a potent inhibitor of phosphatidylinositol. 3-kinase, a tyrosine kinase- regulated enzyme, blocks mitogen-dependent activation of the transfemn receptor promoter ...
Vol. 8, 565-570,
May 1997
Cell Growth & Differentiation
Phosphatidylinositol 3-Kinase Inhibitor Mitogenic Activation of the Transferrin Promoter in Late G1’
W. Keith Robin University
Miskimins,2 Miskimins of
King,
and
component for induction of DNA synthesis. activity of this enzyme resides in a 1 10-kDa
Dakota School of Medicine, Department of
South
Biochemistry
Frank
and Molecular
Biology,
Vermillion,
South
Dakota
associated 57069
Abstract Expression of the transfemn receptor is necessary for cells to progress through S-phase. The transfemn receptor gene promoter is activated as a delayed event following growth factor stimulation of quiescent fibroblasts. Serum stimulation in the presence of vanadate leads to superactivation of the transfemn receptor promoter, suggesting a role for tyrosine phosphorylation. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, a tyrosine kinaseregulated enzyme, blocks mitogen-dependent activation of the transfemn receptor promoter.
Furthermore,
wortmannin
was able to block
activation
of this promoter when added several hours after serum stimulation of quiescent cells. This suggests that phosphatidylinositol 3-kinase may be required in mid to late G1 and that it is directly involved in a pathway leading to activation of the transfemn receptor promoter. This is further supported by the finding that the transferrrin receptor promoter is much less responsive to mitogenic stimulation in cells that have been stably transfected with a dominant negative form of the phosphatidylinositol 3-kinase regulatory subunit. Activation of S6 kinase, an event known to be downstream of phosphatidylinositol 3-kinase activation, appears not to be involved in activation of the transfemn receptor promoter since no effect was observed by treatment of cells with rapamycin.
Introduction P13K3 is activated quiescent
in response to growth factor stimulation
cells and is thought
to be an important
of
signaling
Society.
To whom requests
for reprints should be addressed,
at University
of
South Dakota, School of Medicine, Department of Biochemistry and Molecular Biology, 414 East Clark Street, Vermillion, SD 57069. Phone: (605) 677-5132; Fax: (605) 677-5109. 3 The abbreviations used are: P13K, phosphatidylinositol 3-kinase; PDGF, platelet-derived growth factor EGF, epidermal growth factor TA, transferrin receptor; lL2, interleukin 2; CAT, chloramphenicol acetyltransferase; TBS-T, Tris-buffered saline plus 0.1 % Tween 20.
with
a regulatory
subunit
The catalytic subunit that is
of 85 kDa.
The
regula-
tory subunit contains an SH2 domain which allows the enzyme to be recruited to activated growth factor receptor complexes at the cell surface (1-3). In addition, it is thought that a direct interaction between p21 and the 1 10-kDa catalytic subunit is involved in stimulating its lipid kinase activity (4). At the membrane the catalytic subunit functions to phosphorylate the D3 position in the inositol ring of phosphatidylinositol. The catalytic subunit of P13K has also been shown to have protein kinase activity (5, 6). The downstream events mediated by activated P13K are less certain, and there are indications that multiple signaling pathways may be affected. Cellular responses in which P13K has been implicated include membrane ruffling, targeting of growth factor receptors following endocytosis, cytoskeletal alterations, and activation of protein kinase signaling cascades. Activation of the 56 kinase p7O appears to be downstream of P13K and involves intermediate steps including the activation of a rapamycin-sensitive kinase (7-9). Recently, the product of the proto-oncogene Akt has been shown to be involved in a P13K signaling pathway (10, 11). The Akt protein is a serine/threonine protein kinase and may be directly activated by inositol lipids that are phosphorylated at the D3 position (1 1). P13K has been implicated as a critical factor in the control of cell proliferation because of its nearly universal activation by tyrosine kinase receptors as well as by many viral and cellular oncoproteins (2). Furthermore, mutational analysis of specific autophosphorylation sites has indicated a critical role for P13K in PDGF receptor mitogenic signaling (12). Recently, using microinjection of neutralizing antibodies, Roche et a!. (1 3) demonstrated that P13K is essential for induction of DNA synthesis by PDGF and EGF in 3T3 fibroblasts. Significantly, they found that microinjection of the neutralizing antibodies any time up to 6 h after growth factor addition inhibited entry into the S-phase (13). Thus, P13K is required for critical delayed events in mitogenically stimulated cells. This is of interest because most of the responses associated
Received 5/23/96; revised 2/10/97; accepted 3/10/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact 1 This work was supported by Grant BE-236 from the American Cancer 2
Wortmannin Blocks Receptor Gene
with
P13K activation
are
rapid
and
transient
ef-
fects of growth factor stimulation. Thus, it is possible that P13K is involved in unique signaling mechanisms in mid to late G1 following growth factor stimulation. The TR gene plays an essential role in cell proliferation, as demonstrated by the fact that blocking receptor function causes cells to arrest near the G1-S-phase boundary (1416). The level of cell surface TR is low in quiescent cells but increases in proliferating cells (1 7-1 9). Nearly all tumor cells express elevated levels of the receptor, and the level of TR is often correlated with the degree of malignancy (20-24). In normal lymphocytes and fibroblasts, it has been demon-
565
565
Wortmannin
TR Gene Promoter
Blocks
A
B 7$ -
Activation
Mitogen
Responsive
.
