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INTRODUCTION |
The 44-amino acid E5 oncoprotein of bovine papillomavirus type 1 is the smallest known oncoprotein. The first 30 amino acids are
believed to constitute an
-helical transmembrane domain, and the
C-terminal 14 amino acids are generally hydrophilic, containing two
cysteines that stabilize homodimer formation via disulfide bonds
(1-3). Immunoelectron microscopy studies have demonstrated E5 to be a
type II Golgi polypeptide with its C terminus predominantly facing the
Golgi lumen (4).
The transformation of mouse fibroblasts by E5 has been attributed
mainly to its ability to bind and induce the autophosphorylation of
PDGF-R1 (5-9), although E5
can also interact with the epidermal growth factor receptor (10) and
transform immortalized keratinocytes (11). Other cellular proteins that
associate with E5 include the 16-kDa subunit of the vacuolar proton
pump (16K) and a 125-kDa
-adaptin like protein. Mutagenic analysis
indicates that E5/16K binding may have an important role in cellular
transformation and E5, 16K, and PDGF-R co-precipitate in transfected
cells (12-15).
Specific interactions between transmembrane
-helices are important
for the structure and function of many integral membrane proteins. For
example, interactions between two components of the T-cell receptor
complex, TCR
and CD3
, are mediated through transmembrane domains
and allow for the functional assembly of the complex (16, 17).
Similarly, dimerization of glycophorin A has been shown to be dependent
upon the amino acid sequence of its transmembrane domain (18, 19) and
the assembly of major histocompatibility complex class II molecules
appears to mediated by interactions between the transmembrane domains
of the
and
chains (20). Furthermore, the mutagenic analyses of
glycophorin A have recently been substantiated on a molecular level by
the determination of the structure of glycophorin A dimers using
heteronuclear nuclear magnetic resonance (21). The association between
E5 and the 16K V-ATPase subunit is also mediated by transmembrane interactions, specifically by an interaction between glutamine in the
E5 transmembrane domain and glutamic acid in the fourth TM domain of
16K (22). In addition, we have previously demonstrated the ability of
the isolated E5 TM domain to bind the PDGF-R, suggesting that specific
binding interactions between the TM regions of these molecules might
facilitate their interaction (12). Recent data has defined a threonine
residue in the TM region of PDGF-R that might facilitate hydrogen bond
formation with the E5 TM glutamine residue (position 17) (23).
In order to determine the contribution of amino acids in the E5
transmembrane domain to its biological and binding properties, a series
of alanine mutations was constructed. Twenty-five residues between
positions 4 and 31 of E5 were individually replaced by alanine, and the
resulting proteins were examined for their ability to: 1) associate
with PDGF-R, 2) induce the phosphorylation of PDGF-R, 3) form
homodimers, 4) localize to the Golgi apparatus, and 5) transform NIH3T3
mouse fibroblast cells. Alanine was used in the substitutions since it
neither significantly alters protein conformation nor imposes
electrostatic or steric effects (24).
Our results indicate that the mutants that perturb E5 activities
(e.g. binding to PDGF-R, activation of PDGF-R, homodimer formation) cluster within the TM domain in a pattern that is consistent with this domain being in an
-helical conformation. Thus, E5 mutants
that activate the PDGF-R align along one predicted helical face and E5
mutants that fail to activate PDGF-R align along the opposite helical
face. E5 mutants that fail to bind PDGF-R represent a clustered subset
of mutants that fail to activate the receptor. Most surprisingly, only
two mutants were transformation-defective (positions 17 and 18).
Finally, there were several E5 mutants that retained transforming
activity but failed to bind or activate PDGF-R, suggesting alternative
mechanisms for cellular transformation.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Plasmid Constructions--
COS-1 and NIH3T3
cell lines were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum.
