©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vacuolar H-ATPase Mutants Transform Cells and Define a Binding Site for the Papillomavirus E5 Oncoprotein (*)

(Received for publication, September 23, 1994; and in revised form, December 8, 1994)

Thorkell Andresson (1) Jason Sparkowski (1) David J. Goldstein (1) (2) Richard Schlegel (1)(§)

From the  (1)Departments of Pathology and (2)Obstetrics and Gynecology, Georgetown University Medical School, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The 16K subunit of the vacuolar H-ATPase binds specifically to the bovine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has been suggested that this interaction may contribute to cell transformation (Goldstein, D. J., and Schlegel, R.(1990) EMBO J. 9, 137-146; Goldstein, D. J., Finbow, [Abstract] M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R.(1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., [Medline] and Schlegel, R.(1993) J. Virol. 67, 6170-6178; [Abstract] Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within [Medline] the 16K protein to define binding domains for BPV-1 E5 as well as to characterize the role of 16K in cell transformation. 16K consists predominantly of 4 transmembrane (TM) domains. We showed that mutations within the TM4 domain severely inhibited E5 binding. More specifically, conversion of glutamic acid 143 to arginine within TM4 severely reduced 16K/E5 binding, suggesting that charged interactions facilitated efficient binding. This hypothesis was confirmed by demonstrating that binding to the defective 16K arginine mutant could be restored by complementary charge mutations in E5; conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanced interactions with the 16K arginine mutant. Surprisingly, mutants in TM4 not only bound poorly to wild-type E5 but were converted into an oncoprotein and induced anchorage-independent growth of NIH 3T3 cells. These data define glutamic acid 143 in the 16K TM4 domain and glutamine 17 within E5 as important contributors to E5/16K binding and suggest a role for the 16K protein in the regulation of cell proliferation.


INTRODUCTION

The vacuolar proton ATPase (vacuolar H-ATPase) is a large enzyme complex that is present in several intracellular membrane compartments (such as endosomes, lysosomes, and the Golgi apparatus) and drives the unidirectional flux of protons from the cytoplasmic to lumenal sides of these organelles(5, 6, 7) . This enzyme complex has also been detected in the plasma membranes of renal epithelial cells (5) and, in some cells, may contribute to the intracellular alkalinization observed during cellular transformation(8) . This proton flux causes a limited acidification, which is critical for cell viability(5, 6, 7, 9) as well as for the proper processing, targeting, and function of cellular proteins in these compartments(10, 11, 12, 13, 14, 15) . The vacuolar H-ATPase is composed of approximately 5-10 different proteins ranging in size from 16-115 kilodaltons, and the membrane proton pore appears to be composed of four of these proteins (116K, 39K, 20K, and 16K)(5, 16, 17, 18) . The 16K protein, which is highly hydrophobic and composed almost entirely of four putative transmembrane domains, is believed to be the main component of the proton pore(16, 19, 20) .

Acidification of endosomes appears to be critical for determining the fate of receptor-ligand complexes following endocytosis(5) , making the pump an important regulator of signal transduction pathways. In addition, acidification of the trans-Golgi apparatus may regulate the transport/processing of proteins that proceed through this compartment, including growth factor receptors. Interestingly, activation and altered processing of epidermal growth factor receptors(21) , as well as activation of PDGF (^1)receptors(22, 23) , is a characteristic of cell transformation induced by the bovine papillomavirus type-1 E5 oncoprotein. The E5 gene is the main transforming gene of bovine papillomavirus type-1(24) , and it encodes a 44-amino acid oncoprotein that is composed of two distinct domains: a hydrophobic transmembrane region and a hydrophilic dimerization domain (25) . E5 has recently been shown to exist in an in vivo complex with the PDGF receptor(22, 26, 27) , the epidermal growth factor receptor(26) , and the 16K subunit of vacuolar H-ATPase(1, 2, 22, 28) . It appears that E5 may actively recruit 16K into a ternary complex with PDGF receptor, suggesting a role for 16K in signal transduction.

