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J Virol, January 1998, p. 558-563, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Serine 257 Phosphorylation Regulates Association of Polyomavirus
Middle T Antigen with 14-3-3 Proteins
Xavier
Culleré,1
Paul
Rose,2
Usha
Thathamangalam,3
Alakananda
Chatterjee,1
Karen P.
Mullane,1
David C.
Pallas,4
Thomas L.
Benjamin,3
Thomas M.
Roberts,2 and
Brian S.
Schaffhausen1,*
Department of Biochemistry, Tufts University
School of Medicine,1
Department of
Cellular & Molecular Biology, Dana-Farber Cancer
Institute,2
Department of Pathology,
Harvard Medical School,3 Boston
Massachusetts, and
Department of Biochemistry, Emory
University Medical School, Atlanta Georgia4
Received 27 May 1997/Accepted 7 October 1997
 |
ABSTRACT |
Polyomavirus middle T antigen (MT) is phosphorylated on serine
residues. Partial proteolytic mapping and Edman degradation identified
serine 257 as a major site of phosphorylation. This was confirmed by
site-directed mutagenesis. Isoelectric focusing of immunoprecipitated
MT from transfected 293T cells showed that phosphorylation on wild-type
MT occurred at near molar stoichiometry at S257. MT was previously
shown to be associated with 14-3-3 proteins, which have been connected
to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This
has been demonstrated by comparing wild-type and S257A mutant MTs
expressed with transfected 293T cells or with Sf9 cells infected with
recombinant baculoviruses. The 257 site is not critical for
transformation of fibroblasts in vitro, since S257A and S257C mutant
MTs retained the ability to form foci or colonies in agar. The tumor
profile of a virus expressing S257C MT showed a striking deficiency in
the induction of salivary gland tumors. The basis for this defect is
uncertain. However, differences in activity for the wild type and
mutant MT lacking the 14-3-3 binding site have been observed in
transient reporter assays.
| |
INTRODUCTION |
Polyomavirus middle T antigen (MT)
is necessary (11, 39, 61) and often sufficient
(63) for transformation. It is associated with membranes
(35, 54), and its transforming ability depends on that
association (11). MT has no known enzymatic activity but
instead appears to function by association with cellular proteins involved in signal transduction. It has been a particularly useful model because mutations that affect particular associations have generally had clearly identifiable phenotypes. In contrast, single knockouts in receptors have often not been sufficient to generate clear-cut answers. Among the MT-associated proteins are protein tyrosine kinases of the Src family (Src, Yes, and Fyn) (14, 18,
33, 37). As a consequence of association with activated tyrosine
kinase, MT is phosphorylated on tyrosine residues 250, 315, and 322 (10, 32, 34, 51). Mediated by these tyrosine phosphorylations, MT interacts with SHC (9, 19), PI-3 kinase (16, 36, 59), and phospholipase C
1 (57),
respectively.
Not all MT associations are dependent on tyrosine phosphorylation. The
binding to protein phosphatase 2A (PP2A) is one example of this
(48, 64). This association appears to be important for the
ability of MT to associate with protein tyrosine kinases (8). Not surprisingly, mutants defective in PP2A binding are defective in transformation. All three polyomavirus T antigens contain
sequences resembling a DnaJ domain (12). As expected, MT
also associates with hs70 (47). This interaction apparently occurs when MT either cannot bind PP2A or when the level of expression exceeds that of the cellular PP2A. The significance of the heat shock
interaction is not known.
MT also binds proteins of the 14-3-3 family (45, 46). These
abundant cell proteins clearly function in protein-protein associations. Among important proteins involved in cellular growth regulation and signal transduction, Raf (23, 25, 28), PI-3 kinase (4), and cdc25 phosphatase (17) are all
known to associate with the 14-3-3 proteins. There is also evidence
that 14-3-3 association can modulate apoptosis (67). While
the evidence for association is strong, the role of these proteins has
not been completely clarified (1, 7, 42). Most thinking has
focused on the possibility that these proteins, through their ability
to form dimers (40, 65), could function as adaptors.
In mammalian systems, 14-3-3 associates with Raf in a manner that
depends on serine phosphorylation (44). A similar conclusion has been reached for the association of 14-3-3 with BAD
(67). The work presented here shows that the association of
MT with 14-3-3 is mediated by serine phosphorylation at residue 257. Further, while mutation of the 14-3-3 binding site has no striking
effect on MT transforming ability in vitro, it leads to a specific
alteration in the tumor profile and also alters the action of MT in
assays of promoter activity.
