Vol.
86,
No.
February
BIOCHEMICAL
4, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
28, 1979
1169-1177
RELATIONSHIP BETWEEN CHANGES IN THE CALCIUM DEPENDENT REGULATORY PROTEIN AND ADENYLATE CYCLASE DURING VIRAL TRANSFORMATION David
C. LaPorte,*
Department
of Pharmacology,* University Seattle, Washinqton 98195 of Microbiology,+ University Urbana, Illinois 61801
Department
Received
December
1,
Susan Gidwitz,* ?/!ichael and Daniel R. Storm*
J.
Weber'
of Washington, and of Illinois,
1978
SUMMARY The levels of the calcium dependent regulatory protein in transformed chicken embryo fibroblasts are higher both in soluble fractions and membrane fractions compared to untransformed cells. The kinetics for changes in the calcium dependent regulatory protein, hex&e transport, and adenylate cyclase were compared using a temperature sensitive mutant of Rous sarcoma virus. Decreases in adenylate cyclase activity and increased hexose transport accompanying transformation occurred with half-lives of approximately 7 to 8 hours. Increases in the calcium dependent regulatory protein occurred much slower with a half-life of seventeen hours. It is concluded that the increase in calcium dependent regulatory protein levels is a late event during viral transformation and that the decline in adenylate cyclase activity cannot be due to changes in the amount of calcium dependent regulatory protein. Adenylate
cyclase
activity
is decreased
in many malignant
cell
types
including
, chicken
embryo
Recent (2,3) tory
studies
protein
tumor (CDR).
are
higher
have reported
that
observations,
adenylate
(CEF)'
have shown that
and glial
levels
these
fibroblasts
cyclase
cells
(4)
In addition, in transformed high
levels
transformed
adenylate
Watterson
using
activities
--et al.
(5)
fibroblasts
of CDR inhibit
adenylate
the kinetics
a temperature
virus
dependent
have reported and Brostrom cyclase
for
changes
dependent
(RSV)(l).
in mammalian
by the calcium
normal
we have examined activity
cyclase
are modulated
than
by Rous sarcoma
(4).
brain regula-
that
CDR
-et -*al Because
in CDR levels
mutant
(4) of and
of RSV.
MATERIALS AND METHODS Growth
and Harvesting of Cells Chicken embryo fibroblasts were grown as previously described (6). Secondary cultures were plated at cell densities of 6.25 x 105 cells/lOO-mm plate for normal cells and 1.25 x lo6 cells/lOO-mm plate for cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (RSV-SR-A). Because fewer of the trans1 The abbreviations used are: CEF, chicken sarcoma virus; CDR, calcium dependent regu'latory
embryo fibroblasts; protein.
RSV, Rous
0006-291X/79/041169-09$01.00/0 1169
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
formed cells attached and grew this resulted in similar cell densities at the time of the experiment. Cells were grown at 36°C in Dulbecco's Modified Eagle's Medium, high glucose formula containing penicillin and streptomycin, supplemented with chicken serum. 10% tryptose phosphate broth, 4% calf serum, and 1% heat-inactivated Fibroblasts were harvested on the 3rd day without an intervening culture medium In the temperature shift experiment CEF were infected with the tsNY68 change. mutant of RSV-SR-A or with wild type RSV-SR-A and grown initially at 42°C. Cells were shifted from 42°C to 36°C at various times so that all cells could be harvested within a two hour period. Cells were harvested as previously described (7). Membrane Preparation and Solubilization Crude membranes were prepared as previously described (7) except in the experiment with tsNY68 where the crude homogenate was not diluted prior to centrifugation. Supernatants were assayed for CDR activity. CDR depleted membranes were prepared by a modification of the method of Brostrom et al. (8). One volume crude membranes was homogenized with 9 volumes 1 mM EDTA, 10 mM imidazole pH 7.5 using a tight fitting dounce homogenizer. The suspension was incubated at 4°C for 0.5 hr. with periodic vortexing and then centrifuged at 100,000 x g for 0.75 hr. The pellet was washed twice more with EDTA-Tris buffer and the pellet was finally resuspended in 500 UM EDTA, 1 mM MgC12, 10 mM imidazole pH 7.5. Crude membranes were solubilized in a buffer containing 25 mM Tris.Cl pH 7.5, 0.25 M sucrose, 1 mM MgC12, 1 mM EDTA and 1% Brij 96. The ratio of detergent/membrane protein (w/w) was 2:l. The suspension was incubated at 4" for 0.5 hours with periodic vortexing and then centrifuged at 100,000 x g The 100,000 x g supernatant was used for adenylate cyclase assays. for one hour. Adenylate Cyclase Assay Adenylate cyclase was assayed by the method [a32P]ATP as a substrate and [3H]cAMP to monitor was purified by DEAE Sephadex A-25 chromatography chromatography.