The intent of this investigation was to define the domain(s) of 16K that is responsible for binding E5 and to evaluate the role of 16K in cell transformation. 16K has been cloned and sequenced from several organisms and exhibits strong conservation at the amino acid level. This conservation is especially high in the transmembrane domains, with the carboxyl-terminal fourth transmembrane domain (TM4) showing 100% homology in yeast, nehrops, mouse, and cow. Since previous studies have shown that the yeast 16K can bind efficiently to the E5 oncoprotein(4) , we targeted TM4 for mutagenesis. More specifically, the interaction between the hydrophobic transmembrane domains of 16K and E5 most likely involves the interaction between charged or polar residues since the replacement of the glutamine residue in the transmembrane domain of E5 dramatically inhibits its binding to 16K (as well as its transforming activity)(1, 28) . The 16K TM4 domain is the only transmembrane domain containing a charged amino acid (glutamic acid). This glutamic acid residue is also critical for the biological activity of the vacuolar proton pump, and its conversion to alternative amino acids is lethal for yeast(9) . Our studies indicate that the glutamic acid residue of 16K participates in the binding of E5, that its mutagenic alteration converts the 16K protein into a transforming protein, and that wild-type 16K can suppress the oncogenic activity of E5.


MATERIALS AND METHODS

Plasmid Construction

PCR techniques were used to generate wild-type and mutant 16K expression vectors. The wild-type AU1 16K construct (pTA11) has been described in detail elsewhere(22) . Briefly, primers corresponding to the 5`- (ON11) and 3`- (ON6) end of the bovine 16K cDNA (2) were synthesized encoding XhoI and BamHI restriction sites, respectively. The 5`-primer also included the 6-amino acid epitope (AU1) immediately after the ATG, which allows for efficient recognition by the monoclonal antibody AU1 (29) . A second wild-type 16K construct was generated utilizing the same PCR technique; however, the 5`-primer encoded for the 11-amino acid epitope HA1(30) . The PCR fragment was cloned into the vector pSVL (Pharmacia Biotech Inc.) for transient expression in COS cells. The truncated 16K was generated by using ON11 and a 3`-primer complementary to 12 internal nucleotides, creating a truncated 16K after Ile-136. Arg-143 16K was generated by a two step PCR: two internal primers were made to encode the glutamic acid-to-arginine mutation. These primers were used in the first PCR reaction with ON11 and ON6. The fragments from the first PCR were then used as a template for the second PCR reaction with ON11 and ON6. Both of these mutants were then cloned into the XhoI and BamHI sites of pSVL. Two 16K constructs were made to express separately the alpha helices 1 and 2 (alpha-1,2 16K) and the alpha helices 3 and 4 (alpha-3,4 16K). The alpha-1,2 16K was made by one-step PCR using ON11 and a 3`-primer containing 12 nucleotides complementary to codons 83-86 of 16K followed by stop codon and a BamHI site. The alpha-3,4 16K was made by two-step PCR. The first PCR utilized ON6 and a 5`-primer containing nucleotide sequences corresponding to codons 2-8 from the amino terminus of 16K and nucleotides complementary to codons 91-95. The first PCR product was isolated and used as a template for the second PCR reaction, using ON11 and ON6. The HA1 E5 and E5 mutants were generated by one- or two-step PCR techniques, respectively, using HA1 E5 as a template(22, 31) . All constructs generated by PCR amplification were sequenced to verify their nucleotide composition.

Plasmids used for transformation assay were generated by a two-step cloning procedure. XhoI-BamHI 16K fragments from pSVL constructs were cloned into the SalI-BamHI site of the vector pIC (a gift of M. Finbow). The pIC-16K plasmids were cleaved with HindIII and ClaI and cloned into the HindIII-ClaI sites of the LNCX expression vector (32) .

DNA Transfection

DNA was transfected into NIH 3T3 or COS cells using the CaPO(4)-DNA coprecipitation method (33) . The DNA was mixed in 500 µl of 1 times HEPES-buffered saline followed by the addition of 50 µl of 1.25 M CaCl(2). Following incubation for 30 min, the precipitated DNA was added to 100-mm plates of COS or NIH 3T3 cells at 60% confluence in 5 ml of DMEM. After overnight incubation, the cells were glycerol-shocked for 1 min with 1.5 ml of 15% glycerol in 1 times HEPES, washed 3 times with PBS, and supplemented with 10 ml of DMEM.

