From the
Middle tumor antigen (MT) is the primary transforming protein of
murine Polyomavirus. MT transforms by associating with and
modulating the activities of cellular proteins involved in control of
cell proliferation. MT binds to and is phosphorylated by cellular
tyrosine kinases. The phosphorylated tyrosines become docking sites for
SH2 (Src homology 2) domain-containing molecules. Tyrosine 322 of MT is
known to be phosphorylated but has no known binding protein. We have
found that phospholipase C-
Polyomavirus induces a variety of tumors in rodents and
is capable of transforming primary rodent cell lines. Middle tumor
antigen (MT)
To investigate
the potential role of tyrosine 322 in MT in transformation and in
regulating cellular signaling proteins, we have mutated the tyrosine
322 to phenylalanine (Tyr-322
For anti-PLC-
For
anti-phosphotyrosine immunoblotting, immunoprecipitation was carried
out by incubating 1 µg of anti-PLC-
To test the role of tyrosine 322 in MT function, we mutated
tyrosine 322 to phenylalanine (Tyr-322
Previous work by other laboratories can be reexamined in
light of the data reported here. The reaction catalyzed by PLC-
In experimental
systems for the study of growth factor receptors, it has been difficult
to measure the functional consequences of deleting the binding site for
PLC-
We thank Dr. Steve Dilworth for the generous gift of
the monoclonal antibody PAb 750, and Dr. David Pallas and Dr. Charles
Stiles for critical review of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 (PLC-
1), a SH2 domain-containing
protein, coimmunoprecipitates with MT. Tyrosine phosphorylation of
PLC-
1 is elevated in cells expressing MT, suggesting activation of
this enzyme by MT. A Tyr-322
Phe mutation in MT renders it
defective in MT-PLC-
1 interaction and in transformation. From the
correlation between transformation and MT-PLC-
1 interaction, we
suggest that PLC-
1 may play a role in transformation.
(
)
is the major transforming protein
of Polyomavirus. MT is a membrane protein with no known
enzymatic activity
(1) . It transforms by associating with and
modulating the activities of cellular proteins involved in control of
cell proliferation. MT appears to function much like a growth factor
receptor. It binds to and activates
pp60
and other members of the
src family of tyrosine
kinases
(2, 3, 4, 5, 6) . As a
result, MT is phosphorylated on tyrosines 250, 315, and
322
(7, 8, 9) . When phosphorylated, tyrosines
250 and 315 with adjacent amino acids, respectively, form the binding
sites for the key signaling molecules SHC
(10, 11) and
phosphatidylinositol 3-kinase (PI3-kinase)
(12, 13) .
Mutation of tyrosine 250 or 315 to phenylalanine renders MT defective
both in transformation
(14, 15) and in its ability to
bind SHC or PI3-kinase, respectively. Of the three phosphorylated
tyrosines in MT, the least studied is tyrosine 322.
Phe MT). As judged by
focus-formation assay, the Tyr-322
Phe MT is less transforming
than wild type MT. We have found that phospholipase C-
1
(PLC-
1), a SH2 domain-containing signaling molecule,
coimmunoprecipitates with MT and that tyrosine 322 is important for
this interaction. The tyrosine phosphorylation level of PLC-
1 is
elevated in cells expressing wild type MT but not in cells expressing
Tyr-322
Phe MT. From the correlation between transformation and
MT-PLC-
1 interaction, we suggest that PLC-
1 may play a role
in transformation.
Plasmids and Cell Lines
Site-directed
mutagenesis was performed to create two unique restriction enzyme sites
adjacent to Tyr-315 and Tyr-322 codons in MT cDNA, without altering the
corresponding amino acids. An oligonucleotide-directed mutagenesis
system from Amersham Corp. was used. A StuI site was generated
immediately upstream to the Tyr-315 codon and a XbaI site
downstream to the Tyr-322 codon (Polyomavirus sequence 1176
and 1202, respectively). Excision of the DNA between these two sites
from MT cDNA and insertion of double-stranded oligonucleotides generate
the Tyr-315 Phe and Tyr-322
Phe mutant MTs
(``315F'' and ``322F''). The pLJ retrovirus vector
(16) was used to express the wild type and mutant MTs. The
plasmids were transfected into
2 retrovirus packaging cells by
the CaCl
method
(17) . The transient virus
supernatants were used to infect BALB/3T3 cells. BALB/3T3 cell lines
expressing various MT alleles were isolated as described
previously
(18) . Cells were propagated in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 0.37%
bicarbonate, penicillin (100 units/ml), streptomycin (100 µg/ml),
and 10% calf serum.