of Salomon et al. (9) using recovery. ATP used in assays followed by Dowex AG-50
Calcium Dependent Regulatory Protein CDR was assayed by its ability as previously described (10).
CDR depleted
Assay to stimulate
phosphodiesterase
Polyacrylamide Gel Electrophoresis Supernatant protein was subjected to electrophoresis on non-denaturing discontinuous polyacrylamide slab gels using the procedure of Davis (11). EGTA at a concentration of 100 DMolar was present in the electrophoresis buffers. Transport of [3H]-2-Deoxyglucose Transport of 2-deoxyglucose
was determined
as previously
described
(6).
RESULTS AND DISCUSSION The CDR content The soluble
fraction
CDR activity
higher
the data
from
as normal
CEF transformed times
of normal
reported
the different
transformed
cells.
normal
strain
CEF (5).
by Watterson
Rous sarcoma
cells
Watterson
by the Prague than
and RSV-SR-A
et al.
Our results
reported
are
reported
approximately that
virus
used or the methods
twice
the
in Table as much
CDR content
of
was approximately
qualitatively
The quantitative
1170
CEF are
contained
of Rous sarcoma
et al.
strains
transformed
consistent
differences employed
four with
may reflect for
quantitating
I.
Vol.
86, No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE CDR in
Normal
and
RESEARCH
I Transformed
CEF
r
Sample
COMMUNICATIONS
CDR (units/mg)* Soluble?
Membrane+
36"
42"
36"
Normal
1351
1272
341
RSV-SR-A
2857
2754
443
RSV-tsNY68
2750
1125
ND9
Cells were grown at 36" or 42" and harvested as previously described (6, 7). In the temperature shift experiment, CEF were infected with the temperature sensitive mutant of RSV (RSV-tsNY68) or with wild type RSV-SR-A and grown initially at 42". Cells were then shifted from 42" to 36". f Membranes were prepared as previously described (7). * Soluble refers to the supernatant obtained after centrifuging homogenized cells for one hr at 100,000 x g. * CDR was assayed by stimulation of the CDR-depleted Ca*+ sensitive phosphodiesterase (10). One unit of CDR caused 50% stimulation of 10 units of the phosphodiesterase. The CDR content of membranes was determined after solubilization of membranes with lubrol PX (7).
The
CDR levels. also
determined
shown
in
since
Table
I,
normal
membranes.
mutant
of
were to
CDR content
Rous
grown normal the
data
indicate
Similar
However, is
virus,
these
of obtained
actually
more
of
normal
cells
Watterson contained
et
transformed obtained
grown
approximately twice
by
is
gel
in (5).
half
as
as
as
higher
much
CDR activity
1171
as
sensitive RSV-tsNY68
in
100,000
was
the
normal
CDR
in
normal
below,
agreement
normal
These
CDF
or
cells. show
with
x g supernatant the
comparable
cells.
transformed
to
as
to
temperature,
discussed
the
comparable
CDR content
CDR in
cells
As
with
permissive
compared
transformed Although
infected
high
much
was
were
enzyme.
a temperature
the
the
electrophoresis,
the
al.
CEF
at
twice
is
with
42"C,
membranes
associated
membranes
When
were
transformed
a membrane
temperature,
protein
CDR
RSV-SR-A
RSV-tsNY68.
there the
results
results
is
were
cells
either
activity
cyclase
results
was
that
and
of
nonpermissive When
normal
CDR content
CDR content
intrinsic
there
the
the
CEF.
36"C,
adenylate
sarcoma
at
of
cells,
that the from both
the
BIOCHEMICAL
Vol. 86, No. 4, 1979
I
I
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I
I
I
)O-
A
IO;0IO!O/ o!O)O SOSOIO-
L&&- .