Cell transformation assays by E5 were performed using the transfection procedure described above. Following the glycerol shock, however, the cells were cultured until confluent, transferred into 75-or 162-mm^2 flasks, and then fed every 3 days thereafter. 2-3 weeks later, the cells were washed once with 1 times PBS and then stained with 1% methylene blue in 100% ethanol, and the number of foci were counted.

Immunoprecipitation

Cells were incubated for 1.5 h in methionine- and cysteine-free DMEM medium and then labeled with 0.25 µCi/ml [S]methionine/cysteine mixture (DuPont NEN) for 4 h. Cells were extracted in 1 ml of modified radioimmunoprecipitation assay buffer (20 mM MOPS, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholate, and 0.1% SDS, pH 7.0) containing 0.5 M phenylmethylsulfonyl fluoride (Sigma). Immunoprecipitation was performed by adding 5 µl of AU1 or 5 µl of 12CA5 ascites fluid and 50 µl of a 1:1 suspension of protein A-Sepharose CL-40 beads (Pharmacia) in PBS to the extracts. The immunoprecipitation was incubated for 2 h at 4 °C followed by three washes of the immune complex in 1 ml of extraction buffer. Sepharose beads were then resuspended in 60 µl of sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% beta-mercaptoethanol). Samples were heated at 100 °C for 5 min and then separated on 14% SDS-polyacrylamide gels.

The procedure described above was modified for a double immunoprecipitation. Following 1.5 h of incubation at 4 °C with the first antibody, the immune complex was washed once in extraction buffer and then treated with 200 µl of SOL buffer (50 mM triethylamine HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.4% SDS, and 2 mM beta-mercaptoethanol), heated for 2 min at 100 °C, and then cooled on ice. 4 µl of 0.5 M iodoacetamide was added, and the suspension was centrifuged. The supernatant was collected, and 50 µl of 10% Triton X-100 was added. The second immunoprecipitation was performed using 5 µl of the AU1 antibody plus 50 µl of protein A-Sepharose CL-4B beads. Following 1.5 h of incubation at 4 °C, the immune complex was washed once with PBS. Precipitated proteins were separated on a 14% SDS-polyacrylamide gel.

Generation of Stable NIH 3T3 Cell Lines

60% confluent NIH 3T3 cells on 100-mm plates were transfected with 1 µg of the LNCX-16K DNAs or the LNCX vector alone plus 9 µg of carrier DNA (pUC18) using the CaPO(4)-DNA coprecipitation method as described above. At 16 h post-transfection, cells were glycerol shocked with 1 ml of 15% glycerol in 1 times HEPES-buffered saline, grown to 100% confluence, and then split into 162-mm flasks. At 85% density, the media were supplemented with 1 mg/ml G418. Approximately 50 G418-resistant colonies were pooled and expanded.

Anchorage-independent Growth Assay

1 times 10^4 cells from each cell line were added to DMEM, 0.3% agarose supplemented with 10% fetal calf serum. The mixture was subsequently added to 60-mm dishes coated with DMEM, 10% fetal calf serum, 0.6% agarose. Each cell line was plated in triplicate. The cells were incubated at 37 °C for 4 weeks with weekly medium changes.

Southern Blot Analysis

High molecular weight DNA was isolated from 75-mm^2 flasks of cell lines generated with the vector LNCX, wild-type AU1 16K, and truncated 16K. The DNA was digested with BamHI, which generates a 1.3-kilobase fragment including the CMV promoter and 16K. This same fragment was also used as a probe for Southern analysis. Labeling of the probe was performed by a random priming method, using [P]ATP (Boehringer Mannheim). DNA was electrophoresed on a 1% agarose gel and blotted onto GeneScreen Plus membrane (DuPont NEN). The blot was pre-hybridized in 10% dextran sulfate, 1 M NaCl, 1% SDS, and 50% formamide for 6 h, probed overnight, and exposed to Kodak XAR-5 film.


RESULTS

To identify the E5 binding site on the 16K protein and to study the role of 16K in transformation, six 16K constructs were made using PCR techniques: two wild-type 16K constructs (AU1 16K and HA1 16K), a construct expressing alpha helices 1 and 2 (alpha-1,2 16K), a construct expressing alpha helices 3 and 4 (alpha-3,4 16K), a truncated 16K lacking the fourth transmembrane domain, and a single amino acid substitution (glutamic acid to arginine) in the fourth transmembrane domain (Arg-143 16K) (Fig. 1). Due to the lack of antibodies against native 16K, the AU1 or HA1 epitopes were tagged to the amino terminus of 16K proteins to facilitate immunoprecipitation(29, 30) . All constructs were cloned into and expressed by the pSVL vector.