Focus Formation Assay
Focus formation assay was
performed by infecting BALB/3T3 cells with retroviruses carrying wild
type MT, Tyr-322 Phe MT, or no MT. The retroviruses were
produced using the virus packaging cell line BOSC 23, which is capable
of producing high titer retroviruses
(19) . Ten micrograms of the
plasmids pLJ-MTwt, pLJ-MT322F, or pLJ were transfected into BOSC 23
cells using the CaCl
method
(17) . The resulting
culturing supernatants containing the retroviruses were collected,
aliquoted, and stored at -80 °C. The high titer virus stocks
generated in the BOSC 23 cell line allow freezing of aliquots, a
process that significantly lowers virus titers. Titers of the frozen
aliquots of these retroviruses were determined by infection of BALB/3T3
cells (as described below) and subsequent selection by 400 µg/ml
G418. After 10 days of incubation with medium changed every 2 days, the
G418-resistant colonies were counted. For focus formation assay, an
equal number of each retrovirus (1.0
10
colony-forming units) in 2.5 ml of medium (DMEM supplemented with
0.37% bicarbonate, penicillin (100 units/ml), streptomycin (100
µg/ml), and 10% calf serum) was used to infect the BALB/3T3 cells
(50% confluent) in 100-mm tissue culture dishes. Polybrene were then
added to a concentration of 0.8 µg/ml. After 4 h of incubation at
37 °C and 10% CO
, 7.5 ml of the same medium was added.
Cultures were incubated at 37 °C overnight in the presence of 10%
CO
. The infected cell populations were then split into two
sets of 60-mm tissue culture dishes. One set was cultured in DMEM
medium supplemented with 10% calf serum, and another set was cultured
in DMEM medium supplemented with 3% calf serum. Media were changed
accordingly every 3 days. At 10 days, cultures were fixed and stained
with 0.2% crystal violet in 10% phosphate-buffered formalin (pH 7.0).
Immunoprecipitation and Immunoblotting
Cells were
allowed to grow to confluency and lysed in a lysis buffer containing 1%
Nonidet P-40, 20 mM TrisCl (pH 8.0), 137 mM
NaCl, and 10% glycerol. For anti-p85 immunoblotting,
immunoprecipitation was carried out by incubating anti-MT monoclonal
antibody PAb 750
(20) (40 µl of antibody-containing tissue
culture fluid) or 1 µg of anti-PLC-
1 monoclonal antibody (UBI)
with cell lysates (600 µg of total protein for every cell line)
from BALB/3T3 cells expressing MT and from BALB/3T3 control
(``vector'') cells. After 2 h of incubation at 4 °C,
protein G beads (40 µl of 50% slurry, Pharmacia Biotech Inc.) were
added, and the tubes were rocked at 4 °C for 1 additional hour.
Immune complexes were sequentially washed with Nonidet P-40 lysis
buffer (1% Nonidet P-40, 20 mM Tris
Cl (pH 8.0), 137
mM NaCl and 10% glycerol), 0.5 M LiCl, 20 mM
Tris
Cl (pH 7.6), and TNE (10 mM Tris
Cl (pH 7.6),
0.1 M NaCl, and 1 mM EDTA). Washed immunoprecipitates
were suspended in 15 µl of sample buffer (10% glycerol, 2% SDS, 100
mM dithiothreitol, 50 mM Tris
Cl (pH 6.8), and
0.1% bromphenol blue), heated for 3 min at 95 °C, and analyzed by
8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to
nitrocellulose membranes, and anti-p85 antibody R33 (rabbit serum,
1:1000 dilution) was used for immunodetection. Immunodetection was
performed using the chemiluminescence reagents and the protocol from
DuPont NEN.
1 immunoblotting, immunoprecipitation
was carried out by incubating 2 µl of anti-MT polyclonal antibody
18-8 (rabbit serum) or 2 µl of anti-p85 antibody R33 (rabbit
serum) with cell lysates (700 µg of total protein for every cell
line) from BALB/3T3 cells expressing MT and from BALB/3T3 control
(vector) cells. After 2 h of incubation at 4 °C, protein A beads
(20 µl of 50% slurry, Pharmacia) were added, and the tubes were
rocked at 4 °C for 1 additional hour. Washing and immunoblotting
were performed as described above except anti-PLC-
1 monoclonal
antibody (0.5 µg/ml, UBI) was used for immunodetection.
1 monoclonal antibody (UBI)
with the cell lysates (700 µg of total protein for each lysate).
After 2 h of incubation at 4 °C, protein G beads in a volume of 40
µl (50% slurry, Pharmacia) were added, and the mixtures were
further incubated with rocking at 4 °C for 1 h. Washing and
immunoblotting of the PLC-
1 immunoprecipitates were carried out
essentially the same as described above, except that 0.7 µg/ml
anti-phosphotyrosine antibody 4G10 was used for immunodetection.