The expression vector PJS55 was constructed by adding a polylinker
region to pSG5 (Stratagene) and has been described previously (25). All
alanine point mutants were synthesized via a two-step polymerase chain
reaction (PCR) using oligonucleotides synthesized by Life Technologies,
Inc. In the first step of each PCR reaction, two fragments were
generated. The first corresponded to the 5' end of the E5 mutant and
was constructed with a 5' oligonucleotide (5'-TTACATCTCGAGGCCACCATGGACACCTAT-3') that codes for the initiating methionine of E5 and three residues of the AU1 monoclonal antibody epitope (27) and also contains an XhoI and Kozak consensus
sequence 5' to the translational start site. The 3' oligonucleotide
used in each of these reactions contained the point mutations of
interest and 10-15 nucleotides flanking the codon. They are as
follows: L4A, 5'-CTATATACCAAATGCATGGTTTCTATTG -3'; W5A,
5'-CCAAATCTAGCGTTTCTATTG-3'; F6A, 5'-CAAATCTATGGGCTCTATTGTTC-3'; L7A,
5'-CTATGGTTTGCATTGTTCTTG-3'; L8A,
5'-CTATGGTTTCTAGCGTTCTTGGGAC-3'; F9A, 5'-GTTTCTATTGGCCTTGGGACTAG-3'; L10A, 5'-CTATTGTTCGCGGGACTAGTTG-3'; G11A,
5'-CTATTGTTCTTGGCACTAGTTGCTG-3'; V13A, 5'-CTTGGGACTAGCTGCTGCAATG-3';
M16A, 5'-GTTGCTGCAGCGCAACTGCTG-3'; Q17A,
5'-GCTGCAATGGCACTGCTGCTATTAC-3'; L18A, 5'-GCAATGCAAGCGCTGCTATTAC-3'; L19A, 5'-CAATGCAACTGGCGCTATTACTG-3'; L20A, 5'-CAACTGCTGGCATTACTGTTC-3'; L21A, 5'-CTGCTGCTAGCACTGTTCTTAC-3'; L22A, 5'-CTGCTATTAGCGTTCTTACTC-3'; F23A, 5'-CTATTACTGGCCTTACTCTTG-3'; L24A,
5'-CTATTACTGTTCGCACTCTTGTTTTTTC-3'; L25A, 5'-CTGTTCTTAGCCTTGTTTTTTC-3';
L26A, 5'-GTTCTTACTCGCGTTTTTTCTTG-3'; F27A,
5'-CTTACTCTTGGCTTTTCTTGTATAC-3'; F28A, 5'-CTCTTGTTTGCTCTTGTATAC-3'; L29A, 5'-CTTGTTTTTTGCTGTATACTGG-3'; V30A,
5'-GTTTTTTCTTGCATACTGGGATC-3'; Y31A,
5'-GTTTTTTCTTGTAGCCTGGGATCATTTTG-3'. The second fragment was generated
using a 5' oligonucleotide complementary to the 3' oligonucleotide used
in the above reaction as well as a 3' oligonucleotide complementary to
the last four codons of E5 which included a BamHI
recognition site downstream of the translational stop site
(5'-ATAGCTGGATCCTTAAAAGGGCAGACC-3'). The 5' oligonucleotides are as
follows: L4A, 5'-CAATAGAAACCATGCATTTGGTATATAG-3'; W5A, 5'-
CAATAGAAACGCTAGATTTGG-3'; F6A, 5'-CAATAGAAACGCTAGATTTGG-3'; L7A,
5'-CAAGAACAATGCAAACCATAG-3'; L8A, 5'-GTCCCAAGAACGCTAGAAACCATAG-3'; F9A,
5'-CTAGTCCCAAGGCCAATAGAAAC-3'; L10A, 5'-CAACTAGTCCCGCGAACAATAG-3'; G11A, 5'-CAGCAACTAGTGCCAAGAACAATAG-3'; V13A,
5'-CATTGCAGCAACTGCTCCCAAGAAC-3'; M16A, 5'-CAGCAGTTGCGCTGCAGCAAC-3';
Q17A, 5'-GTAATAGCAGCAGTGCCATTGCAGC-3'; L18A,
5'-GTAATAGCAGCGCTTGCATTGC-3'; L19A, 5'-CAGTAATAGCGCCAGTTGCATTG-3'; L20A, 5'-GAACAGTAATGCCAGCAGTTG-3'; L21A, 5'-GTAAGAACAGTGCTAGCAGCAG-3'; L22A, 5'-GAGTAAGAACGCTAATAGCAG-3'; F23A, 5'-CAAGAGTAAGGCCAGTAATAG-3'; L24A, 5'-GAAAAAACAAGAGTGCGAACAGTAATAG-3'; L25A,
5'-GAAAAAACAAGGCTAAGAACAG-3'; L26A, 5'-CAAGAAAAAACGCGAGTAAGAAC-3';
F27A, 5'-GTATACAAGAAAAGCCAAGAGTAAG-3'; F28A,
5'-GTATACAAGAGCAAACAAGAG-3'; L29A, 5'-CCAGTATACAGCAAAAAACAAG-3'; V30A,
5'-GATCCCAGTATGCAAGAAAAAAC-3'; Y31A,
5'-CAAAATGATCCCAGGCTACAAGAAAAAAC-3'.
Each of the fragments generated above was separated from primers on
1.2% agarose gels run at 100 V for 30-60 min, cut out, and purified
using the Geneclean protocol (Bio 101, Vista, CA). Corresponding
fragments were then combined in another PCR reaction along with the 5'
and 3' oligonucleotides listed above. The resulting fragments were
precipitated following a phenol/chloroform extraction and
restriction-digested with XhoI/BamHI, after which
time they were isolated on agarose gels, purified with Geneclean, and
ligated into pJS55 vector digested with
XhoI/BglII. Conditions for PCR synthesis were as
follows: 30 cycles of denaturing at 94 °C for 1 min, annealing at
50 °C for 1 min, and elongation at 72 °C for 2 min. The plasmid
containing AU1 epitope-tagged wild-type BPV-1 E5, pJS63, was used as a
template in these PCR reactions (25). The fidelity of all constructs
was verified by dideoxy DNA sequencing.