.
Region
-34
0
+1
C
ElementB
0
A
Element Fig. 1.
AGGAAGAGCACJGCCCCC
U
Diagram
of the mitogen-responsive region of the TR gene prostart site is indicated as + 1 . Elements A and B, which are both required for full mitogen responsiveness, are indicated by solid bars. The sequence of both elements is indicated at the bottom. The AP1/CAE/ATF-Iike sequence within the B element and the
motor. The major transcriptional
Spi consensus sequence within the A element are indicated by boxes. The stably transfected 3T3 cells used in these experiments carry the TA promoter from -78 to +261 linked to the CAT gone.
U Serum(20%)
strated
that the mRNA
encoding
TR increases
in response
to
mitogenic activation and that this involves transcriptional activation of the TR gene (25-29). This increase is a delayed response. In lymphocytes, it occurs subsequent to 1L2 and 1L2 receptor expression (25, 26) whereas in fibroblasts it occurs several hours after growth factor treatment but prior to entry into the S-phase (29, 30). Previous experiments have demonstrated that the promoter of the TR gene is also activated in a delayed manner in response to mitogenic activation
of quiescent
3T3 fibroblasts
(29).
This
activation
Results Wortmannin Blocks Mitogen Activation of the TR Gene Promoter. Wortmannin, a fungal metabolite, is a potent inhibitor of P13K with an IC50 in the nanomolar range (32). It
unpublished
data.
(60 iiM)
Wortmannln
Fig. 2.
Wortmannin
(1pM)
-
+
+
+
+
-
-
-
+
+
-
-
+
blocks activation
+
of the TA promoter
in serum-stim-
with serum or plus vanadate in the presence or absence of wortmannin as mdicated at the bottom. After 18 h, the cells were harvested and the level of CAT enzyme activity was determined. CAT activity is expressed as the percentage of chloramphenicol that was converted to the acetylated form by the enzyme. ulated serum
Swiss/3T3
cells.
Quiescent
cells
were
stimulated
re-
quires a mitogen-responsive region that consists of two neighboring elements between nucleotides -37 and -78 (29). Both of these elements have been shown to bind to nuclear factors that are induced in a delayed manner in serum-stimulated 3T3 fibroblasts (31). One of these elements (Element A, Fig. 1) is highly GC-rich and can bind Spl and other Spl family members (31). The other element (Element B, Fig. 1) is related to the consensus sequences for both the Api and CREB/ATF families of transcription factors. However, mitogen-inducible DNA-protein complexes of this element do not appear to contain c-jun or c-fos but may contain other members of these families (31). Element B does bind with specificity to the transcription factor ATF-1 in extracts prepared from mouse melanoma cells but not to other members of the CREB/ATF family that have been tested.4 Other experiments have demonstrated that the TA gene promoter is responsive to treatment with sodium orthovanadate, an inhibitor of tyrosine phosphatases. The response to vanadate is also delayed, as observed with growth factors and serum (29). This and other results have implicated a tyrosine phosphorylation event as an important step in the activation of the TA gene. Because P13K activation is also dependent on tyrosine phosphorylation and is required for up to 6 h after growth factor stimulation of quiescent cells, it is possible that the TA promoter is one target of a delayed P13K-dependent signaling pathway. The experiments described here are aimed at addressing this question.