Phe MT) and conducted a
focus formation assay. When the focus formation assay was carried out
in 10% serum, Tyr-322
Phe MT is only slightly less transforming
than wild type MT (Fig. 1). However, when the assay was conducted
using medium containing 3% calf serum, the difference in transformation
between wild type MT and Tyr 322
Phe MT was pronounced.
Figure 1:
Focus
formation assay of wild type and Tyr-322 Phe MTs. BALB/3T3 cells
were infected with an equal number (1.0
10
colony-forming units) of control retrovirus (Vector),
wild type MT (WildType), and Tyr-322
Phe MT
(Tyr322
Phe) as described under
``Experimental Procedures.'' The lower three cultures were
incubated in DMEM plus 10% calf serum (CS) while the upper
three cultures were incubated in the same medium plus 3% calf
serum.
We
next explored the function of tyrosine 322 in transformation. When
phosphorylated, a tyrosine often forms the core of the binding sites
for SH2 domains of intracellular signaling proteins. Previous work
indicated that tyrosine 322 in MT might be involved in the activation
of PI3-kinase
(21) . Therefore, we analyzed the interaction
between MT and p85 (the SH2 domain-containing subunit of PI3-kinase) in
wild type and Tyr-322 Phe MTs using immunoprecipitation in
conjunction with immunoblotting. BALB/3T3 cells and BALB/3T3 cells
expressing wild type, Tyr-315
Phe, and Tyr-322
Phe MTs
were used for the study. These MT cell lines expressed comparable
levels of MT as judged by anti-MT immunoblotting. Anti-MT
immunoprecipitates were immunoblotted using an anti-p85 antibody to
determine the level of p85 associated with each MT allele. As expected,
Tyr-315
Phe MT is defective in its p85 signal (Fig. 2).
However, the p85 signal of Tyr-322
Phe MT is comparable with
that of wild type MT. Therefore, tyrosine 322 appears unimportant for
p85 binding by MT.
Figure 2:
Effect
of Tyr-322 Phe mutation in MT on its association with p85.
Immunoprecipitation with anti-MT antibody and subsequent immunoblotting
of the immunoprecipitates with anti-p85 antibody were performed as
described under ``Experimental Procedures.'' BALB/3T3 cells
expressing wild type, Tyr-315
Phe and Tyr-322
Phe MTs
were constructed and were used in the experiments. vector represents the pLJ vector for the expression of MT cDNAs (16).
wt, 315F, and 322F represent wild type,
Tyr-315
Phe, and Tyr-322
Phe mutant MTs, respectively.
The arrow indicates the position of p85. The numbers at right represent the positions of the molecular mass
standards (kDa).
While the core of an SH2 binding site is formed
by the phosphotyrosine, each SH2 domain achieves binding specificity by
interacting with the amino acids just carboxyl to the phosphotyrosine.
Amino acids 323, 324, and 325 in MT are Leu, Asp, and Ile. Of the SH2
domains studied to date, only the amino-terminal SH2 domain of
PLC-1 has a preference for Tyr(P)-Leu-Asp-Ile
(22) . Hence,
we investigated the possibility of interaction between PLC-
1 and
MT. Immunoblotting using anti-PLC-
1 antibody was carried out on
anti-MT immunoprecipitates. A distinct band at
145 kDa, the
mobility of which corresponds to PLC-
1, is easily observed in the
MT immunoprecipitate from cells expressing wild type MT (Fig. 3).
The PLC-
1 signal in the Tyr-322
Phe mutant MT (322F)
immunoprecipitate is reduced to the control (vector) level. The Tyr-315
Phe mutant MT (315F), in which the p85 binding site is
abolished, however, gives a comparable PLC-
1 signal to that of
wild type MT. Thus, the data of Fig. 3provide a strong
indication that MT associates with PLC-
1 and that tyrosine 322 in
MT is important for the association.
Figure 3:
Association of MT and PLC-1.
Anti-PLC-
1 immunoblotting was performed on anti-MT
immunoprecipitates. lysate represents the cell lysate (2
µg of total protein from BALB/3T3) that was loaded in the lane to
visualize the exact position of PLC-
1. The arrow indicates the position of PLC-
1. The rest of the lettering is
explained in the legend to Fig. 2.