DNA Transfections--
For focus assays, NIH3T3 cells were
transfected with the indicated plasmids using calcium phosphate-DNA
co-precipitation as described by Graham and Van der Eb (41). Five
micrograms of pJS55-E5 DNA was added along with 5 µg of carrier DNA
(pUC19) in 0.5 ml of 1× HEPES-buffered saline. Fifty microliters of
1.2 M CaCl2 was added slowly with mixing and
incubated at room temperature for 30 min. Each sample was then added to
a 50-70% confluent 100-mm plate of cells in 5 ml of fresh DMEM.
Following overnight incubation at 37 °C, the cells were washed twice
with Dulbecco's phosphate-buffered saline (D-PBS) and 2 ml of 15%
glycerol in 1× HEPES-buffered saline was applied for 1 min. Cells were
immediately washed three times with D-PBS, and 10 ml of DMEM was
applied. After 3 days of growth to confluence at 37 °C, the cells
were trypsinized, transferred to 175-cm2 flasks containing
DMEM, and incubated for 14 days. Flasks were re-fed with fresh growth
medium every 3 days and at the termination of the experiment were
stained with 1% methylene blue in 100% ethanol (w/v) and foci counted.
Transfections to generate stable NIH3T3 lines expressing the above
constructs were carried out as described above. E5 DNAs were
co-transfected with the neomycin R-conferring plasmid, LNCX, at a ratio
of 9:1 (E5 mutant:LNCX). At the time of transfer to 175-cm2
flasks, G418 was added and maintained at a concentration of 0.5 mg/ml.
Neomycin-resistant colonies were allowed to form for a period of 2-3
weeks, after which they were pooled and screened for E5 expression
using the immunoprecipitation/Western blot procedure described below.
Immunoprecipitation and Immunoblotting Analysis--
E5/PDGF-R
protein interactions in NIH3T3 cells were analyzed by a double
immunoprecipitation technique described previously (26). For each
immunoprecipitation, two 15-cm plates of cells at 90% confluence were
utilized. E5 protein was immunoprecipitated using 5 µl of the
monoclonal antibody AU1 (Berkeley Antibody Co. (Babco), Berkeley, CA),
while associated PDGF-R was isolated in the second immunoprecipitation
using 2 µl of the polyclonal antibody 06-131 (Upstate Biotechnology
Inc. (UBI), Lake Placid, NY). Proteins were separated on either 7.5%
(for analysis of PDGF-R) or 14% (for analysis of E5 and 16K) sodium
dodecyl sulfate (SDS)-polyacrylamide gels. All gels were subsequently
fixed in 30% methanol, 10% acetic acid, enhanced with Enlightning
(NEN Life Science Products), dried, and exposed to Kodak XAR-5 film for
1-28 days at
70 °C. Densitometry measurements were made on a PDI
Discovery Series model DNA 35 scanner.
For detecting tyrosine-phosphorylated PDGF-R, two 15-cm diameter plates
at 90% confluence were washed twice with D-PBS and incubated with
serum-free DMEM for 14 to 16 h at 37 °C. Prior to harvesting,
cells were washed once with PBS containing 100 µM
Na3VO4. The cells were then extracted in 750 µl of lysis buffer containing 1% Triton X-100, 50 mM
HEPES (pH 7.5), 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 10 mM NaPPi, 1 mM Na3VO4, 0.5 mM
phenylmethylsulfonyl fluoride (Sigma), 10 mg/ml aprotinin (Boehringer
Mannheim), and 10 µg/ml leupeptin (Boehringer Mannheim). The
procedures for immunoprecipitation were similar to those described
above. Total PDGF-R was isolated in the first immunoprecipitation using
5 µl of polyclonal antibody 06-131. Phosphorylated PDGF-R was then
immunoprecipitated using 2 µl of polyclonal antibody PY-20
(Transduction Laboratories, Lexington, KY). Immunoprecipitates were
washed three times in lysis buffer, solubilized in 30 µl of sample
buffer, and boiled for 4 min. Proteins were then separated on 7.5%
gels, fixed in 30% methanol, 10% acetic acid, enhanced with
Enlightning (NEN Life Science Products), dried, and exposed to Kodak
XAR-1 film for 7-14 days.
In order to determine the competence of E5 mutants to dimerize, two
15-cm plates of the designated cell lines (at 90% confluence) were
harvested in RIPA buffer as described previously. Following immunoprecipitation with 5 µl of anti-AU1 antibody, the
immunoprecipitates were washed three times with RIPA buffer and
solubilized in 30 µl of sample buffer without 2-mercaptoethanol.
These proteins were separated on 14% polyacrylamide gels and then
transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA)
in Tris-glycine buffer, either overnight at 20 V or for 2 h at 80 V. Immunoblotting was performed with a Tropix (Bedford, MA) Western Light protein detection kit according to the described procedures. The
anti-E5 polyclonal antibody antiserum (1:5000 dilution) was used to
detect E5 on the membranes.