Figure 1: Epitope-tagged forms of wild-type and mutant 16K. PCR techniques were used to append the HA1 and AU1 epitopes to the amino terminus of the indicated 16K mutants. These mutants were then expressed by the SV40 late promoter (pSVL vector) for transient expression in COS cells or by the CMV promoter (LNCX vector) for stable expression in cell lines. Each box represents a transmembrane domain, and the soliddot indicates the substitution of glutamic acid with arginine at position 143.



The 16K Binding Site for E5 Maps to alpha Helices 3 and 4

To map the binding site for E5 on 16K, we performed cotransfection and immunoprecipitation experiments in COS cells utilizing the HA1 E5(22) , alpha-1,2 16K, and alpha-3,4 16K constructs (Fig. 1). Double immunoprecipitations were necessary since E5 and the deleted 16K proteins have a very similar mobility on polyacrylamide gels. The first immunoprecipitation was performed using the 12CA5 antibody, which recognizes the HA1 epitope on E5. The second immunoprecipitation, following dissociation of the immune complex, was performed with the AU1 antibody, which recognizes the AU1 epitope on the 16K proteins. As demonstrated in Fig. 2, E5 was able to coprecipitate the alpha-3,4 16K (lane5) efficiently but not the alpha-1,2 16K (lane1). Longer exposure of the gels revealed detectable, low amounts of alpha-1,2 16K associated with E5. However, this difference in binding is actually even more dramatic than visualized because alpha-1,2 16K contains 8 methionine residues, and alpha-3,4 16K contains only 2 methionine residues that can be radioactively labeled. A control immunoprecipitation experiment using antibody 12CA5, which had been pre-absorbed with immunogenic HA1 peptide (1 mg of peptide/1 ml of ascites fluid), was also performed. Fig. 2(lanes2 and 6) shows that the coprecipitation of the 16K proteins in lane5 is dependent on immunoprecipitation of E5. Single immunoprecipitations performed on aliquots of the lysate verified the expression of E5 (lanes3 and 7) and 16K (lane4, alpha-1,2 16K; lane8, alpha-3,4 16K).


Figure 2: The main binding site for E5 on 16K maps to alpha helices 3 and 4. COS cells were cotransfected with 1 µg of the HA1 E5 and 1 µg of either the alpha-1,2 16K (lanes1-4) or the alpha-3,4 16K (lanes5-8). Double immunoprecipitation was performed using 5 µl of either 12CA5 (lanes1 and 5) or pre-absorbed 12CA5 (lanes2 and 6) as the first antibody, followed by a second immunoprecipitation with 5 µl of AU1. To verify expression of the E5 and 16K, a single immunoprecipitation was performed using 5 µl of either 12CA5 for E5 expression (lanes3 and 7) or AU1 antibody for 16K expression (lanes4 and 8).



The Binding Site for E5 Maps Predominantly to the Fourth Transmembrane Domain of 16K and Is Modulated by Charge Interactions

Fig. 2demonstrates that a major binding site for E5 on 16K is contained within alpha helices 3 and 4. To map this binding site more specifically, two mutants were generated within the highly conserved fourth transmembrane domain: a truncated 16K, which has a deletion of alpha helix four, and a point mutant, which has glutamic acid 143 substituted with arginine (Arg-143 16K) (Fig. 1). To evaluate the synthesis and stability of these mutated 16K proteins, the constructs were expressed in COS cells. At 48 h post-transfection, immunoprecipitations of transiently transfected cells demonstrated that the mutant forms of 16K were abundantly synthesized (Fig. 3A), with the deletion mutant exhibiting the anticipated increase in electrophoretic mobility (Fig. 3A, lane3).