!Oo-
1
I ,
I
1
I
I
I
I
I
8
12
16
20
24
28
32
I
I
I I
I
3-
2-
l-
r
I
OO
4
Hours
Fig. 1. Time course of change in (A) Z-deoxyglucose uptake, (B) adenylate cyclase activity, and (C) CDR activity in CEF infected with RSV-tsNY68 following a shift in temperature from 42" to 36". CEF were grown and harvested as preCells were infected with RSV-tsNY68, grown at 42" viously described (6, 7). and then shifted to 36°C (time zero). Membranes were prepared as previously described (7). Transport of 2-deoxyglucose by whole cells was assayed using Adenylate cyclase was assayed by the method of [3H]-P-deoxyglucose (6). Salomon using [o32P]ATP as a substrate and [3H]cAMP to monitor recovery (9). CDR activity in the soluble fraction was assayed by stimulation of CDR depleted phosphodiesterase (10).
1172
Vol. 86, No. 4, 1979
supernatants level
stimulated
these
normal
required
cyclase
changes
tions
6 fold
various
after
occurred
were
cyclase
activity
by wild
type
within
shifting
hours
in CDR levels
shift.
Seventeen
hours
21 hours
were
increase
in CDR activity
required
in CDR is a relatively
adenylate
Because
cyclase
cyclase activities
temperature
No differences
in the
required complete
corresponded
late
gels event
(Fig. during
of CDR are
12 hours
the 2).
These
data
transformation
directly
tsNY68
after
The time
increase
adenylate
indicate and it
In contrast, the temperature and more than course
for
of
of CDR as
that is
typical
cultures
in the amount
responsible
transformed
changes
in CDR levels
in activity.
with
By 12 hours,
or cells
by 12 hours.
50% change
change
cells
and
in activity
shift.
shifted
complete
for
at
cyclase
Morphological
until
cells
50% change
in normal
to occur
sister
frac-
increases
in CDR activity,
temperature
essentially
the
unlikely
increase that
the decrease
in
activity.
of the observations
Ca2+ stimulation adenylate
also
were
in the amount cyclase
after
to 36".
and soluble
Adenylate
course.
course
phenotypically
(which
using
1).
shift.
time
are
uptake
same time the
the
in membrane
(Fig.
occurred
not begin
for
CDR
in CDR levels
when shifted
was measured
the
transport
on polyacrylamide
the changes
measured
a temperature
did
same
brain
changes
RSV-tsNY68
2-deoxyglucose
complete.
and were
with
become transformed
while
apparent
between
infected
7 to 8 hours
were
of purified
upon transformation,
were
with
following
changes
quantitated
Cells rapidly
changed
or hexose
cells
four
occur
from 42" to 36"
essentially
virus
of transformed
which
but
at approximately
amounts
relationship
upon transformation)
uptake
the changes
the
the same cells
approximately
2-deoxyglucose
saturating
and CDR activities
from
times
activity
at 42",
cyclase
obtained
define
were compared.
when grown
Adenylate
with
This
Ca*+.
to more fully
and adenylate
PDE to the same maximum level.
was obtained
stimulations
In order
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CDR-depleted
of stimulation
and all
for
BIOCHEMICAL
of brain
adenylate
in normal were
cited
above and the reports
cyclase
(2,
and RSV transformed
assayed
as a function
1173
3),
that
the calcium
membranes
CDR mediates sensitivity
was examined.
of Ca 2+ concentration
of Adenylate
in the
Fig. 2. Polyacrylamide slab gel of 100,000 x g supernatant from CEF infected with RSV-SR-tsNY68 shifted from 42" to 36" at (A) 0, (B) 6, (C) 12, (D) 18, (E) 24, (F) 30 hours following temperature shift, (G), cells grown at 36", (H) 3 ug of purified bovine brain CDR (10). Supernatant protein was subjected to electrophoresis on non-denaturing discontinuous polyacrylamide slab gels (11). Slots A-G each contained 250 ug of protein.
Vol.
86,
No.