Immunofluorescence Assays--
COS-1 cells were grown on glass
coverslips and at 60% confluence were transfected with the pJS55
constructs as described above. Twenty-four hours following glycerol
shock, the cells were washed twice with PBS and fixed in 3.7%
formaldehyde for 20 min. The coverslips were then washed three times
with PBS and incubated for 20 min with 10% normal goat serum (NGS),
0.1% saponin in PBS. Following two more washes in PBS, cells were
incubated for 1 h with AU1 antibody that was diluted 1:100 in PBS
containing 10% NGS, 0.1% saponin. Coverslips were washed three times
with PBS and then incubated for 1 h with rhodamine conjugated to
goat anti-mouse antibody (Jackson Laboratories, Bar Harbor, ME), 1:50
in PBS, 10% NGS, 0.1% saponin. The cells were again washed in PBS
three times and mounted on slides using Fluoromount (PanData,
Rockville, MD) mounting solution. Cells were observed with a Zeiss
Axioskop fluorescence microscope.
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RESULTS |
Construction of Epitope-tagged E5 Transmembrane Mutants--
In
order to probe the E5 transmembrane domain for residues or domains
important in the interactions and functions of E5, 26 of the 30 residues in the putative TM domain of E5 were changed independently to
alanine (Fig. 1). The immunological
detection and isolation of the mutant E5 proteins was facilitated by
the addition of an N-terminal, six-amino acid AU1 epitope, which has been shown to have no effect on E5 biological activity (25). Each of
the E5 mutants was cloned into pJS55, a derivative of pSG5 (Stratagene)
that utilizes the SV40 early promoter for efficient expression in mouse
fibroblasts.

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Fig. 1.
Scanning alanine mutagenesis of the E5
transmembrane domain. Each amino acid in the proposed
transmembrane domain of E5 was individually converted to alanine by a
two-step PCR technique as described under "Experimental
Procedures." The wt E5 sequence is indicated at the top of
the figure with the TM region delineated in capital
letters. The position of the mutant alanine residues is
indicated in bold capital letters.
Residues 14 and 15 were not mutagenized since they were already
alanine. The PCR products were cloned into pJS55, a modified pSG5
vector (Strategene) that contains an inserted polylinker and utilizes
the SV40 early promoter for gene expression. The fidelity of the mutant
E5 genes was verified in the final plasmid constructs by DNA
sequencing.
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All E5 Transmembrane Mutant Proteins Localize Normally to the Golgi
Apparatus--
Previously, it has been shown that the accumulation of
E5 in the Golgi apparatus is essential for its transforming activity (26). To determine whether alterations in the E5 TM domain might alter
its targeting to the Golgi (and indirectly its ability to transform
cells), we transfected each of the designated mutants into COS-1 cells
and evaluated their intracellular localization by immunofluorescence
microscopy. The cells were fixed and reacted with the AU1 epitope
antibody as described under "Experimental Procedures." In all
cases, the expressed E5 mutant proteins were detected in the
perinuclear Golgi region, identical to the pattern observed for
wild-type E5 (data not shown). This indicates that no single amino acid
residue in the E5 TM region is required for proper Golgi targeting and
that any perturbations in the biological activity of the E5 mutants
cannot be ascribed to altered intracellular localization.
All Alanine E5 Mutants Are Expressed Similarly in Cell Lines, but
Some Fail to Form Dimers--
To determine the protein stability and
level of expression of each of the E5 mutants, we utilized a combined
immunoprecipitation/Western blot analysis. Neomycin-selected NIH3T3
cell lines expressing each of the E5 mutant proteins were solubilized
in detergent and subjected to immunoprecipitation with the monoclonal
antibody recognizing the appended AU1 epitope at the E5 N terminus
(27). The immunoprecipitated proteins were then separated by PAGE,
transferred to nitrocellulose membranes, and detected by Western blot
analysis using antiserum generated against the C terminus of E5
(28).
Fig. 2A shows an SDS-PAGE gel
of the E5 alanine mutant proteins that were immunoprecipitated and
blotted from NIH3T3 cell lines. All of the mutant proteins were stably
expressed in these cell lines, although there was some variation in the
absolute level of E5 protein. While the majority of the wild-type E5
(wt E5) was present in cells as a dimer, alanine substitutions at positions 7(Leu), 13(V), 18(Leu), 20(Leu), 25(Leu), and 26(Leu) generated mutants that failed to form dimers and preferentially formed
tetramers. These tetramers were not observed when the samples were
treated with reducing agent, indicating that disulfide linkages were
responsible for tetramer stabilization (data not shown). When the
dimerization-defective E5 mutants were plotted as a helical net diagram
(Fig. 2B), they sorted into two distinct zones or faces of
the predicted E5 helix (residues 7, 18, and 25, and residues 13, 16, and 20), indicating that two non-contiguous regions regulated the
interaction of adjacent E5 molecules in dimers. In addition, E5 mutants
at positions 16 (Met) and 31 (Tyr) formed an abundance of higher order
oligomers, up to and including hexamers.