Figure 3: Specific amino acids within 16K and E5 transmembrane domains mediate their interaction. COS cells were transfected with the DNAs indicated at the top of each gel and then metabolically labeled with [S]methionine, lysed in radioimmunoprecipitation assay buffer, and immunoprecipitated with either AU1 or 12CA5 antibody. Immunoprecipitated proteins were separated on SDS-polyacrylamide gels and visualized by fluorography. Positions of molecular mass standards (in kilodaltons) are indicated to the left of each gel. A, expression of two mutant forms of 16K. COS cells were transfected with pSVL (lane1), wild-type 16K (lane2), truncated 16K (lane3), or Arg-143 16K (lane4) and immunoprecipitated with 5 µl of AU1. B, mutations in the fourth transmembrane domain of 16K affect binding to the E5 oncoprotein. Coprecipitations using 5 µl of 12CA5 were performed on COS cells cotransfected with the DNAs indicated at the top. Since 16K constructs express more efficiently than E5 constructs, 0.1 µg of wild-type AU1 16K and 0.25 µg of the truncated and Arg-143 16K DNAs were used to cotransfect COS cells with 1 µg of E5 DNA. C, E5/16K binding is governed by charge interactions. COS cells were cotransfected with 1 µg of either of the two E5 mutants (glutamic acid and aspartic acid substitutions) and 1 µg of the Arg-143 16K, as indicated at the top. Labeled cell extracts of cotransfected cells were immunoprecipitated with 5 µl of 12CA5 antibody. D, binding efficiency between E5 and wild-type 16K is reduced by substitution of glutamine 17 with glutamic or aspartic acid. COS cells were transfected with 1 µg of the indicated E5 constructs. Labeled cell extracts were then immunoprecipitated with 5 µl of the monoclonal antibody 12CA5. Positions of molecular mass standards (in kilodaltons) are indicated to the left of each gel.



The ability of the two mutated 16K proteins to form a complex with E5 protein was evaluated by coprecipitation (Fig. 3B). Plasmids encoding the 16K proteins and the HA1 E5 protein were cotransfected into COS cells and, 48 h later, immunoprecipitated with a monoclonal antibody (12CA5) against the HA1 epitope on E5. The amounts of immunoprecipitated E5 protein and coprecipitated 16K protein were quantitated by film densitometry, and the ratio of 16K/E5 was calculated from three different experiments (Table 1A). The deleted form of 16K protein bound 8% (Fig. 3B, lane2), and the arginine mutant bound 28% of wild-type levels of E5 protein (Fig. 3B, lane3). These results demonstrate the importance of TM4 and, more specifically, the glutamic acid in TM4 for E5 binding and indicate that E5 targets an amino acid critical for the function of the proton pump(9) .



Previous studies have implicated glutamine 17 in the hydrophobic domain of E5 as the main binding site for 16K(1, 28) . Our current results indicate that 16K/E5 binding is mediated by polar glutamine 17 (partial positive charge) in E5 and the negatively charged glutamic acid 143 in 16K. To demonstrate the apparent charge dependence of these interactions, coprecipitation experiments were performed in COS cells using Arg-143 16K, which binds with reduced efficiency to wild-type HA1 E5. The COS cells were also transfected with E5 constructs containing either glutamic acid or aspartic acid at position 17 (31) to provide complementary charge interactions with the arginine residue in 16K. These E5 mutants exhibited an anticipated decreased binding to the endogenous wild-type 16K (Fig. 3D, lanes3 and 4, and Table 1C) when compared with wild-type E5 (lane 2). On the other hand, when coprecipitation was performed using the E5 mutants and Arg-143 16K (Fig. 3C, lanes2 and 3), they bound more 16K than wild-type HA1 E5 (Fig. 3C, lane1 and Table 1B). These results demonstrated that the binding of 16K/E5 was regulated by charged intramembrane amino acids and that the interaction was optimal when the 16K and E5 amino acids were of opposite charge.

The striking mobility shift of the E5 mutants (Fig. 3, C and D) was unexpected since only one amino acid was mutated in the transmembrane domain of E5. Conformational changes in the mutated E5 or different interactions between the E5 TM and the detergent might account for this aberrant mobility.