Tyrosine phosphorylation of
PLC-1 is required for its activation in vivo and can
increase its catalytic activity in
vitro(23, 24) . MT complexes contain activated
members of the src family tyrosine
kinases
(2, 3, 4, 5, 6) , and
thus the level of tyrosine phosphorylation of PLC-
1 in MT
transformed cells might be expected to be elevated. As shown in
Fig. 4
, the tyrosine phosphorylation level of PLC-
1 is
indeed increased in cells expressing MTs that bind PLC-
1, i.e. wild type MT and Tyr-315
Phe MT. Cells expressing Tyr-322
Phe MT, which is defective in PLC-
1 binding, showed a level
of PLC-
1 tyrosine phosphorylation comparable with the background
level found in control cells (Fig. 4). The same membrane was
striped and immunoblotted by anti-PLC-
1 antibody in order to
determine the position and the relative quantity of PLC-
1 in these
immunoprecipitates. The PLC-
1 signals appear exactly at the same
position of the signals in the anti-phosphotyrosine blot, and equal
intensities of PLC-
1 signals were observed in each lane.
Figure 4:
Tyrosine phosphorylation of PLC-1.
Anti-phosphotyrosine immunoblotting was carried out on anti-PLC-
1
immunoprecipitates. Immunoprecipitation and immunoblotting were
performed as described under ``Experimental Procedures.'' The
lettering is explained in the legend to Fig.
2.
Since
PLC-1 and p85 both bind MT, we analyzed the interaction of the two
enzymes in cells expressing MT. Immunoblotting with anti-PLC-
1
antibody was conducted on anti-p85 immunoprecipitates from cells
expressing the various alleles of MT. High levels of PLC-
1 were
detected only in the anti-p85 immunoprecipitates from cells expressing
wild type MT and not from control cells or cells containing MTs, which
were mutant in either the PLC-
1 or p85 binding site
(Fig. 5). The reverse of the above experiment was also carried
out. Anti-p85 immunoblotting was performed on anti-PLC-
1
immunoprecipitates. Similar results were obtained (Fig. 6). Since
previous studies have shown that each MT
src complex
contains only one molecule of MT
(25) , the data of
Fig. 5
and Fig. 6raise the possibility that PLC-
1 and
p85 might bind to the same MT molecule. However, the data in
Fig. 2
and Fig. 3suggest that no positive or negative
cooperativity occurs between PLC-
1 and p85 in their binding to MT.
This follows from the fact that when MT loses the binding site for one
protein, its binding to the other one is similar to wild type MT
(Fig. 2, lanes3 and 4; Fig. 3,
lanes2 and 3).
Figure 5:
Anti-PLC-1 immunoblotting of anti-p85
immunoprecipitates. Immunoprecipitation and immunoblotting were
conducted as described under ``Experimental Procedures.'' The
lettering is explained in the legend to Fig.
2.
Figure 6:
Anti-p85
immunoblotting of anti-PLC-1 immunoprecipitates.
Immunoprecipitation and immunoblotting were performed as described
under ``Experimental Procedures.'' The lettering is
described in the legend to Fig. 2. Cell lysate (2 µg of total
protein from BALB/3T3) was loaded in the last lane (labeled as
lysate) to visualize the exact position of
p85.
1
produces two second messenger molecules: inositol 1,4,5-trisphosphate
and diacylglycerol. It has been observed that the expression of wild
type MT caused an increase in the inositol 1,4,5-trisphosphate
level
(26) . Diacylglycerol activates protein kinase
C
(27) . It has been shown that protein kinase C is activated in
cells by the expression of MT
(28, 29) . Both of these
published results are consistent with our conclusion that PLC-
1
may be involved in MT-induced transformation.
1 on the receptors. Tyr
Phe mutations in the PLC-
1
binding sites of the platelet-derived growth factor, fibroblast growth
factor, and nerve growth factor receptors had no detectable biological
effects
(30, 31, 32) . However, restoring the
PLC-
1 binding site also restores a mitogenic response in a
platelet-derived growth factor receptor with multiple Tyr
Phe
mutations
(33) . We observed a pronounced difference in
transformation ability of wild type MT and Tyr-322
Phe MT in 3%
calf serum (Fig. 1). In 10% serum, the effects of the Tyr-322
Phe mutation are less obvious. Presumably, when certain factors
in serum are less available, the PLC-
1 pathway becomes limiting in
MT-induced transformation. Wild type Polyomavirus raises, as
its name would suggest, a wide variety of tumors in mice. Thus
installation of a PLC-
1 binding site mutation into the polyoma
genome will create an excellenttest system to evaluate the
role of PLC-
1 activation in a broad spectrum of tumor types in
vivo.
1, phospholipase C-
1; DMEM, Dulbecco's modified
Eagle's medium; SH2, Src homology 2; PI3-kinase,
phosphatidylinositol 3-kinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.