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Fig. 2.
A, expression and oligomerization of wt
and mutant E5 proteins. G418-selected NIH3T3 cell lines were extracted
and immunoprecipitated with AU1 monoclonal antibody and separated on
14% SDS-PAGE gels in the absence of reducing agent. The proteins were
then transferred to PVDF membranes and detected using a polyclonal
antiserum generated against the C terminus of E5 and a
chemiluminescence detection kit (Tropix). The total expression level of
wt and mutant proteins is similar, although the proportions of monomer,
dimer, and tetramer vary. For wt E5, the dimeric form predominates.
Several mutant E5 proteins (e.g. L7A, L25A, and L26A) fail
to form dimers but still form tetramers, whereas one can only form
dimers (e.g. L22A). B, helical net plot of E5
mutants that are dimerization-incompetent. The position of dimerization-incompetent E5 alanine mutants is
indicated in the two shaded areas of a
helical web plot of the E5 TM domain. These defective mutants
segregated into two distinct domains or faces: (domain 1, residues 7, 18, and 25; domain 2, residues 13, 16, and 20).
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Three E5 Mutants Define a Potential Binding Site for the PDGF-R
Complex--
In most cases, the ability of E5 to transform fibroblast
cells has been linked to its ability to bind and induce the
phosphorylation of PDGF-R (5-9) and to associate with the 16K protein
(12-15). It is believed that E5-mediated activation of PDGF-R
constitutively stimulates signal transduction pathways, which results
in a mitogenic response and consequent cellular transformation. In
order to define those residues that were important for the binding of
E5 to the PDGF-R complex, immunoprecipitates of the indicated E5 mutant proteins from NIH3T3 cell lines were evaluated for the presence of
co-precipitated PDGF-R (Fig.
3A). As reported previously,
wt E5 predominantly associates with the immature form of the receptor (6, 8). Substitution at positions 17 (Gln), 21 (Leu), and 24 (Leu)
resulted in mutant proteins that were unable to co-precipitate PDGF-R.
While previous studies had identified position 17 as being critical for
E5/receptor association (29), the current study identifies two
additional residues (Leu-21 and Leu-24) that also regulate interactions
between E5 and the PDGF-R complex. Assuming that the transmembrane
domain of E5 forms an
helix with a periodicity of 3.6 residues per
turn, these residues reside in a restricted area on the same face of
the helix (Fig. 3B).

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Fig. 3.
A, binding of PDGF-R by wt and mutant E5
proteins. NIH3T3 stable cell lines were metabolically labeled with
[35S]cysteine/methionine, extracted, and
immunoprecipitated with AU1 monoclonal antibody. 10% of the lysate was
separated by 14% SDS-PAGE, while the remaining lysate was subjected to
a second immunoprecipitation using a monoclonal antibody against PDGF-R
and separated by 7.5% SDS-PAGE. No E5-associated PDGF-R was detected
in cells transfected with the control plasmid, pJS55, whereas cells
expressing wt E5 show significant amounts of co-precipitated receptor.
While the majority of E5 alanine mutant proteins were able to associate
with PDGF-R, two are shown that are negative (L21A, L24A). Q17A was
also unable to associate with PDGF-R. B, helical net plot of
the apparent E5 binding site for PDGF-R The E5 alanine mutants that
were defective for binding PDGF-R (shaded area)
are displayed as a helical net plot of the E5 TM domain. The three
binding-defective mutants (Q17A, L21A, and L24A) mapped to a discrete
region on one face of the predicted E5 helix, the presumed binding site
for PDGF-R.
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An Entire E5 TM Helical Face Regulates PDGF-R Tyrosine
Phosphorylation--
While the above experiments clearly indicated
that three distinct residues of E5 modulated the association of E5 with
the PDGF-R complex, it was important to determine whether these same sites affected receptor phosphorylation. Therefore, each of the alanine
mutants was screened for its ability to induce PDGF-R phosphorylation
in NIH3T3 cell lines. Cells were grown in the absence of exogenous PDGF
ligand (serum-free media), labeled with [35S]methionine/cysteine, and the amount of total and
phosphorylated PDGF-R determined by immunoprecipitation as described in
Fig. 4A. Comparison of the
ratio of phosphorylated PDGF-R to total PDGF-R revealed that E5 mutants
at positions 7 (Leu), 10 (Leu), 13 (Val), 17 (Gln), 18 (Leu), 20 (Leu),
21 (Leu), 24 (Leu), 25 (Leu), 26 (Leu), 30 (Val), and 31 (Tyr) failed
to induce significant phosphorylation of PDGF-R. When these mutants
(positions 7-25) and their biological activities were plotted as a
helical wheel diagram, it was apparent that all E5 mutants defective
for inducing receptor phosphorylation were aligned along one helical
face and those that retained biochemical activity were aligned along
the opposite helical face (Fig. 4B). Mutants at residues 26, 30, and 31 did not conform precisely to this localization. However, the 19 amino acids between positions 7 and 25 are sufficient to span the
plasma membrane as a TM domain and the amino acids distal to this,
especially residues 30 and 31, have been proposed to constitute a
non-helical, "juxtamembrane" region rather than a transmembrane
domain (28).