Mutant 16K Proteins Retain Normal Binding to the Wild-type 16K

The pore of the vacuolar proton ATPase is predicted to be formed by six molecules of the 16K protein(16, 18, 34, 35) . The interaction between multiple 16K molecules would therefore regulate the integrity of the enzyme pore. The finding that E5 binds with high specificity to 16K and potentially alters the function of the vacuolar proton ATPase led us to speculate that a mutated 16K protein might also perturb the pore complex and function in a dominant-negative manner. As a first step in evaluating this possibility, we investigated the ability of two TM4 mutants to bind wild-type epitope-tagged 16K (HA1 16K). Wild-type HA1-tagged 16K was cotransfected with AU1-tagged wild-type or mutant forms of 16K into COS cells, and a double immunoprecipitation performed, first with antibody (12CA5) against the HA1 16K and then, following dissociation of the immune complexes, with AU1 antibodies against the AU1-tagged wild-type and mutant 16K proteins. Both wild-type and mutant forms of AU1 16K were coprecipitated in a complex with the HA1 16K protein with comparable efficiency (Fig. 4A), indicating that TM4 was not directly involved in mediating the interactions between 16K proteins. To verify the specificity of the double immunoprecipitation, another cotransfection was performed on COS cells using the same 16K DNAs. This time, however, a double immunoprecipitation was performed using 12CA5 antibody, which had been pre-absorbed by the HA1 peptide. Fig. 4B demonstrates that coprecipitated AU1 16K protein observed in Fig. 4A was due to its ability to interact with HA1 16K, since blocking of the HA1 antibody prevented the appearance of the AU1 16K proteins (Fig. 4B, lanes2-4).


Figure 4: Mutant 16K proteins retain normal binding to wild-type 16K protein. A, Double immunoprecipitations were performed on [S]methionine-labeled COS cells, which had been cotransfected with 1 µg of HA1 16K and 1 µg of the AU1 16K, truncated 16K, or Arg-143 16K as indicated at the top of each lane. Cotransfected COS cells were labeled, extracted in lysis buffer, and immunoprecipitated first with 5 µl of 12CA5. Immune complexes were dissociated as described under ``Materials and Methods'' and reprecipitated with 5 µl of AU1. The final immune complex was dissociated in SDS sample buffer and separated on 14% polyacrylamide gels. B, coprecipitation of AU1 16K proteins is dependent upon immunoprecipitation of the HA1 16K. Verification of the specificity of the double immunoprecipitation method was performed using a 12CA5 antibody pre-absorbed with an HA1 peptide as the first antibody on COS cells cotransfected with HA1 16K and AU1 16K constructs as described in A. Precipitated proteins were electrophoretically separated on 14% SDS-polyacrylamide gels. Positions of molecular mass standards (in kilodaltons) are indicated to the left of each gel.



Mutant 16K Proteins Transform Mouse Cells

Since mutant 16K proteins retained normal interaction with wild-type 16K proteins, it was possible that these mutant proteins might act dominantly to alter or perturb normal 16K protein function and mimic some of the activities of E5. To evaluate this possibility, the wild-type, truncated, and point mutants of 16K were cloned into the LNCX vector (32) (containing a neomycin resistance gene) to permit expression in immortalized murine fibroblasts (NIH 3T3 cells). NIH 3T3 cells were transfected with the wild-type and mutant 16K expression vectors and selected in G418. Approximately 50 drug-resistant clones from each transfection were pooled and evaluated for their ability to form anchorage-independent colonies (Fig. 5). Cell lines containing only LNCX vector or wild-type 16K did not induce NIH 3T3 cells to form colonies. However, both the deletion and arginine mutant 16K constructs induced colony formation. Quantitation of colony formation in two independent experiments indicated that the mutant 16K proteins induced colony formation at a frequency of 0.3% (Table 2). Even though the transformation ability of mutated 16K was rather low, it is similar to that recently reported for the HPV 6 E5 (0.2%) and HPV 16 E5 (0.4%) proteins(36) .


Figure 5: Mutant 16K proteins exhibit cell transformation activity. NIH 3T3 cell lines generated with AU1 16K constructs were evaluated for anchorage-independent growth by plating the cell lines into soft agar and allowing them to grow for 4 weeks. Panela, cell line generated with the vector alone; panelb, cell line generated with wild-type 16K; panelc, cell line generated with truncated 16K; paneld, cell line generated with Arg-143 16K.





