels in most tissues as a 4.1-kb transcript. However, high level expression in lactating mammary gland is accompanied by a switch from one transcriptional start site to another, which is more proximal to the translational start site, giving rise to the expression of a 3.9-kb transcript (43). The longer transcript is predicted to form a stable hairpin loop structure in the 5'-untranslated region, which may reduce translation efficiency. The shorter transcript is predicted to be unable to form any significant 5' structure and therefore is thought to be translated with greater efficiency. More direct evidence of this kind of regulation can be found in a comparison between alpha (2,6)-sialyltransferase cDNAs from different tissues. The cDNAs from transcripts encoding an alpha (2,6)-sialyltransferase isolated from placenta and B cells encode identical proteins, but their 5'-untranslated regions diverge from each other approximately 180 bp upstream from the ATG. The 5'-untranslated regions confer different in vitro translational efficiencies on the respective mRNAs. The placental transcript is translated with what appears to be a 10-fold greater efficiency than is the B cell transcript. Furthermore, an artificial cDNA construct lacking the 400-bp 5'-untranslated region is translated with an apparent 10-fold greater efficiency than the B cell transcript, indicating that both 5'-untranslated regions function with different efficiencies as translational suppressors (44).

Another mechanism for the regulation of glycosylation is the alteration in the stability of the mRNAs that encode glycosyltransferases. F9 teratocarcinoma cells treated with retinoic acid plus dibutyryl cyclic AMP differentiate into parietal endoderm-like cells and express 20-fold greater specific activity of the beta (1,4)-galactosyltransferase. This increased expression of activity was found to be accompanied by a similar increase in the expression levels of the mRNA encoding the enzyme. However, promoter activity determinations based on luciferase reporter activity, as well as nuclear run-off assays both indicated there to be no significant increase in the transcription rate of the gene. The elevated expression of the mRNA was found to be associated with a marked increase in the half-life of both the short and long transcripts encoding the enzyme (45). In a similar study an effect on the stability of the mRNA for N-acetylglucosaminyltransferase V has been shown to account for an increase in mRNA levels in B-16 mouse melanoma cells treated with transforming growth factor-beta (2).

Finally, control of glycan structure through control of the transcriptional activity of glycosyltransferase genes has now been described in several systems. Early indications of this regulatory mechanism were found in a functional analysis of a promoter of the alpha (2,6)-sialyltransferase gene. This transferase is highly expressed in liver, and its promoter was found to contain binding sites for liver-enriched transcription factors. These sites and their cognate transcription factors were found to be functional in luciferase reporter transactivation assays in Hep G2 cells but were not functional in Chinese hamster ovary cells. These transcription factors and their binding sites are thought to be responsible for the liver-restricted expression of this message (46, 47). Also, retinoic acid-induced differentiation of F9 teratocarcinoma cells has been documented to be associated with an increase in the activity of the murine alpha (1, 3)-galactosyltransferase and a switch in the expression of terminal capping structures from alpha (2, 3)-linked sialic acid to galactose. The increase in the activity of the transferase is associated with an elevation in the steady-state levels of the mRNA and a similar increase in the transcription rate of the gene (1).

The association between oncogenic transformation and increases in GlcNAc-T V enzyme activity and expression of GlcNAcbeta (1,6)Man branches on glycoproteins raises the question concerning the mechanism by which oncogenes and proliferative signals in general up-regulate the synthesis of GlcNAcbeta (1,6)Man branches. From our results it is reasonable to conclude that the increases in GlcNAc-T V enzyme activity seen in src-transformed cells are not due to an increase in the catalytic efficiency of the glycosyltransferase; rather, they are due to an increase in the expression of the GlcNAc-T-V mRNA and subsequently an increase in the number of GlcNAc-T V enzyme molecules/cell. The elevation in steady-state mRNA levels could be a result of increased mRNA stability or an increase in the transcription rate of the gene. Our results demonstrate that a 5' fragment of the GlcNAc-T V gene is able to confer src-transcriptional responsiveness upon a heterologus reporter gene, indicating that the mechanism by which src increases steady-state mRNA levels is most likely transcriptional activation. Also, the 5'-flanking region of the GlcNAc-T V gene was found to contain AP-1 and PEA-3 binding sites, the presence of at least two being required for src-stimulated transcriptional enhancement. These data suggest that cooperation may be required between AP-1 and Ets transcription factors to render the gene fully responsive to oncogenic stimulation. Finally, transcriptional stimulation by src was found to be significantly dependent on the Ras-Raf pathway, again implicating transcription factors that are activated by the MAPK pathway and suggesting a general association, at least in some cell types, between GlcNAcbeta (1,6)Man branching and cellular proliferation. Other studies have shown that particular glycosyltransferase activities are increased after ras-transformation; notably, alpha (2,6)-sialyltransferase activity in NIH3T3 cells is specifically increased by ras expression, and this increase is due to increased levels of the mRNA encoding this enzyme (49, 50).