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Fig. 4.
A, induction of PDGF-R
phosphorylation by wt and mutant E5 proteins. NIH3T3 stable cell
lines were metabolically labeled with
[35S]cysteine/methionine and immunoprecipitated with
anti-PDGF-R monoclonal antibody. 10% of the lysate was then separated
by 7.5% SDS-PAGE to determine the total amount of PDGF-R present in
each sample. The remaining lysate was subjected to a second
immunoprecipitation using a monoclonal antibody against phosphotyrosine
residues and separated by 7.5% SDS-PAGE to determine the amount of
phosphorylated PDGF-R in each sample. Approximately half of the
alanine mutant proteins failed to induce PDGF-R
autophosphorylation. B, helical wheel plot of E5 mutants
that induce PDGF-R phosphorylation. When plotted as a helical web, the
E5 alanine mutants that induce PDGF-R phosphorylation all reside on the
same helical face. Conversely, all of the mutants that fail to induce
PDGF-R phosphorylation reside on the opposite E5 helical face. Most of
these phosphorylation-defective mutants fall into two categories: 1)
those that fail to dimerize and 2) those that fail to bind to
PDGF-R.
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The above experiments on receptor binding and activation indicate that
there are two distinct classes of E5 mutants that are defective for
inducing PDGF-R phosphorylation. First, there are those mutants that
fail to bind to the receptor and therefore cannot subsequently induce
receptor phosphorylation (i.e. mutants at positions 17, 21 and 24). Second, there are those mutants that can bind to the receptor
but still cannot induce receptor activation (i.e. mutants at
positions 7, 13, 18, 20, 25, and 26). Interestingly, this second class
of mutants is incapable of forming dimers, suggesting that this might
represent their defect in mediating receptor activation. Evidently both
receptor binding and homodimer formation are essential for E5-induced
phosphorylation of the PDGF-R complex.
TM Mutants Further Dissociate E5 Transforming Activity from PDGF-R
Binding/Activation--
Although the ability of E5 to bind and
activate PDGF-R has been thought to be essential for fibroblast
transformation (5-9), a recent study of E5 mutations at position 17 indicated that the substitution of glutamine 17 with serine resulted in
a biologically active protein that failed to activate the PDGF-R (29).
This suggested that PDGF-R phosphorylation was not essential for
mitogenic signaling by E5. Our current study reinforces these findings. When the alanine TM mutants were evaluated for transforming activity (Fig. 5), only two mutants (at position
Gln-17 and Leu-18) were transformation-defective. All other mutants
were transformation-competent, most displaying transformation
efficiencies above 75% wt activity. Two mutants, at Leu-7 and Val-13,
had slightly reduced transforming efficiencies of 42% and 52%,
respectively (Table I). Most importantly, a large number of substitutions resulted in E5 oncoproteins with higher
transformation efficiencies than wt E5. Three of these mutants,
substitutions at positions 24 (Leu), 25 (Leu), and 26 (Leu), did so
without inducing phosphorylation of PDGF-R (Fig. 4). In addition,
mutation of Leu-24 produced an E5 protein that showed little or no
ability to bind PDGF-R, yet it transformed fibroblasts 3-fold better
than wt E5. Thus, these studies establish several new E5 mutants that
are unable to induce the phosphorylation of PDGF-R yet retain
transforming activity.

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Fig. 5.
Transforming activity of wt and mutant E5
proteins. Each of the E5 mutant plasmids was transfected into
recipient NIH3T3 cells and evaluated for focus formation after 2 weeks as described under "Experimental Procedures." The
percentage of transforming efficiency of each mutant
(compared with (wt E5) is indicated by the numbers adjacent
to each amino acid in the helical wheel plot of the E5 TM region. Only
2 of the 25 alanine mutants (positions 17 and 18) showed greatly
reduced transforming activity (0% and 2%, respectively) and are
indicated in bold lettering.