4B. A. Moore and W. K. Miskimins,
Vanadate
binds irreversibly to the 1 10-kDa catalytic subunit of P13K. Wortmannin is considered to be a highly selective inhibitor of this enzyme and most of its cellular effects are thought to be the result of this inhibition (32). However, there is some evidence that, at much higher doses, wortmannin can inhibit other kinases (32). In addition, it may have effects on other phospholipid metabolizing enzymes (33). The data shown in Fig. 2 demonstrate that wortmannin blocks activation of the TR gene promoter in Swiss/3T3 fibroblasts. In this experiment, a stably transfected cell line carrying the CAT gene linked to the mitogen-responsive region of the TR promoter
was allowed
to enter into a quiescent
state followed
by
stimulation with serum in the presence or absence of wortmannin. As documented previously (29), serum stimulation leads to a significant increase in TR promoter activity. However, in the presence of wortmannin CAT activity is only slightly elevated above basal levels. This suggests that wortmannin blocks a critical signaling pathway that leads to enhanced TR promoter activity but does not affect basal promoter activity. Fig. 2 also shows that the TR promoter can be superactivated by the addition of 60 M sodium orthovanadate in combination with serum, implying a role for tyrosine
phosphorylation.
The
presence
of
wortmannin
blocks the superactivating effect of vanadate. Fig. 3 shows that the inhibitory effects of wortmannin on the TA promoter are dose dependent, with significant inhibition in the low n range. It is known that wortmannin has a relatively short half-life in the presence of serum. Thus, the actual concentration of the drug within the cell in these experiments is likely to be much lower than the initial dose. Wortmannin Blocks a Delayed Signaling Pathway in Mitogen-activated Cells. The effect of wortmannin on the activation of the TR promoter could be due to inactivation of an immediate/early signal transduction mechanism that is necessary for subsequent processes in mid to late G1 How.
Cell Growth
& Differentiation
A
a 0
a 0
I
U C C
.,1
FU
Serum
(aM)
Wortmannin
-
+
-
-
+ 2000
+
+
+
+
200
125
50
10
I
Fig. 3. The effect of wortmannin on TA promoter activation is dose dependent. Quiescent cells were stimulated with serum in the presence of the indicated concentrations of wortmannin. The cells were harvested and assayed for CAT activity 1 8 h later. Data indicate the means from at least three samples. Bars, SE.
Time After
Serum
-
+
-
-
+ 3
Stimulation
B ‘i’ .
ever, growth
factors
until
late
must be present throughout most of G1 the restriction point. If growth factors are removed any time prior to the restriction point, the cells will not progress into the S-phase. Thus, important growth factor-dependent signals are generated at later times a point
in mitogen-stimulated
sponds
cells.
to mitogens
Because
in a delayed
inhibits
wortmannin
a signaling
the
manner,
pathway
promoter
re-
it is possible
that
TR
initiated
than
6 h after
stimulation.
In these
begins about 12 h after serum promoter shows the greatest
serum-stimulated quiescent In the experiment shown stimulated
with
promoter.
Wortmannin
following
vested the
serum
and vanadate
stimulation.
For
of CAT
activity
synthesis
each was
quiescent
Serum./Vanadate
cells were
to superactivate
was added
p.M)
18 h after treatment
level
DNA
TR
stimulation (29). Thus, the TR activity in mid to late G1 in
fibroblasts. in Fig. 4B,
(0.2
cells,
culture,
the
with serum assayed.
the TR
at various cells
times
were
har-
and vanadate,
and
When
I
subsequent
to the immediate early phase. Fig. 4A shows that the serum-induced increase in promoter-driven CATgene expression occurs predominantly later
C
U
in G1 called
wortmannin
Tune
Womnannit
Aided
+ 0
+ 6
+ 9
+ 12
+ 15
Fig. 4. Wortrnannin can block activation ofthe TR promoter when added hours after serum stimulation. A, Quiescent cells were stimulated with serum and at various times afterward were harvested and assayed for TR promoter-driven CAT activity. B, Quiescent cells were stimulated with serum in the presence of 60 M vanadate. At various times after stirnulation, as indicated at the bottom (in hours), wortrnannin was added at a concentration of 200 riM. All cells were harvested 1 8 h after addition of serum and vanadate and assayed for CAT activity. Bars, SE.
is
added
3 h after stimulation, it is still nearly as effective in inhibiting TR promoter activation as when added at the zero time
point.