4, 1979
BIOCHEMICAL
100
200
300
AND
400
500
BIOPHYSICAL
600
700
RESEARCH
600
900
COMMUNICATIONS
EC 0
CaCIe (,uM) Fig. 3. Effect of CaC12 on adenylate cyclase activity from normal (I) and RSV-SR-A infected CEF (a). Membranes were prepared as previously described (7). Adenylate cyclase was assayed by the method of Salomon (9) using 1 mM [a32P]ATP 3 mM MgC12, 200 pM EGTA, and indicated amounts of CaCl in the assay cocktail. A, membranes; B, membranes solubilized with Brij 56 (7 5 .
presence
of 3 mM Mg2+ and 200 LIM EGTA (Fig.
membranes
showed
40% at low calcium In contrast, at all observed
a biphasic
concentrations,
adenylate
concentrations
response
cyclase of calcium
3A).
to calcium. followed
activity
Adenylate This
tested.
activity
by inhibition
in transformed Similar
when 1 mM EGTA was used to complex
1175
in normal
was stimulated
at higher membranes
calcium
endogenous
cyclase
concentrations.
was inhibited
dependencies
Ca 2+ ,
Solubilization
were of
Vol. 86, No. 4, 1979
adenylate
cyclase
calcium
BIOCHEMICAL
from normal
sensitivity
branes
(Fig.
EDTA containing
cyclase
was lost
was mediated
membranes
unable
The data levels
Therefore,
calcium
sensitivity
cyclase
adenylate
report
extensively
of adenylate
of adenylate
adenylate
cyclase
of CDR present cyclase
demonstrate
that
the
of CEF by RSV is a late cyclase
activity
in
in transformed
of CDR. It seems likely in transformed
membrane that
or membrane
in CDR
which hexose
cannot
are
trans-
cyclase
not attributable
preparations
contained
Ca2+ .CDR cannot because
occurs
be due to
in adenylate
membranes
membranes
event
activity
The differences
these
increase
and increased
cyclase
and transformed
of CDR since
system
stimulation
levels
that
in adenylate
in normal
were washed
mem-
Ca '+CDR.
of CDR present.
activity
cyclase
with
in this
in the amount
comparable levels
imply
and the
and transformed
stimulation
are sufficient
data
the decline
in the amount
late
these
in adenylate
changes
to changes
there
stimulation
membranes
the calcium
transformation
the decrease
port.
Since
presented
calcium
from normal
When normal
that
to interact
accompanying
after
abolished
to remove CDR, calcium
suggesting
membranes, is
3B).
buffers
by CDR.
transformed
membranes
of the enzyme solubilized
was comparable
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
modulate
of alterations
adeny-
in the
environment.
ACKNOWLEDGMENT We wish technical Heart
to express
assistance.
Association,
Career
Development
and Research Public
Health
our appreciation This
National Award
Career Service
work
was supported
Science A100120
Development Predoctoral
to Diane
Foundation
to D.R.S. Award
by a grant Grant
for
from
her skillful the
PCM 73-001245,
and National
CA00092
Fellowship
G. Toscano
to M.J.W.
Cancer S.G.
Grant
Illinois and Research CA12467
is a recipient
5T 32 GM07110-03.
REFERENCES 1. 2. 3.
Anderson, W.B. and Pastan, I. (1975) Advances in Cyclic Nut. Res. 5, 681-698. Brostrom, C.O., Huang, Y.C., Breckenridge, B.M. and Wolff, D.J. (1975) Proc. Natl. Acad. Sci. USA 72, 64-68. Cheung, W.Y., Bradham, L.S., Lynch, T.J., Lin, Y.M. and Tallant, E.A. (1975) Biochem. Biophys. Res. Conmun. 66, 1055-1062.
1176
of
Vol.
4.
86, No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Brostrom,
M.A., Brostrom, C.O., Breckenridge, B.M. and Wolff, D.J. (1976) Chem. 251, 4744-4750. Watterson, D.M., Van Eldik, L.J., Smith, R.E. and Vanaman, T.C. (1976) Proc. Natl. Acad. Sci. USA 73, 2711-2715. Weber, M.J. (1973) J. Biol. Chem. 248, 2978-2983. Gidwitz, S., Weber, M.J. and Storm, D.R. (1976) J. Biol. Chem. 251, 79507951. Brostrom, C.O., Brostrom, M.A. and Wolff, D.J. (1977) J. Biol. Chem. 252,
J. Biol.
5. F: 8.
5677-5685. 9. 10. 11.
Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 3, 541-548. LaPorte, D.C. and Storm, D.R. (1978) J. Biol. Chem. 253, 3374-3377. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427.