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MATERIALS AND METHODS |
Cells, plasmids, and viruses.
NIH 3T3 and derived cell lines
were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented
with 10% calf serum (HyClone). 10.1 are p53-negative murine 3T3 cells
(31) and were maintained in 10% fetal calf serum. 293T
cells were grown in Dulbecco's medium supplemented with 10% calf
serum. Sf9 (Spodoptera frugiperda) insect cells were
maintained in Grace's complete medium at 27°C as described by
Summers and Smith (58a).
MT cDNA was reconstructed into an expression vector bearing the human
cytomegalovirus (CMV) immediate-early promoter. To do this,
5'-GCGCGGATCCATCATGGATAGAGTTCTGAGC-3' and
5'-GCGCGGATCCCTAGAAATGCCGGGAACGTT-3' were used as outside
primers for PCR. PCR was carried out by using Vent polymerase (New
England Biolabs) in a Perkin-Elmer thermal cycler for 25 cycles of 1 min at 96°C, 2 min at 55°C, and 3 min at 72°C. After
BamHI digestion, the fragment was ligated into the
BamHI site of the pCMV NeoBam parent vector (49).
Serine 257 was converted to alanine by using overlap PCR with wild-type CMV MT as a template. The 5' primer
(5'-GCGCGGATCCATCATGGATAGAGTTCTGAGC-3') and a 257 noncoding
primer (5'-TCGGGTTGGGGGATAGGCGTGGCTCCTCATAAC-3') were used
as one pair and a 257 coding primer
(5'-GTTATGAGGAGCCACGCCTATCCCCCAACCCGA-3') and the 3' primer
(5'-GCGCGGATCCCTAGAAATGCCGGGAACGTT-3') were used as the
other pair for the initial PCR. To regenerate virus bearing a mutation
at 257, residue 257 was also converted to a cysteine so that the large
T antigen reading frame could be conserved. The primer
5'-ATGAGGAGCCACTGCCTATCCCCCAACCCGA-3' was used for this
construction as described previously (26).
Myc-tagged 14-3-3 was made in a PRK5 (21, 41) background by
amplification of ovine zeta cDNA from plasmid pHAF603 by PCR. Glutathione S-transferase (GST)-14-3-3 was made in
Escherichia coli DH5
. Bovine zeta cDNA from plasmid
pHAF603 was amplified by PCR and ligated into pGEX-2T cleaved with
BamHI and EcoRI. The GST-14-3-3 protein was
purified on glutathione agarose by standard methods.
Rous sarcoma virus
-galactosidase (RSV
-gal) (
2,
22)
was kindly provided by Amy Yee.
Metabolic labeling and MT Immunoprecipitation.
In vivo
labeling was carried out by published procedures (51).
Briefly, cells were rinsed with phosphate-free medium for [32P]orthophosphate labeling or with methionine-free
medium or Hanks' salts for [35S]methionine labeling, and
then medium containing the label was added for 2 h. Typically, 25 to 100 µCi of [35S]methionine (Express label)
(Dupont-NEN) or 200 to 1,000 µCi of [32P]orthophosphate
(Dupont-NEN) was used in a volume of 1.5 to 2 ml on a 100-mm-diameter
dish of cells.
Immunoprecipitations were done as previously described (
50).
T antigens were extracted with TEB (T-Ag extraction buffer)
consisting
of 0.137 M NaCl, 0.020 M Tris (pH 9.0), 0.00092 M CaCl
2,
0.00049 M MgCl
2, 1% (vol/vol) Nonidet P-40 (NP-40), and
10% (vol/vol)
glycerol. After extraction for 20 min at 4°C, the
lysate was spun
for 15 min at 10,000 ×
g. After
incubation with antibody and protein
A-Sepharose for 60 min at 4°C
with mixing, the beads were washed
twice with phosphate-buffered
saline, twice with 0.5 M LiCl-0.1
M Tris (pH 8.0), and once with
distilled water, with 5 ml for
each wash.
Electrophoresis.
Samples were routinely analyzed on
discontinuous buffer-sodium dodecyl sulfate (SDS) gels (38).
Western blot analysis (62) was carried out after transfer to
nitrocellulose membranes. The blot was then blocked in Tris-buffered
saline-Tween (500 mM Tris-Cl [pH 7.5], 1.5 M NaCl, 0.5% [vol/vol]
Tween-20) containing 5% (wt/vol) dried milk (Carnation) for 1 h
at room temperature. The blot was incubated with 1:50 PN-116 monoclonal
anti-T antibody followed by 1:5,000 anti-mouse horseradish
peroxidase-conjugated antibody (Amersham). Protein was detected by
enhanced chemiluminescence (Amersham).