Anchorage-independent cell lines were also evaluated for expression of the mutant 16K proteins. In Fig. 6A, cell lines generated with the LNCX vector, wild-type AU1 16K, truncated 16K, or Arg-143 16K were immunoprecipitated with AU1 antibody. Both the wild-type and arginine point mutant cell lines synthesized detectable levels of 16K protein (Fig. 6A), whereas the deletion mutant cell line did not. Since it was not possible to detect any protein expressed by the truncated 16K, the cause of cell transformation observed with this mutant remains uncertain. The lack of detectable truncated protein might reflect protein stability since this protein appeared less stable in COS cells than wild-type or arginine E5 proteins (Fig. 3A). Southern blot analysis clearly demonstrated that the transformed cell line contained the deleted 16K gene (Fig. 6B, lane3). Therefore, the activity of the 16K truncated protein might be sufficiently high to induce cell transformation in the presence of very low protein levels, a situation that is analogous to a potent Fos recombinant protein(37, 38) .


Figure 6: Evaluation of 16K expression in wild-type and mutant 16K cell lines. A, NIH 3T3 cell lines generated with the AU1 16K constructs were plated onto 100-mm plates, metabolically labeled with [S]methionine, extracted, and immunoprecipitated with 5 µl of AU1 antibody. Lane1, vector alone; lane2, AU1 16K; lane3, Arg-143 16K; lane4, cell line generated with deleted 16K. Proteins were resolved on 14% polyacrylamide gels. AU1 16K (lane2) and Arg-143 16K (lane3), but not the truncated 16K (lane4), expressed detectable levels of 16K proteins. Positions of molecular mass standards (in kilodaltons) are indicated to the left. B, Southern blot analysis of cell lines generated with the wild-type and deleted 16K. High molecular weight DNA from the cell lines generated with the LNCX vector, AU1 16K, and truncated 16K were isolated. DNA was cut with BamHI, separated on a 1% agarose gel, blotted onto GeneScreen Plus membrane, and probed with DNA labeled with [P]ATP harboring the 16K cDNA and the CMV promoter. Lane1, cell line generated with vector alone; lane2, cell line generated with the AU1 16K construct; lane3, cell line generated with deleted 16K.



Cotransfection of Wild-type 16K with E5 Significantly Inhibits the Ability of E5 to Transform Murine Fibroblasts

It has been suggested that one of the mechanisms by which E5 might transform cells is to bind to 16K and interfere with the function of the vacuolar proton pump. By interrupting the normal function of the pump, the processing of critical cellular proteins (such as growth factor receptors) might be altered. If this assumption were correct, providing the cell with an excess amount of wild-type 16K might restore the normal function of the pump and partially inhibit the transformation function of E5. However, providing the cell with a mutated, defective form of 16K would not be expected to inhibit E5-mediated transformation.

To evaluate these possibilities, the mouse fibroblast cell lines NIH 3T3 and C127 were plated at 50% confluence on 100-mm plates and cotransfected with 9 µg of wild-type AU1 16K, Arg-143 16K, or truncated 16K expressed from the LNCX vector and 1 µg of wild-type E5. After the plates had reached 100% confluence, they were split into either 75- or 162-cm^2 flasks and allowed to form foci. 2-3 weeks later, the number of foci formed with E5 alone (Fig. 7, panelA) was compared with the number of foci formed when the 16K genes were cotransfected with E5 (Fig. 7, panelsB-D). These studies ( Table 3and Fig. 7) revealed a 75% inhibition of E5-mediated transformation by wild-type 16K (Fig. 7, panelB), 58% inhibition by the Arg-143 16K (Fig. 7, panelC), and only 25% inhibition by the truncated 16K (Fig. 7, panelD), demonstrating that the ability of 16K to inhibit E5 transformation correlated directly with its ability to bind E5.


Figure 7: Inhibition of E5 transformation by coexpression of 16K. Mouse fibroblast cell lines, plated at 50% confluence on 100-mm plates, were cotransfected with 1 µg of wild-type E5 and 9 µg of pUC (panelA), 1 µg of wild-type E5 and 9 µg of AU1 16K (panelB), 1 µg of wild-type E5 and 9 µg of Arg-143 16K (panelC), or 1 µg of wild-type E5 and 9 µg of truncated 16K (panelD). Cells were grown for 3 weeks and stained with 1% Trypan blue; the number of foci were then counted.