When
the
inhibitor
is added
at later
time
points,
there is progressively less effect on the level of TR promoterdriven CAT activity. When added at 1 5 h after serum and vanadate addition, there is no effect. The profile of inhibition shown in Fig. 4B is very similar to the profile of the CAT enzyme induction shown in Fig. 4A. This suggests that the target of wortmannin must be active in mid to late G1 and that this target
functions
activation
of the TA promoter.
Expression
in a manner
of a Dominant
temporally
proximate
to the
coding
a dominant
mined
(Fig.
stably
transfected
struct.
In the
single
colony
5). The
P13K Regulatory Subunit Inhibits Mitogenic Activation of the TR Promoter. Stable transformants from independent transfections of Swiss/3T3 cells with both the TA promoter construct and an expression vector carrying either the cDNA encoding the wild-type p85 regulatory subunit of P13K or the cDNA en-
form
of the p85 subunit
and
shows
results
labeled
was isolated a Western that
of two
(34) were
independently
cell lines are shown
experiment
Fig. SC shows
Negative
negative
isolated. The transfected cells were grown to confluence, allowed to become quiescent, and then treated with either serum (20%) or vanadate (60 MM). After 1 8 h, the cells were harvested and the level of CAT enzyme activity was deter-
both
Transfection
and expanded blot that
wild-type
was and
derived
for each
1 in Fig.
5, a
for the analysis.
probed zp85
p85 con-
with are
anti-p85
significantly
overexpressed in these colonies compared with control cells. In the experiment labeled Transfection 2, all of the resistant colonies resulting from a separate transfection were pooled, expanded, and then analyzed. Expression of the wild-type p85 subunit appeared to have little effect on TA promoter
567
568
Wortmannin
Blocks
TR Gene
Promoter
Activation
B
A
C
I
:
-+
$en* Vaiia&*
-+
-
+
-
-+-
Sera*
-
+
-
VIJI2Z
+
Fig. 5. A dominant negative P13K regulatory subunit inhibits mitogen activation of the TR promoter. Quiescent Swiss/3T3 cells stably transfected with the TA promoter construct and a cDNA encoding either a wild-type (A) or dominant negative form (B) of p85 were stimulated with either serum or vanadate as indicated at the bottom. After 1 8 h, the cells were harvested and the level of CAT activity was determined. For each construct, the results shown are from two completely independent transfections. For Transfection 1, a single colony was isolated and then expanded for the analysis. For Transfection 2, all of the resistant colonies from a single plate were pooled, expanded, and then analyzed. A Western blot of p85 from the colonies from transfection 1 and from control cells is shown in C. Arrow, position of p85. The Western blot was performed on the same passage of cells used for CAT analysis.
activity which by vanadate, 29). However, subunit,
was
induced
crease
only in serum-stimulated
in CAT activity
vanadate.
These
target
of
by serum
and 2-3-fold
a
results (see Fig. 1 and Ref. the dominant negative p85 a 2-fold increase in TA promoter-
there was CAT activity
driven
5-10-fold
similar to previous in cells expressing
was
results
wortmannin
observed
support that
cells,
is
after
the
involved
and
treatment
conclusion in
no
C
a 0 U
in-
with
that
activation
0
.?
the
of
U
the
,,t
TR promoter is P13K. Rapamycin Does Not Block TR Promoter Activation by Mitogens. One of the known downstream events of a P13Kdependent signaling (9), and this pathway
pathway is sensitive
is the activation to wortmannin.
for S6 activation involves additional steps tivation of a rapamycin-sensitive kinase.
U Serum
of 56 kinase The pathway
including Aapamycin,
the achow-
ever, has no effect on P13K activity or on the putative P13K AKT (1 0, 1 1). Fig. 6 shows that rapamycin, at concen-
Vanadate
-
+
+
+
+
Rapamycin
-
-
20
100
200
+
Fig. 6. Rapamycin does not block mitogenic activation of the TR promoter. Quiescent cells were stimulated with serum (20%) and vanadate (60 .ai) in the presence of the indicated concentration of raparnycin. The level of CAT activity was determined 1 8 h later. Bars, SE.
target
trations
from
promoter ment
20 to 200
activation
of cells
increase no effect
with
ng/ml,
does
by serum rapamycin
not negatively
and vanadate.
causes
a small
affect
TA
In fact, treatbut reproducible
mannin
in CAT activity in stimulated cells. Aapamycin has on the basal level of expression from the TA pro-
moter
(data
naling
pathway
not shown). that
mitogen-stimulated
These
results
leads
to activation
cells
does
not
suggest
that
the sig-
of the TR promoter involve
activation
in
of 56
kinase.