Isoelectric focusing (IEF) samples were prepared by treatment with
Garrels' sample buffer (9.5 M urea, 2% ampholytes [Pharmacia-LKB]
[pH 3 to 10: pH 5 to 7: pH 6 to 8 in a 1:2:2 ratio], 100 mM
dithiothreitol,
4% NP-40) (
29). After incubation on ice for
30 min the samples
were loaded onto IEF cylinders. First-dimension IEF
gels composed
of 4% bis/acrylamide (1.62/28.3 [wt/wt]), 8.0 M urea,
4% NP-40,
and 2% ampholytes in the ratios described above were poured
to
a height of 15 cm. Samples were run at 400 to 500 V per h for
9,000 V · h. At that point, the gels were turned up to 800 V for
1 h. Second-dimension electrophoresis of the focused proteins
was
done on 5 or 7.5% acrylamide discontinuous buffer SDS slab
gels.
Protein analysis.
To examine phosphorylations of various
wild-type and mutant MTs, partial V8 protease digestion (51,
53) was performed. Immunoprecipitated proteins separated on a
cylindrical gel (15 cm by 2 mm) were digested with Staphylococcus
aureus V8 protease (U.S. Biochemicals, Cleveland, Ohio) prior to a
second dimension of SDS electrophoresis. To prepare samples for Edman
degradation, cyanogen bromide digestion was carried out as described
previously (10).
Manual Edman degradation was performed according to the method of
Sullivan and Wong (
58). The peptide to be sequenced was
dissolved in 30 µl of 50% acetonitrile and spotted onto a
1,4-phenylene
diisothiocyanate-Sequelon (Millipore) disc. The disc was
placed
on a Mylar sheet on top of a heating block set at 50°C for 5 min.
Covalent linkage was accomplished by adding a solution of
N-methylmorpholine
to the diisothiocyanate disc. After 30 min at room temperature
the disc was washed extensively with water,
extracted five times
with trifluoroacetic acid (TFA) to remove unbound
peptide, and
then extracted with methanol. Edman degradation of the
immobilized
peptide was carried out in 1.5-ml centrifuge tubes with
washing
done by gentle vortexing. The following cycles were performed:
(i) incubation with 0.5 ml of coupling reagent
(methanol-water-triethylamine-phenylisothiocyanate;
7:1:1:1,
vol/vol/vol/vol) for 10 min at 50°C, (ii) five washes
with 1 ml of
methanol, (iii) drying under vacuum for 5 min, (iv)
incubation with 0.5 ml of TFA for 6 min at 50°C, (v) extraction
with 1 ml of TFA-42.5%
phosphoric acid (9:1, vol/vol), and (vi)
Cerenkov counting of disc and
of washes from steps 4 and 5. The
disc was washed six times with 1 ml
of methanol before the next
cycle was started.
Reporter assays.
10.1 cells were transfected (13)
at 15 to 25% confluence with pCMV-based MT plasmids and either RSV
-gal or a chloramphenicol acetyltransferase (CAT) reporter plasmid
by
N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid-buffered saline-mediated-CaPO4 precipitation in a
total volume of 1.5 ml. A total of 1 ml of each precipitate was added
to a 100-mm-diameter dish for the CAT assay. For transient expression, BES-buffered saline CaPO4 transfection of NIH 3T3 cells was
employed. Cells were harvested 48 h posttransfection, and -gal
was measured by standard techniques. CAT activity was measured
chromatographically (30). Thin-layer plates were quantitated
with ImageQuant software (Molecular Dynamics) to determine the
percentage of acetylated [14C]chloramphenicol versus all
forms.
| |
RESULTS |
MT is phosphorylated on serine 257.
It has long been known
that MT is phosphorylated on serine residues as well as tyrosine
residues (43, 50, 51, 53, 55). In fact, the principal
phosphoamino acid recovered after in vivo labeling is phosphoserine.
Figure 1a shows that the major site(s) of
serine phosphorylation in vivo resides in V8 fragment C; this fragment
has been previously mapped as arising from residues 225 to 275 (53). To localize the site of phosphorylation further, a
cyanogen bromide 32P-labeled fragment starting at residue
253 (10) was isolated from acrylamide gels. This peptide was
then subjected to manual Edman degradation (Fig. 1b). There was
specific release of label at turn 4 indicating the phosphorylation of
MT at serine residue 257.