The regulation of the expression of the GlcNAc-TV gene is quite complex, involving the use of multiple promoters and alternative splicing. The brain transcript described in this paper begins at a promoter about 3 kb upstream of the translation initiation site, generating a long 5'-untranslated sequence that is unspliced and colinear with the genomic sequence. This transcript and its promoter are different from those observed in HuCC-T1 human bile duct carcinoma cells (48). The GlcNAc-T V mRNAs in these cells appear to be initiated from multiple promoters, one of which is about 1.4 kb downstream of the brain promoter. These mRNAs are also differentially spliced in the 5'-UTR to generate messages with much shorter 5'-UTRs. There are also data to suggest that there are additional promoters and alternative splices used by A431 cells when expressing this gene.2 The precise identification of the cis-acting sequences that mediate the activation of GlcNAc-T V transcription from these alternate promoters will require further investigation. Other regulatory mechanisms may also be operative in some cell types, including translational control. The GlcNAc-T V transcript expressed in human brain and in HepG2 cells is approximately three times larger than the coding region, indicating the presence of extensive 5'- and 3'-untranslated sequences. The transcript from human brain was found to have a 5'-untranslated region of approximately 3 kb, the longest 5'-UTR of any glycosyltransferase observed to date. This 5'-UTR may function as a regulator of translation efficiency, as has been suggested in the case of the mammary-specific transcript for the beta (1,4)-galactosyltransferase and demonstrated for the placental and B cell forms of the alpha (2,6)-sialyltransferase.

Of what consequence is the increased expression of GlcNAcbeta (1,6)Man branches during proliferation? One possibility is that these branches expressed on cell surface proteins that function in cell adhesion can significantly alter the adhesiveness of cells to the extracellular matrix or to each other, allowing them to become more migratory. Evidence in support of this hypothesis has been observed in the case of mink lung epithelial cells stabily transfected with and overexpressing mouse GlcNAc-T V (51). The cells overexpressing GlcNAc-T V show an altered morphology show altered rates of migration in an in vitro assay. Moreover, these cells show less adhesion to laminin-coated surfaces, compared with the nontransfected controls, suggesting an effect of GlcNAc-T V expression on laminin adhesion.


FOOTNOTES

*   This research was supported by the NCI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF004882.

In memoriam, E.A.P.


§   Present address: The Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231.
par    To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, Life Science Bldg., University of Georgia, Athens, GA 30602. Tel.: 706-542-1702; Fax: 706-542-1759; E-mail: pierce{at}bscr.uga.edu.
1   The abbreviations used are: GlcNAc-T V, N-acetylglucosaminyltransferase V; BHK, baby hamster kidney; RSV, Rous sarcoma virus; PEA, polyoma enhancer activator; GAPDH, glyceraldehyde phosphate dehydrogenase; RACE, rapid amplification of cDNA ends; GlcNAc-T I, N-acetylglucosaminyltransferase I; MES, 4-morpholineethanesulfonic acid; kb, kilobase pair(s); PCR, polymerase chain reaction; bp, base pair(s); UTR, untranslated region.
2   N. Fregien, unpublished observation.

ACKNOWLEDGEMENT

We are grateful to Dr. Kelley Moremen for many insightful discussions and suggestions.