Discussion It was
demonstrated
that
wortmannin
is a potent
TR gene
promoter activation in serum-stimulated This drug also was shown to inhibit superactivation promoter
most directly
the presence due to inhibition
in
likely
involved
in a signaling
of vanadate. These of P13K and suggest pathway
leading
inhibitor
of
fibroblasts. of the TA results are that P13K is to activation
of the TA promoter. is an
This
effective
is supported
the low nanomolar range. fact that in cells expressing the
P13K
regulatory
of TA
inhibitor
It is further a dominant
subunit,
the
by the fact promoter
that
wort-
activation
in
substantiated by the negative form of the
promoter
is much
less
re-
sponsive to serum and has no response to vanadate alone. Another possibility is that some of the observed effects of wortmannin on TA promoter activation are due to other targets that are downstream from a P13K-dependent step. For example, P13K is a member of a large family of related kinases that also includes a DNA-dependent protein kinase and the product of the ATM gene (35, 36). The DNA-dependent protein kinase was found in vitro but at concentrations although tive
it is possible
to the drug
at lower
that
to be sensitive to wortmannin in the micromolar range (37), other
family
concentrations.
members
are sensi-
Cell Growth & Differentiation
An important finding is that wortmannin can block activation of the TR promoter when added several hours after serum stimulation of quiescent cells. Because TA promoter activation is a delayed response in mitogen-stimulated cells,
P13K most probably
is involved
in a signaling
pathway
that
directly leads to activation of the promoter. Roche et a!. (13) have shown that microinjection of neutralizing antibodies to the catalytic subunit of P13K into 3T3 cells blocks PDGF or EGF induction of DNA synthesis. Moreover, they have shown that the antibodies could be injected hours after growth factor addition and still block entry into the S-phase, demonstrating that functional P13K is necessary in mid to late G1.
The timing
of inhibition
of DNA synthesis
by neutralizing
antibodies to P13K observed by Roche et al. (1 3) is very similar to the timing of inhibition of TR promoter activation by wortmannin shown in Fig. 4B. This signaling cascade may be critical for cell cycle progression since TA expression is required for the S-phase. An important question is how P13K activation in late G1 leads to transcriptional activation of gene expression. P13K has been shown to be both a lipid kinase and a protein kinase, and it is possible that either of these activities could
be necessary.
In any event, TA promoter
activation
in growth
factor-stimulated cells does not appear to require activation of 56 kinase, a well-known downstream event of P13K activation. This is indicated by the fact that rapamycin has no inhibitory effect on TA promoter activation in stimulated cells. It is likely that activation of the promoter involves phosphorylation
events
that modulate
transcription
factor
interactions
within the mitogen-responsive region of the promoter. This could require activation of the AKT protein kinase which has recently been shown to be dependent on P13K (1 0, 1 1). It will be of interest to determine whether AKT is required for delayed G1 events in growth factor-stimulated cells. This will require the development of neutralizing antibodies or dominant negative forms of the enzyme.
Materials Materials.
and OMEM,
Methods penicillin/streptomycin,
wortmannin,
puromycin,
and
sodium orthovanadate were purchased from Sigma Chemical Co. Newborn calf serum was purchased from Atlanta Biologicals. Raparnycin was purchased from LC Laboratories. Acetyl CoA was purchased from Pharmacia and [14C]chloramphenlcol was purchased from Arnersham. Cell culture. The cell line used in most of the experiments is a stably transfected line of Swiss/3T3 cells that carries the region of the TR gene from -78 to + 261 linked to the bacterial CAT gene. This line has previously been fully characterized for responsiveness to mitogens (29). The cells were maintained in OMEM containing 1 0% newborn calf serum, 100 units/mI penicillin, and 100 pg/mI stroptomycin in 5% CO2 in a
humidified
atmosphere
Stable
Transfections.
at 37#{176}C.
ysis. Again the analysis was performed sion of the resistant
on the first passage
after expan-
cells.
Western Blotting of
p85.