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FIG. 1.
(a) Phosphorylation of MT. MT was labeled in vivo with
32PO4 (right) or in vitro with
[32P]ATP (left). After resolution on cylindrical gels,
the MT was digested with S. aureus V8 protease in the second
dimension. The major peptides are indicated by the arrows, and their
positions in the MT sequence are indicated below the gel. (b) Edman
degradation of phosophrylated MT produced in Sf9 cells. The
32P released by each turn of Edman degradation of the
cyanogen bromide fragment was measured by Cerenkov counting. Each cycle
of Edman degradation is shown with the corresponding Py MT sequence
indicated underneath.
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To confirm that serine 257 was a phophosphorylation site, the residue
was converted to an alanine by site-directed mutagenesis
using overlap
PCR. Figure
2 shows a mapping of
wild-type and 257
mutant MT labeled in vivo with
32P. While
the metabolic labeling of peptides A and B, which contain
the tyrosine
phosphorylation sites at residues 315 and 322, respectively,
was
similar for the two proteins, there was a dramatic decrease
in
phosphorylation of peptide C. This means that mutation of serine
257 gave the expected decrease of phosphorylation. The remaining
phosphorylation of peptide C could well arise from tyrosine 250,
a
known phosphorylation site (
32).
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FIG. 2.
In vivo phosphorylation of wild-type and 257 mutant MT.
3T3 cells were transfected with CMV vectors expressing wild-type or 257 mutant MT. Cells were labeled with 32PO4 for
2 h. MT immunoprecipitates were resolved on cylindrical gels and
then digested with V8 protease in the second dimension. The arrows
indicate the positions of the V8 peptides shown in Fig. 1. WT, wild
type.
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IEF suggested that the phosphorylation of MT at residue 257 was almost
stoichiometric in 293T cells. MT does not focus on
IEF gels, perhaps
because of the membrane anchor. However, there
is a fragment somewhat
greater than 40 kDa known to lack the C
terminus (
52) that
can be focused. In Fig.
3, the focusing
patterns
for this fragment are compared for 257 mutant and wild-type
labeling.
The multiple forms came from phosphorylation. When labeled
with
32PO
4, increasingly acidic forms had a
higher specific activity
(not shown). For wild-type MT, the most
abundant fragment corresponded
to a singly phosphorylated species. By
contrast, when the 257
mutant was analyzed, it was clear that the
pattern shifted one
step to the basic direction compared to wild type.
In other words
the 257 mutant MT had on average one less mole of
phosphate than
the wild type per mole of protein. The predominant 257 mutant
species was an unphosphorylated form.
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FIG. 3.
IEF of a proteolytic fragment of MT. 293T cells were
transfected with CMV vectors expressing wild-type or 257 mutant MT.
Cells were labeled with 35S-Express label for 2 h. MT
immunoprecipitates were resolved on IEF gels as described previously
(15, 16). Portions of the IEF gels showing the resolved
forms of the MT fragments for wild-type (left) and S257A (right) MTs
are shown. Arrows indicate the major species; from left to right are
increasingly acidic forms. For comparison of the two gels, the
positions of two nonspecific cellular bands are shown by the lines.
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The 257 mutant is defective in association with 14-3-3 proteins but
retains associated tyrosine kinase activity.
MT is known to bind
to 14-3-3 proteins (45, 46). Analysis of 14-3-3 binding
suggested a consensus RSXSXP binding sequence (44),
consistent with the MT sequence around residue 257. Both in vivo and in
vitro binding assays show that the 257 phosphorylation site is
important for binding of 14-3-3.
Recombinant baculoviruses were used to express MTs for the in vitro
binding assay (Fig.
4). GST-14-3-3 zeta
fusions bound
to agarose beads were incubated with extracts from wild
type,
dl8 (carrying a deletion of residues 253 to 282), or 257A
baculovirus-infected
cells. Wild type bound to the 14-3-3 fusion but
not GST; neither
the point mutant 257A or the deletion mutant dl8
showed significant
binding.
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FIG. 4.
Binding of MT to 14.3.3 in vitro. Extracts from Sf9
cells infected with baculoviruses expressing control, wild-type, dl8,
or 257 mutant MT were incubated with GST or a GST fusion with 14-3-3 bound to GST beads. After incubation for 90 min the beads were washed
and the proteins were resolved on an SDS gel and blotted with antibody
(F4) recognizing MT. WT, wild type.
