[CANCER RESEARCH 47, 1081-1086, February 15, 1987]
Growth State-dependent Regulation of Protein Kinase C in Normal and
Transformed Murine Cells1
Patricia G. McCaffrey and Marsha Rich Rosner2
Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
ABSTRACT
We determinedwhether growth state can influence the action of protein
kinase C by measuring protein kinase C activity in growingand stationary
cultures of normal and transformed cells. Two approaches were used to
measure protein kinase C: assay of intact cells for inhibition of epidermal
growth factor (EGF) binding in response to phorbol dibutyrate (HDBu);
and assay of detergent extracts for total calcium, phospholipid-dependent
kinase activity. In extracts of growing and stationary Swiss 3T3 cells,
the total amount of protein kinase C activity was similar, indicating that
growth state does not alter the level of enzyme in the cell. The shortterm response of Swiss 3T3 cells to an activator of protein kinase C also
appeared to be independent of growth state, since the 50% effective dose
for PDBu inhibition of EGF binding to its receptor was approximately 7
UMfor both growth conditions. In contrast, the response of cells to longterm treatment with PDBu was significantly different depending upon
the initial growth state of the cells. In both growth states, PDBu caused
loss of protein kinase C activity, which reflected a loss in protein mass
as determined by immunoblotting with antiserum to protein kinase C.
However, the maximum decrease approached 100% in stationary cultures
versus approximately 75% in growing cells. Protein kinase C levels in
several transformed cell lines were subject to down modulation in a
similar growth state-dependent manner. Further, the inhibition of EGF
binding by tumor promoters following long-term treatment of Swiss 3T3
cells with PDBu also varied with growth state. In down modulated
growing cells, PDBu caused almost complete inhibition of EGF binding,
whereas in down modulated stationary cells, minimal inhibition of EGF
binding by PDBu was observed. These results suggest that prolonged
treatment with tumor promoters alters the sensitivity of cells to activators
of protein kinase C in a growth state-dependent manner.
INTRODUCTION
There is increasing evidence that protein kinase C plays an
important role in the control of cell growth. First described by
Takai et al. (1), protein kinase C is a calcium- and phospholipiddependent kinase that phosphorylates serine and threonine
residues (reviewed in Ref. 2). Protein kinase C is also the major
cellular receptor for tumor promoters, which activate the en
zyme both in vitro and in vivo by substituting for diacylglycerol,
the endogenous activator. A variety of effectors, including cer
tain growth factors (3, 4), induce phosphatidylinositol break
down upon binding to their specific receptors, resulting in the
transient production of diacylglycerol and activation of protein
kinase C. Thus, this enzyme is activated in response to both
tumor promoters and growth factors, and may be essential to
their mitogenic action.
Further evidence for a role of protein kinase C in cell growth
comes from work of our laboratory and others which indicates
that protein kinase C can modulate growth-regulatory path
ways. A variety of activators of protein kinase C such as tumor
promoters, synthetic diacylglycerols, and platelet-derived
Received 6/24/86; revised 10/28/86; accepted 11/17/86.
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 indicate this fact.
1This work was supported by NIH Grants CA35S4 and CA40407 to M. R.
R., and by Training Grant 2T32ES07020 to P. G. M.
2To whom requests for reprints should be addressed, at Department of Applied
Biological Sciences, Bldg. £18-506,Massachusetts Institute of Technology, Cam
bridge, MA 02139.
growth factor can affect EGF3 action by altering the phosphorylation and binding of the EGF receptor (5-10). Tumor pro
moters have been shown to cause loss of EGF binding to a class
of high affinity receptors in a protein kinase C-dependent
process (11). This loss of EGF binding has been correlated with
an increase in phosphorylation of the EGF receptor at serine
and threonine residues (6, 7, 12, 13), suggesting that protein
kinase C can mediate phosphorylation of the EGF receptor in
vivo and alter its binding properties.
It is apparent that activation of protein kinase C can have
profound effects on cell growth through the ability of the
enzyme to mediate or modulate the action of growth-promoting
factors. Conversely, it is possible that the growth state of the
cell could influence the action of protein kinase C. To test this
possibility, we determined the effect of growth state on protein
kinase C activity before and after exposure of cells to long-term
tumor promoter treatment. The results indicate that growth
state has no effect on the level of protein kinase C in Swiss 3T3
cells before treatment with the tumor promoter PDBu. How
ever, the extent of down modulation of protein kinase C after
PDBu treatment is affected by growth state in both normal and
transformed cells. Further, in down modulated cells containing
a low level of protein kinase C, tumor promoters mediate a
biological response, inhibition of EGF binding, in growing but
not stationary Swiss 3T3 cells. Thus, growth state has an effect
not only on the extent of down modulation of C kinase, but
also on the resultant biological response to activators of protein
kinase C.
These results suggest that, for certain biological targets,
effective desensitization of cells to tumor promoters occurs in
stationary, but not growing, cells. This point is particularly
relevant to the question of whether down modulation can be
utilized to eliminate from cells the response to activators of
protein kinase C.