1177
86,
No.
February
BIOCHEMICAL
4, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
28, 1979
1169-1177
RELATIONSHIP BETWEEN CHANGES IN THE CALCIUM DEPENDENT REGULATORY PROTEIN AND ADENYLATE CYCLASE DURING VIRAL TRANSFORMATION David
C. LaPorte,*
Department
of Pharmacology,* University Seattle, Washinqton 98195 of Microbiology,+ University Urbana, Illinois 61801
Department
Received
December
1,
Susan Gidwitz,* ?/!ichael and Daniel R. Storm*
J.
Weber'
of Washington, and of Illinois,
1978
SUMMARY The levels of the calcium dependent regulatory protein in transformed chicken embryo fibroblasts are higher both in soluble fractions and membrane fractions compared to untransformed cells. The kinetics for changes in the calcium dependent regulatory protein, hex&e transport, and adenylate cyclase were compared using a temperature sensitive mutant of Rous sarcoma virus. Decreases in adenylate cyclase activity and increased hexose transport accompanying transformation occurred with half-lives of approximately 7 to 8 hours. Increases in the calcium dependent regulatory protein occurred much slower with a half-life of seventeen hours. It is concluded that the increase in calcium dependent regulatory protein levels is a late event during viral transformation and that the decline in adenylate cyclase activity cannot be due to changes in the amount of calcium dependent regulatory protein. Adenylate
cyclase
activity
is decreased
in many malignant
cell
types
including
, chicken
embryo
Recent (2,3) tory
studies
protein
tumor (CDR).
are
higher
have reported
that
observations,
adenylate
(CEF)'
have shown that
and glial
levels
these
fibroblasts
cyclase
cells
(4)
In addition, in transformed high
levels
transformed
adenylate
Watterson
using
activities
--et al.
(5)
fibroblasts
of CDR inhibit
adenylate
the kinetics
a temperature
virus
dependent
have reported and Brostrom cyclase
for
changes
dependent
(RSV)(l).
in mammalian
by the calcium
normal
we have examined activity
cyclase
are modulated
than
by Rous sarcoma
(4).
brain regula-
that
CDR
-et -*al Because
in CDR levels
mutant
(4) of and
of RSV.
MATERIALS AND METHODS Growth
and Harvesting of Cells Chicken embryo fibroblasts were grown as previously described (6). Secondary cultures were plated at cell densities of 6.25 x 105 cells/lOO-mm plate for normal cells and 1.25 x lo6 cells/lOO-mm plate for cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (RSV-SR-A). Because fewer of the trans1 The abbreviations used are: CEF, chicken sarcoma virus; CDR, calcium dependent regu'latory
embryo fibroblasts; protein.
RSV, Rous
0006-291X/79/041169-09$01.00/0 1169
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
formed cells attached and grew this resulted in similar cell densities at the time of the experiment. Cells were grown at 36°C in Dulbecco's Modified Eagle's Medium, high glucose formula containing penicillin and streptomycin, supplemented with chicken serum. 10% tryptose phosphate broth, 4% calf serum, and 1% heat-inactivated Fibroblasts were harvested on the 3rd day without an intervening culture medium In the temperature shift experiment CEF were infected with the tsNY68 change. mutant of RSV-SR-A or with wild type RSV-SR-A and grown initially at 42°C. Cells were shifted from 42°C to 36°C at various times so that all cells could be harvested within a two hour period. Cells were harvested as previously described (7). Membrane Preparation and Solubilization Crude membranes were prepared as previously described (7) except in the experiment with tsNY68 where the crude homogenate was not diluted prior to centrifugation. Supernatants were assayed for CDR activity. CDR depleted membranes were prepared by a modification of the method of Brostrom et al. (8). One volume crude membranes was homogenized with 9 volumes 1 mM EDTA, 10 mM imidazole pH 7.5 using a tight fitting dounce homogenizer. The suspension was incubated at 4°C for 0.5 hr. with periodic vortexing and then centrifuged at 100,000 x g for 0.75 hr. The pellet was washed twice more with EDTA-Tris buffer and the pellet was finally resuspended in 500 UM EDTA, 1 mM MgC12, 10 mM imidazole pH 7.5. Crude membranes were solubilized in a buffer containing 25 mM Tris.Cl pH 7.5, 0.25 M sucrose, 1 mM MgC12, 1 mM EDTA and 1% Brij 96. The ratio of detergent/membrane protein (w/w) was 2:l. The suspension was incubated at 4" for 0.5 hours with periodic vortexing and then centrifuged at 100,000 x g The 100,000 x g supernatant was used for adenylate cyclase assays. for one hour. Adenylate Cyclase Assay Adenylate cyclase was assayed by the method [a32P]ATP as a substrate and [3H]cAMP to monitor was purified by DEAE Sephadex A-25 chromatography chromatography.