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The same results were obtained from in vivo experiments. IEF
represents a convenient method for analyzing MT-associated proteins.
IEF of the 257A mutant showed that the 14-3-3 polypeptides were
absent,
while the spots for heat shock and the PP2A 63- and 36-kDa
subunits
were present (Fig.
5). In confirmation of
this result,
wild-type, but not mutant, MT was precipitated with 9E10
antibody
recognizing a Myc epitope tag when a Myc-14-3-3 fusion was
cotransfected
(not shown).
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FIG. 5.
Binding of MT to 14.3.3 in vivo. 293T cells were
transfected with CMV vectors expressing wild-type (left) or 257 mutant
(right) MT. Cells were labeled with 35S-Express label for
2 h. MT immunoprecipitates were resolved on IEF gels as described
previously (15, 16). Proteins specifically associated with
MT are indicated by the arrows. These include hsc70 (HS70), the 36 and
63-kDa subunits of PP 2A, as well as the 14-3-3 proteins.
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The association of MT with members of the Src tyrosine kinase
family is restricted and occurs in a relatively slow fashion
(
3,
15). Given the high apparent level of serine 257 phosphorylation
and the known abundance of 14-3-3 proteins, it was important to
check
MT-associated tyrosine kinase activity. Figure
6 shows that
there was little difference
in the amount of tyrosine phosphorylation
of wild-type and mutant MTs.
Peptide mapping confirmed that wild
type and mutant were phosphorylated
at similar sites (not shown).
One difference that was noted was a
reduction in the amount of
the 58-kDa form of MT (Fig.
6). This was not
surprising, since
the 58-kDA form has previously been shown to depend
on serine
phosphorylation in this part of the MT molecule
(
51). Support
of this interpretation can also be seen in the
in vivo labeling
shown in Fig.
2, where there is a reduction in the
amount of the
peptide derived from the 58-kDa form migrating more
slowly than
peptide C.
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FIG. 6.
Comparison of in vitro kinase activity for wild-type and
257 mutant MTs. MT immunoprecipitates from control (lane (M) 1) and
cells expressing 257 mutant (lanes 2 and 3) or wild type (lane 4 and 5)
were labeled in vitro with [32P]ATP. Proteins were then
resolved on a 7.5% acrylamide-SDS gel. The 56 and 58-kDa forms of MT
are indicated by the arrows.
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MT mutant in 14-3-3 binding retains the ability to transform cells
and to induce most tumors.
The most direct functional test of the
associated tyrosine kinase activity is transformation. To test
transforming activity, NIH 3T3 cells were transfected with empty vector
DNA, wild-type MT, or mutant DNAs (S257A or NG59, a classic
nontransforming mutant), and dense foci were scored 9 days later. The
S257A mutant was rather similar to wild type in the number of foci
induced (43 versus 77, respectively, per 100-mm-diameter dish). Cells
transfected with empty vector DNA or NG59 did not induce foci. It did
appear that the foci were perhaps slightly smaller with the mutant.
Whether this very modest difference resulted from slightly lower levels of MT is uncertain.
To provide a more complete test of 14-3-3 association, polyomavirus was
reconstructed by using the PTA strain bearing a mutation
changing
serine 257 to a cysteine. This alteration was used instead
of alanine
so that the large T antigen reading frame remained
intact. The virus
obtained grew similarly to wild type as judged
by the virus titers
obtained after infection of baby mouse kidney
cells. Table
1 shows the results of transformation
assays on
F111 rat embryo fibroblast cells. Assayed either by focus
formation
or growth in soft agar, the S257C virus behaved like wild
type.
The mutant virus was then examined for its ability to induce tumors in
mice. Newborn animals were infected with mutant virus
as described
previously (
27). As shown in Table
2, all infected
animals developed tumors.
Virus recovered from tumor-bearing animals
contained the expected
mutation. The data for S257C can be compared
to that previously
obtained with wild type. Overall, the profiles
are similar, with tumors
of both epithelial and mesenchymal origin
arising at approximately the
same time and at similar frequencies
at most sites. One notable
exception was in the salivary glands,
where the mutant failed to induce
any tumors despite the fact
that such tumors occurred in over half of
the wild-type-infected
animals. Fibrosarcomas were induced by the
mutant but at a lower
frequency than wild type. These findings are
reminiscent of results
with other MT mutants showing differential
effects on tumors at
different sites (
6,
27,
66).
MT mutant in 14-3-3 binding showed altered behavior in promoter
assays.