REFERENCES

  1. Cho, S. K., Yeh, J., Cho, M., and Cummings, R. D. (1996) J. Biol. Chem. 271, 3238-3246 [Abstract/Free Full Text]
  2. Miyoshi, E., Nishikawa, A., Ihara, Y., Saito, H., Uozumi, N., Hayashi, N., Fusamoto, H., Kamada, T., and Taniguchi, N. (1995) J. Biol. Chem. 270, 6216-6220 [Abstract/Free Full Text]
  3. Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) J. Biol. Chem. 269, 10637-10643 [Abstract/Free Full Text]
  4. Finne, J., Castori, S., Feizi, T., and Burger, M. M. (1989) Int. J. Cancer 43, 300-304 [Medline] [Order article via Infotrieve]
  5. Kawano, I., Takasaki, S., Tao, T.-W., and Kobata, A. (1993) Int. J. Cancer 53, 91-96 [Medline] [Order article via Infotrieve]
  6. Buck, C. A., Glick, M. C., and Warren, L. (1971) Biochemistry 10, 2176-2180 [Medline] [Order article via Infotrieve]
  7. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. (1987) Science 236, 582-585 [Medline] [Order article via Infotrieve]
  8. Buck, C. A., Glick, M. C., and Warren, L. (1970) Biochemistry 9, 4567-4576 [Medline] [Order article via Infotrieve]
  9. Tuszynski, G. P., Baker, S. R., Fuhrer, J. P., Buck, C. A., and Warren, L. (1978) J. Biol. Chem. 253, 6092-6099 [Abstract]
  10. Warren, L., Fuhrer, J. P., and Buck, C. A. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1838-1842 [Abstract]
  11. Warren, L., Critchley, D., and Macpherson, I. (1972) Nature 235, 275-278 [Medline] [Order article via Infotrieve]
  12. Glick, M. C. (1979) Biochemistry 18, 2525-2532 [Medline] [Order article via Infotrieve]
  13. Glick, M. C., and Buck, C. A. (1973) Biochemistry 12, 85-90 [Medline] [Order article via Infotrieve]
  14. Meezan, E., Wu, H. C., Black, P. H., and Robbins, P. W. (1969) Biochemistry 8, 2518-2524 [Medline] [Order article via Infotrieve]
  15. Yamashita, K., Ohkura, T., Tachibana, Y., Takasaki, S., and Kobata, A. (1984) J. Biol. Chem. 259, 10834-10840 [Abstract/Free Full Text]
  16. Arango, J., and Pierce, M. (1988) J. Cell. Biochem. 37, 225-231 [Medline] [Order article via Infotrieve]
  17. Santer, U. V., DeSantis, R., Hard, K. J., van Kuik, J. A., Vliegenthart, J. F., Won, B., and Glick, M. C. (1989) Eur. J. Biochem. 181, 249-260 [Abstract]
  18. Yamamura, K., Takasaki, S., Ichihashi, M., Mishima, Y., and Kobata, A. (1991) J. Invest. Dermatol. 97, 735-741 [Abstract]
  19. Yamashita, K., Tachibana, Y., Ohkura, T., and Kobata, A. (1985) J. Biol. Chem. 260, 3963-3969 [Abstract]
  20. Yousefi, S., Higgins, E., Daoling, Z., Pollex-Kruger, A., Hindsgaul, O., and Dennis, J. W. (1991) J. Biol. Chem. 266, 1772-1782 [Abstract/Free Full Text]
  21. Lu, Y., and Chaney, W. (1993) Mol. Cell. Biochem. 122, 85-92 [Medline] [Order article via Infotrieve]
  22. Palcic, M. M., Ripka, J., Kaur, K. J., Shoreibah, M., Hindsgaul, O., and Pierce, M. (1990) J. Biol. Chem. 265, 6759-6769 [Abstract/Free Full Text]
  23. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. (1987) Science 236, 582-585 [Medline] [Order article via Infotrieve]
  24. Lu, Y., Pelling, J. C., and Chaney, W. G. (1994) Clin. Exp. Metastasis 12, 47-54 [Medline] [Order article via Infotrieve]
  25. Fernandes, B., Sagman, U., Auger, M., Demetriou, M., and Dennis, J. W. (1991) Cancer Res. 51, 718-723 [Abstract]
  26. Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 615-619 [Abstract]