of Salomon et al. (9) using recovery. ATP used in assays followed by Dowex AG-50
Calcium Dependent Regulatory Protein CDR was assayed by its ability as previously described (10).
CDR depleted
Assay to stimulate
phosphodiesterase
Polyacrylamide Gel Electrophoresis Supernatant protein was subjected to electrophoresis on non-denaturing discontinuous polyacrylamide slab gels using the procedure of Davis (11). EGTA at a concentration of 100 DMolar was present in the electrophoresis buffers. Transport of [3H]-2-Deoxyglucose Transport of 2-deoxyglucose
was determined
as previously
described
(6).
RESULTS AND DISCUSSION The CDR content The soluble
fraction
CDR activity
higher
the data
from
as normal
CEF transformed times
of normal
reported
the different
transformed
cells.
normal
strain
CEF (5).
by Watterson
Rous sarcoma
cells
Watterson
by the Prague than
and RSV-SR-A
et al.
Our results
reported
are
reported
approximately that
virus
used or the methods
twice
the
in Table as much
CDR content
of
was approximately
qualitatively
The quantitative
1170
CEF are
contained
of Rous sarcoma
et al.
strains
transformed
consistent
differences employed
four with
may reflect for
quantitating
I.
Vol.
86, No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE CDR in
Normal
and
RESEARCH
I Transformed
CEF
r
Sample
COMMUNICATIONS
CDR (units/mg)* Soluble?
Membrane+
36"
42"
36"
Normal
1351
1272
341
RSV-SR-A
2857
2754
443
RSV-tsNY68
2750
1125
ND9
Cells were grown at 36" or 42" and harvested as previously described (6, 7). In the temperature shift experiment, CEF were infected with the temperature sensitive mutant of RSV (RSV-tsNY68) or with wild type RSV-SR-A and grown initially at 42". Cells were then shifted from 42" to 36". f Membranes were prepared as previously described (7). * Soluble refers to the supernatant obtained after centrifuging homogenized cells for one hr at 100,000 x g. * CDR was assayed by stimulation of the CDR-depleted Ca*+ sensitive phosphodiesterase (10). One unit of CDR caused 50% stimulation of 10 units of the phosphodiesterase. The CDR content of membranes was determined after solubilization of membranes with lubrol PX (7).
The
CDR levels. also
determined
shown
in
since
Table
I,
normal
membranes.
mutant
of
were to
CDR content
Rous
grown normal the
data
indicate
Similar
However, is
virus,
these
of obtained
actually
more
of
normal
cells
Watterson contained
et
transformed obtained
grown
approximately twice
by
is
gel
in (5).
half
as
as
as
higher
much
CDR activity
1171
as
sensitive RSV-tsNY68
in
100,000
was
the
normal
CDR
in
normal
below,
agreement
normal
These
CDF
or
cells. show
with
x g supernatant the
comparable
cells.
transformed
to
as
to
temperature,
discussed
the
comparable
CDR content
CDR in
cells
As
with
permissive
compared
transformed Although
infected
high
much
was
were
enzyme.
a temperature
the
the
electrophoresis,
the
al.
CEF
at
twice
is
with
42"C,
membranes
associated
membranes
When
were
transformed
a membrane
temperature,
protein
CDR
RSV-SR-A
RSV-tsNY68.
there the
results
results
is
were
cells
either
activity
cyclase
results
was
that
and
of
nonpermissive When
normal
CDR content
CDR content
intrinsic
there
the
the
CEF.