In searching for a possible role for 14-3-3 interaction
with MT, the possibility that it modulates transcription was tested with 10.1 3T3 cells. Figure 7 shows that
cotransfected wild-type MT can reduce expression from an RSV -gal
reporter. Wild-type MT increases expression from some synthetic
promoters developed by Taylor and colleagues (60); Fig. 7
shows the results for one containing an octamer binding site, but
similar results have been obtained for AP1 or ATF (not shown). The
S257A mutant MT gave higher promoter activity than wild type, reflected
as either loss of repression of the RSV construct or greater activation of the octamer construct. It could be imagined that wild-type MT simply
titrates cellular 14-3-3. However, the difference between wild type and
mutant was not overcome by the additional coexpression of 14-3-3 (not
shown).
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FIG. 7.
Reporter assays. 3T3 10.1 cells were transfected with an
RSV -gal (2, 22) or an octamer/heat shock CAT (OCT/HSC
CAT) (60) reporter and CMV empty vector (CON) CMV wild type
(WT), or S257A mutant MT. Activity was measured 48 h after
transfection. Values are averages from 10 experiments (-gal) or 5 experiments (CAT) done in duplicate or triplicate and are normalized to
100%.
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| |
DISCUSSION |
This work establishes that serine residue 257 is a major site of
phosphorylation of MT. Further, phosphorylation on serine residue 257 is the primary basis for association of MT with the 14-3-3 family of
proteins. Based on the IEF experiments, phosphorylation of serine 257 appears to be stoichiometric. The kinase responsible for this
phosphorylation is unknown. Given the extreme overexpression in 293T
cells, it must have a very high capacity. In the same cells, it appears
that the binding of 14-3-3 to MT appears to be less than
stoichiometric. This conclusion is based on the observation that
cotransfection of 14-3-3 increases the amount of protein associated
with MT immunoprecipitates. Since 14-3-3 proteins are reported to be
very abundant, this result might seem surprising. However, it can be
imagined that much of the pool of cellular 14-3-3 is already associated
with cellular phosphoproteins.
While the biochemistry of 14-3-3 association is clear, the consequences
of the binding to MT complexes are less clear. There is a restriction
in the amount of wild-type MT associated with Src family members and
therefore downstream targets such as SHC and PI 3'-kinase (3, 9,
15). Evidence suggests that the NPTY motif starting at 248 is the
SHC binding site (9). A little further upstream (residues
185 to 210) are elements required for Src binding (5, 20).
It is therefore natural to ask whether 14-3-3 is part of the
restriction. However, there is no indication that MT of mutant 257 has
associated kinase activity significantly greater than wild type (Fig.
6). Similarly, 14-3-3 binding does not prevent association with SHC,
since SHC can be found in 14-3-3 immunoprecipitates from MT transformed
cells (not shown). Transformation assays in fibroblasts also support
the conclusion that association with tyrosine kinases or their targets
such as SHC does not depend substantially on the 14-3-3 interaction.
257 mutants were neither defective nor hyperactive in transformation. A
very recent report raises the possibility that this region of MT is
involved in multimerization of MT complexes (56).
The ability of 257 mutant MT to transform in vitro was broadly
reflected in the ability of virus to induce tumors in animals. All
infected animals had tumors with a broad range of tumor types represented. However, the absence of salivary gland tumors and the
reduction in the subcutaneous connective tissues was dramatic. This
underlines the fact that viral tumorigenesis is a complex process,
reflecting contributions of the viral control regions, viral capsid
proteins, and the transforming proteins. This differential effect on
certain tumor types by mutation in MT in particular repeats a theme
that has been seen before in examination of MTs with mutations at
residues 315 (27) or 250 (6, 66). In those cases,
the specific molecular defect can be traced to loss of binding of a
specific signalling pathway. For 257, we can show that the 14-3-3 association alters the ability of MT to affect the activity of at least
some promoters. Sorting out the precise basis for the effect represents
an interesting problem for the future.
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ACKNOWLEDGMENTS |
This work was supported by NIH grants to B.S.S. (PO-CA50661 and
R37-CA34722), T.R. (PO1-CA50661), T.B. (PO1-CA50661 and R35-CA44343) and D.P. (CA57327) as well as by the Markey Foundation (B.S.S.).
We thank Ken Dower and John Carroll for their assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6876. Fax: (617) 636-6409. E-mail:
bschaffh_pol{at}opal.tufts.edu.
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J Virol, January 1998, p. 558-563, Vol. 72, No. 1
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