Control
cultures
or cultures
stably
trans-
with either Wp85 or tp85 as described in the preceding section were grown to confluence in 60-mm culture dishes. The culture medium was removed and the cells were rinsed with Oulbecco’s PBS. The cells were lysed by addition of 200 l of SOS-PAGE sample buffer directly to the culture dish. The cell lysate was transferred to a microfuge tube and fected
briefly
sonicated
to shear
DNA. The lysate
was centrifuged
for 5 mm, and
an equal amount from each culture was applied to a SOS-polyacrylamide gel (8% acrylarnide). After electrophoresis, the proteins were transferred to Imrnobilon P (Millipore) membranes using a Bio-Rad semi-dry transfer apparatus
according
to the
rnanufacturer’s
instructions.
The
filter
was
blocked for 1 h in 10 m Tris-CI (pH 7.8), 100 m NaCI, and 0.1 % Tween 20 rBS-T) containing 5% nonfat dry milk. It was then incubated for 1 h with a 0.1 &g/mI mouse monoclonal anti-P13-kinase (Transduction Laboratories) in the TBS-T. The filter was washed six times for 5 mm in TBS-T and then incubated for 1 h with secondary antibody (1 :2500 dilution of
goat anti-mouse lgG-horseradish peroxidase; Santa Cruz filter was washed as described
Biotech.).
The
and then bands were detected by enhanced chemiluminescence (Amersham). Mitogen Stimulation and CAT Assays. For the experiments doscribed here, the cells were grown to confluence in DMEM containing 10% calf serum. After reaching confluence and entering quiescence, the cells were further incubated in serum-free medium consisting of a 1:1 mixture of OMEM and Wayrnouth’s medium for 2 days. They were then stimulated or treated with the reagents described In each figure. Unless noted otherwise in the figure legends, the cells were harvested 18 h after above
stimulation. Extract preparation and out as reported previously (29).
CAT enzyme
analysis
were
carried
References 1 . Panayotou, G., and Waterfield, M. 0. Phosphatidylinositol 3-kinase: a key enzyme in diverse signalling processes. Trends Cell Biol., 2: 358-360, 1992.
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3-ki-
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kinase
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EMBO
J., 13:
6. Lam, K., Carpenter, C. L, Auderman, N. B., Friel, J. C., and Kelly, K. L The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-i. Stimulation by insulin and inhibition by wortmannin. J. Biol. Chom., 269: 20648-20652,
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(Sra-ip85)
8. Sabatini, 0. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycmn-dependent fashion and is homologous to yeast TORs. Cell, 78:
Kasuga
35-43,
Wild-type
(Sra-wp85)
and dominant
negative
constructs (34) were provided by Wataru Ogawa and Masato (Kobe University School of Medicine). Using the calcium phosphate precipitation method, these constructs (20 g) were cotransfected with pPur (1 p.9) into Swiss/3T3 cells that were plated in 10-cm culture dishes.
colonies
The cells
appeared
were
selected using 1 .tg/mI puromycin and resistant after 2-3 weeks. For transfection 1 (in Fig. 5), single
resistant colonies were isolated using cloning rings and expanded
in T25 flasks. When the T25 flask was confluent, the cells were split into dishes for analysis of mitogenic activation of promoter activity and for Western blotting for p85. Therefore, there was no extended passaging of cells prior to analysis. For transfection 2 (Fig. 5), all of the resistant colonies on one plate were pooled and expanded as a mixed population for further anal-
1994.
9. Downward,
J. Regulating
56 kinaso. Nature (Lond.), 371:
378-379,
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T. F., Yang,
S-I.,
Chan,
T. 0.,
Datta,
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A., kinase of the POGF-activated 1995.
P. N. The protein
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TR Gone Promoter
Blocks
12. Valius, M., and Kazlauskas, dylmnositol mitogenic
Activation
A. Phospholipaso
C-yi
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13. Roche, S., Koegl, M., and Courtneidge, 3-kinase a is required growth factors. Proc.
14. Neckers,
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S. A. The phosphatidylinositol
human
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is required
by interleukin
induction
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2. Proc. NatI. Aced. Scm.
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ulated
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40-46,
1982.
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antibodies.
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BioI., 5: 1814-1821
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virus transformed
rat lymphoblasts.
, 1985.
in concanavalin A stimJ. Cell. Physiol., 113:
P. Modulation
receptors by cellular density 11: 579-586, 1979.
and
state
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