36"C,
adenylate
sarcoma
at
of
cells,
that the from both
the
BIOCHEMICAL
Vol. 86, No. 4, 1979
I
I
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I
I
I
)O-
A
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I ,
I
1
I
I
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I
I
8
12
16
20
24
28
32
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I
3-
2-
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4
Hours
Fig. 1. Time course of change in (A) Z-deoxyglucose uptake, (B) adenylate cyclase activity, and (C) CDR activity in CEF infected with RSV-tsNY68 following a shift in temperature from 42" to 36". CEF were grown and harvested as preCells were infected with RSV-tsNY68, grown at 42" viously described (6, 7). and then shifted to 36°C (time zero). Membranes were prepared as previously described (7). Transport of 2-deoxyglucose by whole cells was assayed using Adenylate cyclase was assayed by the method of [3H]-P-deoxyglucose (6). Salomon using [o32P]ATP as a substrate and [3H]cAMP to monitor recovery (9). CDR activity in the soluble fraction was assayed by stimulation of CDR depleted phosphodiesterase (10).
1172
Vol. 86, No. 4, 1979
supernatants level
stimulated
these
normal
required
cyclase
changes
tions
6 fold
various
after
occurred
were
cyclase
activity
by wild
type
within
shifting
hours
in CDR levels
shift.
Seventeen
hours
21 hours
were
increase
in CDR activity
required
in CDR is a relatively
adenylate
Because
cyclase
cyclase activities
temperature
No differences
in the
required complete
corresponded
late
gels event
(Fig. during
of CDR are
12 hours
the 2).
These
data
transformation
directly
tsNY68
after
The time
increase
adenylate
indicate and it
In contrast, the temperature and more than course
for
of
of CDR as
that is
typical
cultures
in the amount
responsible
transformed
changes
in CDR levels
in activity.
with
By 12 hours,
or cells
by 12 hours.
50% change
change
cells
and
in activity
shift.
shifted
complete
for
at
cyclase
Morphological
until
cells
50% change
in normal
to occur
sister
frac-
increases
in CDR activity,
temperature
essentially
the
unlikely
increase that
the decrease
in
activity.
of the observations
Ca2+ stimulation adenylate
also
were
in the amount cyclase
after
to 36".
and soluble
Adenylate
course.
course
phenotypically
(which
using
1).
shift.
time
are
uptake
same time the
the
in membrane
(Fig.
occurred
not begin
for
CDR
in CDR levels
when shifted
was measured
the
transport
on polyacrylamide
the changes
measured
a temperature
did
same
brain
changes
RSV-tsNY68
2-deoxyglucose
complete.
and were
with
become transformed
while
apparent
between
infected
7 to 8 hours
were
of purified
upon transformation,
were
with
following
changes
quantitated
Cells rapidly
changed
or hexose
cells
four
occur
from 42" to 36"
essentially
virus
of transformed
which
but
at approximately
amounts
relationship
upon transformation)
uptake
the changes
the
the same cells
approximately
2-deoxyglucose
saturating
and CDR activities
from
times
activity
at 42",
cyclase
obtained
define
were compared.
when grown
Adenylate
with
This
Ca*+.
to more fully
and adenylate
PDE to the same maximum level.
was obtained
stimulations
In order
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CDR-depleted
of stimulation
and all
for
BIOCHEMICAL
of brain
adenylate
in normal were
cited
above and the reports
cyclase
(2,
and RSV transformed
assayed
as a function
1173
3),
that
the calcium
membranes
CDR mediates sensitivity
was examined.
of Ca 2+ concentration
of Adenylate
in the
Fig. 2. Polyacrylamide slab gel of 100,000 x g supernatant from CEF infected with RSV-SR-tsNY68 shifted from 42" to 36" at (A) 0, (B) 6, (C) 12, (D) 18, (E) 24, (F) 30 hours following temperature shift, (G), cells grown at 36", (H) 3 ug of purified bovine brain CDR (10). Supernatant protein was subjected to electrophoresis on non-denaturing discontinuous polyacrylamide slab gels (11). Slots A-G each contained 250 ug of protein.
Vol.
86,
No.