DISCUSSION

We have shown that the fourth transmembrane domain of 16K is critical for its interaction with the E5 oncoprotein. More specifically, the glutamic acid residue within this domain, which is essential for the activity of the proton pump, regulates the binding of E5. Mutation of glutamic acid 143 reduced the binding to 28% of wild-type, which suggested that other residues within the domain were also involved in the interaction. Accordingly, the truncation of the fourth transmembrane domain virtually abolished binding, suggesting that the predominant binding site for E5 may be the fourth transmembrane domain. The importance of charged amino acids for the interaction between E5 and 16K was supported by the demonstration that binding between the two proteins can be restored by reversing the charges in TM4 of 16K and E5 ( Fig. 3and Table 1). These results strongly indicated that the interaction between E5 and 16K was governed by charge interaction. The targeting of such a critical domain of 16K by E5 suggests that modulation of proton pump activity may represent one of the mechanisms by which E5 contributes to cellular transformation.

Even more suggestive of the relevance of TM4 for E5 action is the finding that mutation of the glutamic acid residue converts 16K into a transforming protein. This might indicate that disrupting the function of the critical fourth transmembrane domain either by mutation or by binding E5 will cause abnormalities in the function of the pump, thereby leading to cell transformation. However, although the Arg-143 16K mutant is transforming, it does not exactly mimic the activity of E5. For example, while both E5 and the Arg-143 16K induce anchorage-independent growth, only E5 induces efficient focus formation on immortalized mouse cells. (^2)This suggests that the 16K mutant is only able to partially mimic E5 transformation. While mutant 16K might only be able to interfere with the function of the proton pump, E5 may also activate tyrosine kinase growth factor receptors, a function that has not yet been shown for the mutated 16K. These combined functions carried out by the E5 protein may be necessary to attain full transformation. However, it is also possible that the observed biologic differences between mutant 16K and E5 might reflect variations in the level of protein expression since different vectors and promoters have been used to express 16K and E5 proteins in mouse cells.

The simplest hypothesis for the mechanism by which E5 and 16K might mediate cell transformation involves their direct interaction with growth factor receptors. Thus, the dimeric form of E5 (39) might facilitate cross-linking of the PDGF receptor. This cross-linking would then initiate receptor transphosphorylation and the consequent hierarchy of signaling events leading to a mitogenic response. In this scheme, 16K, via its ability to bind E5 protein and receptors(22) , would facilitate the cross-linking of receptors or their activation. Finally, there is also the possibility that E5 transforms cells by mediating both receptor activation and altered receptor processing (proton pump activity)(3) .

It is presumed that the 16K mutant proteins transform cells by interfering with the proton pump, although direct evidence for this hypothesis has not been obtained. Mutant 16K proteins retain their ability to interact with wild-type forms of 16K and could potentially interfere with the acidification of intracellular compartments such as endosomes and consequently inhibit the degradation of internalized, activated receptors. Three reports have demonstrated that the E5 proteins of both the bovine and human papillomaviruses inhibit the degradation of internalized growth factor receptors(21, 36, 40) .

The high specificity and the nature of the binding site on 16K strongly suggest that E5-16K interaction is important for cell transformation. Verification of a direct interaction between E5 and 16K within cells was also inferred from the ability of wild-type 16K, but not non-binding mutants, to efficiently inhibit the transforming activity of E5 ( Fig. 7and Table 3). These results suggest that providing the target cell with excess wild-type 16K might supply enough 16K to restore normal proton pump activity. An alternative explanation for these results might be that excess 16K sequesters E5 away from other cellular targets, such as the PDGF receptor.

Our experiments with reciprocal charge exchange have demonstrated that E5 and 16K interact via hydrophilic, charged residues within their transmembrane domains. Not only do these experiments suggest new mechanisms for conferring specific protein interactions, they also supply the first evidence that E5 binds directly to the 16K cellular protein target. In contrast, it has not been possible to show that E5 binds directly to the PDGF receptor, although they are present within the same immunoprecipitate. Future experiments will be necessary to define the complex formed between E5, 16K, and PDGF receptor in transformed cells.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Georgetown University Medical School, 3900 Reservoir Rd., NW, Washington, D. C., 20007.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TM, transmembrane; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
T. Andresson, and R. Schlegel, unpublished results.


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