MATERIALS AND METHODS
Cell Culture. Swiss 3T3 cells were obtained from the American Type
Cell Culture Collection. The v-aW-transformed cell line ANN-1 (14)
was provided by G. Foulkes and D. Baltimore. Ha-r<u-transformed
NIH 3T3 cells were from D. Stern and R. Weinberg. All cells were
grown in DME supplemented with 10% PCS or calf serum (Gibco).
Stationary cultures of Swiss 3T3 cells were obtained by incubating
confluent dishes in DME containing 1 mg/ml bovine serum albumin
(Fraction V, essentially fatty acid free; Sigma) or 0.1% PCS for 24 h.
Transformed cell lines were growth arrested by serum starvation. When
high density cultures of either ANN-1 or ros-transformed cells were
placed in DME/0.1% BSA, no increase in cell number was seen after
48 h (data not shown). Growing cultures were subconfluent cells which
received fresh medium containing serum every other day.
[3H]Thymidine Uptake. Swiss 3T3 cells in 35-mm dishes were labeled
for 24 h with [JH]thymidine (2 pCi/ml; specific activity, 50 to 80 Ci/
mmol) in DME or DME containing 10% FCS. After the labeling
period, which corresponds to one round of DNA synthesis, cells were
' The abbreviations used are: EGF, epidermal growth factor, OME, Dulbecco's
modified Eagle's medium; PCS, fetal calf serum; BSA, bovine serum albumin;
POBu, phorbol dibutyrate; EGTA, ethyleneglycol-bis(0-aminoethylether)AyvyV'-Af'-tetraacetic acid.
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REGULATION
OF PROTEIN
washed with phosphate-buffered saline, lysed in hypotonie Tris/1%
sodium dodecyl sulfate buffer, and precipitated in ice-cold 10% trichloroacetic acid. The precipitates were collected by filtration, washed, and
counted.
Cell Fractionation and Protein Kinase C Isolation. Cells grown on
150-mm dishes were treated with 200 nM PDBu in dimethyl sulfoxide
alone. After treatment, the cells were washed with phosphate-buffered
saline and then scraped in 20 HIMTris (pH 7.5)/l% Triton X-100/2
mM EDTA/0.5 mM EGTA/10 fig/ml aprotinin/1 mM phenylmethylsulfonyl fluoride/5 mM dithiothreitol. These extraction conditions have
been shown to solubilize both cytoplasmic and paniculate protein
kinase C (15). Cell extracts were centrifugea at 100,000 x Kfor 60 min.
Aliquots of the supematants (generally 0.5 to 1.0 mg protein) were
loaded onto I-ml I) KAli-cellulose columns (DE-52; Whatman), which
had been equilibrated with 20 mM Tris (pH 7.5)/2 mM EDTA/0.5 mM
EGTA/1 mM dithiothreitol. The columns were washed with 15 ml of
this buffer and then eluted with a linear gradient of 20 ml of buffer and
20 ml of buffer plus 0.15 M NaCl. One-mi fractions were collected, and
50-nl aliquots were assayed for protein kinase C as described below.
Protein Kinase C Assay. Calcium- and phospholipid-stimulated pro
tein kinase C activity was assayed in a reaction mix containing 20 mM
Tris (pH 7.4), 10 mM MgCl2, 1 mM CaCl2, 400 /ig/ml histone (type
III-S; Sigma), 50 MM [7-î2P]ATP(2 »iCi/sample), 160 ^g/ml 1-«phosphatidylserine (Avanti Polar Lipids), and 400 nM PDBu. Control
reactions were run in the absence of phosphatidylserine and PDBu.
Samples were incubated for 10 min at 30*C and aliquots were spotted
on Whatman P81 phosphocellulose paper. Assays were washed in 30
mM phosphoric acid, dried, and counted. The activity of protein kinase
C was quantItated in two ways, (a) The area under the peak of the
column profile was determined. This value was then adjusted for the
amount of protein loaded onto the columns, and the protein kinase C
activity was expressed as cpm of 32Pincorporated into histone/min//¿g
of cell protein. Alternatively, the peak fractions were pooled and assayed
for protein kinase C and protein to yield a specific activity for the
enzyme in the column eluant.