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DISCUSSION |
Previous mutagenic studies of the E5 oncoprotein have suggested
several cellular targets that may be important for the biological activity of E5, including the PDGF-R (5-9), the epidermal growth factor receptor (10), the 16K V-ATPase subunit (12-15), and an
-adaptin-like protein (30). Earlier studies with E5 indicated that
its C-terminal region might be a critical determinant of its biological
activity and that the E5 TM domain could be replaced with other
hydrophobic residues, suggesting that it did not confer specificity for
target interactions or cell transformation (3, 31). Since the E5 C
terminus was suggested to show homology to PDGF, it was hypothesized
that E5 stimulated cell proliferation via mimicry of this mitogenic
ligand (8). However, more recent studies have demonstrated that the E5
TM domain is a critical regulator of binding to target proteins
(including PDGF-R) and biological activity (12) and that there are
residues within the PDGF-R and E5 TM domains that can alter these
interactions (23). In this study we have performed scanning alanine
mutagenesis of the E5 TM domain in order to define those residues that
were critical for regulating E5 transforming activity and ability to interact with target proteins. Our results demonstrate for the first
time that the E5 TM domain contains functionally distinct domains that
separately regulate homologous (E5/E5) interactions and heterologous
(E5/PDGF-R) interactions. In addition, our studies define two new
classes of mutants, which transform by a mechanism that is independent
of PDGF-R phosphorylation, significantly extending our previous studies
with mutants at position 17 (29).
Translocation of E5 to the Golgi Complex Is Not Dependent upon Any
Single TM Residue--
None of the E5 alanine mutants generated in
this study was defective for Golgi localization, suggesting that there
is no single residue within the TM domain which is required for proper
intracellular translocation. However, this does not eliminate the
possibility that a series or combination of TM amino acids regulates
this processing. Several other possibilities might explain Golgi
localization (via retention or recycling): (a) The first is
the length of the E5 TM domain. Variation in the length of
transmembrane
-helical domains has been proposed as a mechanism of
differentially regulating membrane protein flow from the Golgi to other
compartments such as the plasma membrane (32). Since there is a
gradual, cholesterol-dependent increase in the thickness of
membranes during the progression from the ER to the Golgi to the plasma
membrane, a membrane embedded protein with a long TM
-helix could
make the full transit from the ER to the plasma membrane. Proteins with
shorter TM domains would become selectively sequestered in intermediate
compartments such as the Golgi depending upon the length of their TM
domain (33). If this hypothesis were correct, only a subset of the 30 amino-terminal hydrophobic residues of E5 could be involved in forming
a membrane-spanning
-helix. This would be consistent with our
experimental results suggesting that only 19 amino acids of E5
(residues 7-25) constitute the transmembrane domain. (b) The second possibility is oligomer formation. Another hypothesis for
Golgi retention proposes that the formation of oligomers interferes with normal protein traffic through the various membrane compartments, thereby restricting access to the plasma membrane (34, 35). E5
certainly has the ability to form both homo-oligomers and well as
hetero-oligomers, which could specify its retention in the Golgi.
However, our finding that mutant E5 proteins that are incapable of
binding PDGF-R still accumulate in the Golgi indicates that E5/PDGF-R
hetero-oligomer interactions cannot be required for this sequestration.
(c) The third possibility is non-TM sequences. A linear
sequence might exist outside the TM domain, in the C-terminal hydrophilic region, which is responsible for Golgi localization.
Three E5 TM Residues Are Important for the Association with
PDGF-R--
Only 3 of the 25 alanine scanning mutants failed to
associate with PDGF-R in NIH3T3 lines. These three residues (positions 17, 21, and 24) lie on the same face of the
-helix and include the
glutamine at position 17, previously shown to be important for receptor
association (15, 29, 31). In addition, alanine mutants at positions 21 and 24 were still able to transform mouse fibroblast cells, indicating
that the association of E5 with PDGF-R cannot be the sole determinant
of E5 transforming activity. Most likely, residues 17, 21, and 24 represent components of a specific binding site for PDGF-R.
Alternatively, these three residues may be essential for the structure
of E5 and the substitution of alanine at these positions could perturb
normal E5 protein conformation. This seems unlikely, however, since
mutants 21 and 24 retain normal transforming activity.
Phosphorylation of PDGF-R Correlates with E5 Dimerization, Not
Tetramerization--
Prior to this study, most experimental data
regarding the role of E5 oligomerization in cell transformation relied
upon the analysis of E5 mutants mutated in the two C-terminal cysteine residues. Substitution of either cysteine (in the Cys-X-Cys
sequence) with methionine or arginine diminished E5 transforming
activity (3). The most profound inhibition was achieved when both
cysteines were simultaneously replaced with methionine. This double
cysteine mutant could not form dimers, could not activate the PDGF-R,
and could not transform cells (3). These previous studies, therefore, concluded that E5 disulfide bond formation was critical for E5 function.
Our current study indicates that the ability to form dimers and
oligomers is strongly regulated by the TM region and perhaps may be the
critical domain in initiating oligomer formation. Nearly all alanine TM
mutants that retain the ability to form dimers can also induce the
phosphorylation of the PDGF-R (e.g. mutants at position 8, 9, 11, 16, 19, 22, and 23). As expected, alanine mutants in the PDGF-R
binding domain (positions 17, 21, and 24) form dimers but cannot induce
PDGF-R phosphorylation. Conversely, alanine mutants that cannot form
dimers are unable to activate the PDGF-R (e.g. mutants at
position 7, 13, 25, 26). Thus, it appears that the ability of E5 to
form stable dimers is essential for inducing the phosphorylation of the
PDGF-R. Interestingly, oligomerization of E5 molecules is clearly
insufficient for promoting PDGF-R phosphorylation since mutants at
positions 25 and 26 can readily form tetramers but cannot activate
PDGF-R. Since these tetrameric forms of E5 still bind PDGF-R, it
appears that their inability to induce receptor phosphorylation may be
the result of inappropriate spatial configurations, which do not allow
appropriate receptor trans-phosphorylation.