  27. Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1991-1995 [Abstract]
  28. Shoreibah, M., Perng, G.-S., Adler, B., Weinstein, J., Basu, R., Cupples, R., Wen, D., Browne, J. K., Buckhaults, P., Fregien, N., and Pierce, M. (1993) J. Biol. Chem. 268, 15381-15385 [Abstract/Free Full Text]
  29. Graham, F. L., and van der Eb, A. J. (1973) Virology 54, 536-539 [Medline] [Order article via Infotrieve]
  30. Bruder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes & Dev. 6, 545-556 [Abstract]
  31. Aperlo, C., Pognonec, P., Stanley, R., and Boulukos, K. E. (1996) Mol. Cell. Biol. 16, 6851-6858 [Abstract]
  32. Uehara, Y., Hori, M., Takeuchi, T., and Umezawa, H. (1985) Jpn. J. Cancer Res. 76, 672-675 [Medline] [Order article via Infotrieve]
  33. Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S. (1989) Cancer Res. 49, 780-785 [Abstract]
  34. Uehara, Y., Murakami, Y., Mizuno, S., and Kawai, S. (1988) Virology 164, 294-298 [Medline] [Order article via Infotrieve]
  35. Murakami, Y., Mizuno, S., Hori, M., and Uehara, Y. (1988) Cancer Res. 48, 1587-1590 [Abstract]
  36. Suzukake-Tsuchiya, K., Moriya, Y., Hori, M., Uehara, Y., and Takeuchi, T. (1989) J. Antibiot. (Tokyo) 42, 1831-1837 [Medline] [Order article via Infotrieve]
  37. Qureshi, S. A., Joseph, C. K., Hendrickson, M., Song, J., Gupta, R., Bruder, J., Rapp, U., and Foster, D. A. (1993) Biochem. Biophys. Res. Commun. 192, 969-975 [CrossRef][Medline] [Order article via Infotrieve]
  38. Qureshi, S. A., Rim, M., Bruder, J., Kolch, W., Rapp, U., Sukhatme, V. P., and Foster, D. A. (1991) J. Biol. Chem. 266, 20594-20597 [Abstract/Free Full Text]
  39. Perng, G. S., Shoreibah, M., Margitich, I., Pierce, M., and Fregien, N. (1995) Glycobiology 4, 867-871 [Abstract]
  40. Hahn, T. J., and Goochee, C. F. (1992) J. Biol. Chem. 267, 23982-23987 [Abstract/Free Full Text]
  41. Wasylyk, B., Wasylyk, C., Flores, P., Begue, A., Leprince, D., and Stehelin, D. (1990) Nature 346, 191-193 [CrossRef][Medline] [Order article via Infotrieve]
  42. Brew, K., Vanaman, T. C., and Hill, R. L. (1968) Biochemistry 59, 491-497
  43. Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359 [Abstract/Free Full Text]
  44. Aasheim, H. C., Aas-Eng, D. A., Deggerdal, A., Blomhoff, H. K., Funderud, S., and Smeland, E. B. (1993) Eur. J. Biochem. 213, 467-475 [Abstract]
  45. Kudo, T., and Narimatsu, H. (1995) Glycobiology 5, 397-405 [Abstract]
  46. Svensson, E. C., Soreghan, B., and Paulson, J. C. (1990) J. Biol. Chem. 265, 20863-20868 [Abstract/Free Full Text]
  47. Svensson, E. C., Conley, P. B., and Paulson, J. C. (1992) J. Biol. Chem. 267, 3466-3472 [Abstract/Free Full Text]
  48. Saito, H., Gu, J., Nishikawa, A., Ihara, Y., Fujii, J., Kohgo, Y., and Taniguchi, N. (1995) Eur. J. Biochem. 233, 18-26 [Abstract]
  49. Easton, E. W., Bolscher, J. G. M., and van den Eijnden, D. H. (1991) J. Biol. Chem. 266, 21674-21680 [Abstract/Free Full Text]
  50. Le Marer, N., Laudet, V., Svensson, E. C., Cazlaris, H., Van Hille, B., Lagrou, C., Stehelin, D., Montreuil, J., Verbert, A., and Delannoy, P. (1992) Glycobiology 2, 49-56 [Abstract]
  51. Demetriou, M., Nabi, I. R., Coppolino, M., Dedhar, S., and Dennis, J. W. (1995) J. Cell. Biol. 130, 383-392 [Abstract]

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