4, 1979
BIOCHEMICAL
100
200
300
AND
400
500
BIOPHYSICAL
600
700
RESEARCH
600
900
COMMUNICATIONS
EC 0
CaCIe (,uM) Fig. 3. Effect of CaC12 on adenylate cyclase activity from normal (I) and RSV-SR-A infected CEF (a). Membranes were prepared as previously described (7). Adenylate cyclase was assayed by the method of Salomon (9) using 1 mM [a32P]ATP 3 mM MgC12, 200 pM EGTA, and indicated amounts of CaCl in the assay cocktail. A, membranes; B, membranes solubilized with Brij 56 (7 5 .
presence
of 3 mM Mg2+ and 200 LIM EGTA (Fig.
membranes
showed
40% at low calcium In contrast, at all observed
a biphasic
concentrations,
adenylate
concentrations
response
cyclase of calcium
3A).
to calcium. followed
activity
Adenylate This
tested.
activity
by inhibition
in transformed Similar
when 1 mM EGTA was used to complex
1175
in normal
was stimulated
at higher membranes
calcium
endogenous
cyclase
concentrations.
was inhibited
dependencies
Ca 2+ ,
Solubilization
were of
Vol. 86, No. 4, 1979
adenylate
cyclase
calcium
BIOCHEMICAL
from normal
sensitivity
branes
(Fig.
EDTA containing
cyclase
was lost
was mediated
membranes
unable
The data levels
Therefore,
calcium
sensitivity
cyclase
adenylate
report
extensively
of adenylate
of adenylate
adenylate
cyclase
of CDR present cyclase
demonstrate
that
the
of CEF by RSV is a late cyclase
activity
in
in transformed
of CDR. It seems likely in transformed
membrane that
or membrane
in CDR
which hexose
cannot
are
trans-
cyclase
not attributable
preparations
contained
Ca2+ .CDR cannot because
occurs
be due to
in adenylate
membranes
membranes
event
activity
The differences
these
increase
and increased
cyclase
and transformed
of CDR since
system
stimulation
levels
that
in adenylate
in normal
were washed
mem-
Ca '+CDR.
of CDR present.
activity
cyclase
with
in this
in the amount
comparable levels
imply
and the
and transformed
stimulation
are sufficient
data
the decline
in the amount
late
these
in adenylate
changes
to changes
there
stimulation
membranes
the calcium
transformation
the decrease
port.
Since
presented
calcium
from normal
When normal
that
to interact
accompanying
after
abolished
to remove CDR, calcium
suggesting
membranes, is
3B).
buffers
by CDR.
transformed
membranes
of the enzyme solubilized
was comparable
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
modulate
of alterations
adeny-
in the
environment.
ACKNOWLEDGMENT We wish technical Heart
to express
assistance.
Association,
Career
Development
and Research Public
Health
our appreciation This
National Award
Career Service
work
was supported
Science A100120
Development Predoctoral
to Diane
Foundation
to D.R.S. Award
by a grant Grant
for
from
her skillful the
PCM 73-001245,
and National
CA00092
Fellowship
G. Toscano
to M.J.W.
Cancer S.G.
Grant
Illinois and Research CA12467
is a recipient
5T 32 GM07110-03.
REFERENCES 1. 2. 3.
Anderson, W.B. and Pastan, I. (1975) Advances in Cyclic Nut. Res. 5, 681-698. Brostrom, C.O., Huang, Y.C., Breckenridge, B.M. and Wolff, D.J. (1975) Proc. Natl. Acad. Sci. USA 72, 64-68. Cheung, W.Y., Bradham, L.S., Lynch, T.J., Lin, Y.M. and Tallant, E.A. (1975) Biochem. Biophys. Res. Conmun. 66, 1055-1062.
1176
of
Vol.
4.
86, No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Brostrom,
M.A., Brostrom, C.O., Breckenridge, B.M. and Wolff, D.J. (1976) Chem. 251, 4744-4750. Watterson, D.M., Van Eldik, L.J., Smith, R.E. and Vanaman, T.C. (1976) Proc. Natl. Acad. Sci. USA 73, 2711-2715. Weber, M.J. (1973) J. Biol. Chem. 248, 2978-2983. Gidwitz, S., Weber, M.J. and Storm, D.R. (1976) J. Biol. Chem. 251, 79507951. Brostrom, C.O., Brostrom, M.A. and Wolff, D.J. (1977) J. Biol. Chem. 252,
J. Biol.
5. F: 8.
5677-5685. 9. 10. 11.
Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 3, 541-548. LaPorte, D.C. and Storm, D.R. (1978) J. Biol. Chem. 253, 3374-3377. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427.
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