E5 Tetramers Can Transform Cells--
E5 alanine mutants that
exist as stable tetramers (i.e. mutants at positions 25 and
26) are transformation-competent but cannot induce the phosphorylation
of PDGF-R. Clearly this revises previous conclusions that only the
dimeric form of E5 has transforming activity. Potentially tetrameric E5
might signal through the PDGF-R in a phosphorylation-independent
mechanism. More likely, however, tetrameric forms of E5 might have
distinct biological activities. Indeed, wt E5 also exists in tetrameric
forms, although it is the minority of total E5 protein. The observation
that E5 can form biologically active oligomers is suggestive that it
might display membrane pore-forming activities. The M2 protein of
influenza protein is a similar, small hydrophobic polypeptide, which is localized in the Golgi apparatus, assembles into tetramers, and forms
proton pores in the membrane (36-40). To date, however, no pore-forming activity has been ascribed to E5. It will also be important in future experiments to evaluate the functional consequences of E5/16K interactions; it is possible that the
transformation-competent E5 mutants that cannot activate PDGF-R might
still bind 16K protein and interfere with V-ATPase activity.
Substitution of Alanine along One Side of the E5 Helix Interferes
with Activation of the PDGF-R--
Glutamine 17 in the E5 TM region
has previously been identified as being critical not only for
transforming activity but also for binding to cellular target proteins;
it is essential for binding to the 16K V-ATPase subunit (15) and the
PDGF-R (23, 29). It is also required for the induction of PDGF-R
phosphorylation (15, 29, 31). When viewed in the context of the helical wheel plot in Fig. 4 and the helical net plot in Fig. 3, it is evident
that this glutamine residue lies near the middle of the E5 helical face
along which alanine substitutions perturb PDGF-R phosphorylation.
Alanine substitutions along the opposite helical face have no effect on
the ability of E5 to induce PDGF-R activation. The residues surrounding
the glutamine residue are also critical for regulating PDGF-R binding
and E5 dimerization.
Preliminary Model for E5 TM Interactions--
Collation of our
experimental results suggests a preliminary model for explaining the
various activities of the mutant E5 proteins (Fig.
6). The model is based upon the
hypothesis that the E5 TM domain forms homo- and hetero-oligomers via
intermediates of increasing stability. Thus, when two E5 TM domains
first come in contact, the preliminary complex does not have the lowest
energy level possible. Only after the two TM domains realign and
optimize binding interactions (including the formation of hydrophobic
interactions, hydrogen bonding, charge interactions, and disulfide
bonds) is a complex of low energy state achieved. It is also postulated that the accessibility of binding sites for 16K and PDGF-R in the TM
domain varies with the structure of the various energy state
intermediates. In the case of wt E5 interactions, therefore, the
formation of a stable dimeric E5 complex would present a binding site
for PDGF-R with appropriate structure to allow for receptor transphosphorylation. On the other hand, mutation of the helical domain
involved in dimer formation would result in an alternate low energy
state in which further interactions with E5 would be favored over those
with PDGF-R, resulting in tetramer formation and the loss of
appropriate conformation to facilitate PDGF-R transphosphorylation.
Further dissection of these E5 alanine mutants will assist in defining
a new mechanism for fibroblast transformation as well as provide a
general model for specific intramembrane
-helical interactions.

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Fig. 6.
Preliminary model of E5 TM interactions.
The helical barrels of the E5 TM domain are shown with the domains
critical for dimerization (yellow) and PDGF-R binding
(red). The disulfide bonds are located near the E5 C
terminus and reside within the Golgi lumen. The PDGF-R TM domain is
presented in blue with the attached internal kinase domains
(small cylinders) and external ligand-binding
domain (five ovals). Upon initial contact, the E5
TM domains are predicted to undergo a series of sequential
rearrangements to optimize binding (achieve a minimum energy state). In
wt E5 (upper figure), an initial complex of E5
(step 1) rearranges to permit the interaction of adjacent,
non-identical E5 dimerization sites (step 2) such that the PDGF-R
binding site is available (step 3). Dimeric E5 then forms a complex
with PDGF-R, resulting in receptor transphosphorylation (step 4). This
is not the case with E5 mutants (e.g. L18A) with an alanine
in the dimerization domain (lower figure). This
mutant cannot form stable dimers and therefore seeks another low energy
state (step 2), which does not present the PDGF-R binding site in a
conformation conducive to transphosphorylation (step 3). Rather, these
mutants proceed to tetramer formation, a state that is observed less
frequently in wt E5 protein (due to stable dimer formation).
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