Binding of IISI-EGF. Receptor-grade EGF from Collaborative Re
search was iodinated using lodogen (16). For the dose-response studies,
cultures of growing and stationary Swiss 3T3 cells in 35-mm dishes
were washed with DME containing 1 mg/ml BSA. PDBu was added at
concentrations from 0.02 to 2000 nM for 10 min at 37'C. I25I-EGF was
then added at a final concentration of approximately 1 ng/ml. Scatchard
analyses indicate that EGF at this concentration binds primarily to the
high affinity receptor population in both untreated and PDBu-down
modulated cells (26). After a 40 min binding period, cells were placed
on ice and rapidly washed 6 times with ice-cold DME/0.1% BSA. The
monolayers were then lysed with 0.5 N NaOH, and the lysates were
collected and counted. For long-term studies, cells were treated with
200 nM PDBu or dimethyl sulfoxide in DME with 10% FCS (growing
cultures) or 0.1% FCS (stationary cultures) for up to 96 h. After this
treatment, the cells were washed with DME/0.1% BSA for 2 h, which
removed approximately 90% of the bound PDBu. Cells were then
rechallenged with 200 nM PDBu or dimethyl sulfoxide for 10 min at
37*C, and I25I-EGF binding was measured as above. In all cases, binding
KINASE C LEVELS
min, and the supematants were removed to fresh tubes. After addition
of polyclonal antiserum to porcine brain protein kinase C (18) (approx
imately 4 ii\ per 5 x 10s cells), the extracts were incubated at 4*C for 4
h. Immune complexes were absorbed to Staphylococcus aurcu.v-Sepharose CL4B beads. After washing several times with the lysis buffer,
absorbed complexes were eluted by boiling in sodium dodecyl sulfate/
/3-mercaptoethanol, and the eluted proteins were separated on 10%
polyacrylamide gels and visualized by autoradiography.
Immunoblotting Analysis. Growing and stationary Swiss 3T3 cells
were treated with 100 ng/ml PDBu for 120 h. Cells were then washed
and lysed in boiling 2% sodium dodecyl sulfate containing 20 mM Tris
(pH 7.5), 2 HIMEDTA, and 0.5 mM EGTA. Cell lysates were spun for
15 min at 12,000 x g. Proteins were fractionated on 10% polyacryl
amide gels and transferred to Zeta-probe blotting membranes. After
transfer, the membranes were placed in blocking buffer [20 mM Tris
(pH 7.4)/150 HIMNaCl/5% casein] for 12 h at 50'C. The blots were
probed overnight at 4'C with antiserum diluted 1:400. After washing,
the blots were probed with I25I-Protein A (New England Nuclear),
washed, and exposed to X-ray film. Molecular weights were determined
using prestained markers. Rat brain protein kinase C (a M, 80,000
protein) migrated as 87,000 to 89,000 in this system.
RESULTS
In order to assess the regulation of protein kinase C as a
function of growth state, enzyme activity in growing and sta
tionary cells was compared. Growing cells were defined as
subconfluent, serum-fed cultures actively traversing the cell
cycle as indicated by rapid rates of DNA synthesis. Stationary
(Go) cultures were quiescent, serum-starved cells not undergoing
DNA synthesis. The growth state of cultures of Swiss 3T3 cells
was determined by measuring incorporation of [3H]thymidine
into trichloroacetic acid-precipitable material over a 24-h period
prior to measurement of protein kinase C activity or EGF
binding. Serum-fed, subconfluent cultures incorporated |'H)
thymidine rapidly, while serum-starved confluent cultures
showed little uptake (Table 1). Under these conditions, the
stationary cells were still able to respond to serum stimulation,
as indicated by the ability of 10% FCS to stimulate thymidine
uptake in these cells.
No significant difference was observed in the amount of
protein kinase C activity in growing and stationary cells. Protein
kinase C was extracted from Swiss 3T3 cells in a hypotonie
buffer containing 1% Triton X-100 and chromâtograplied on
DEAE-cellulose to remove inhibitors of the enzyme. When
enzyme activity was corrected for protein loaded onto the
column, the relative amount of protein kinase C activity per mg
of protein in the cells was similar regardless of growth state
(Fig. 1). In this assay, the calcium, phospholipid-stimulated
kinase activity detected in the column eluate is a measure of
the total cellular protein kinase C, both membrane bound and
cytoplasmic, and does not necessarily reflect the state of acti
vation of the enzyme in intact cells.
To assess the ability of kinase C to be activated in intact cells,
we measured the inhibition of EGF binding to its receptor
following tumor promoter treatment. Loss of EGF binding to
was measured in triplicate wells, with a SD of less than 10%. Nonspe
cific binding, measured in the presence of 1 pg/ml of unlabeled EGF,
was approximately 10% of total binding. The extent of inhibition of
EGF binding observed in cells which have been pretreated with PDBu
is expressed as a percentage of that seen in cells which have been
pretreated with dimethyl sulfoxide only.
Protein determinations were done by the method of Bradford (17),
using BSA as a standard. The dose-response curves and protein kinase
Table 1 fHJThymidine incorporation by Swiss 3T3 cells
C decay curves were fit using a nonlinear regression program developed
The basal and serum-stimulated rates of [3H]thymidine incorporation into
by M. Böiger.
trichloroacetic acid-insoluble material were measured in subconfluent, serum-fed,
Immunoprecipitation of Protein Kinase C. Subconfluent Swiss 3T3
or confluent, serum-starved cells.
cells were labeled for 24 h with 100 ¿iCi/ml[39S]methionine in met hi10~3)Growth
[3H]Thymidine incorporation (cpm x
onine-free DME containing 10% dialyzed fetal calf serum. The cells
stateGrowing
medium
change139
FCS115
10%
were then washed and scraped in 10 mM Tris (pH 7.4)/5 mM EDTA/
±39°
150 mM NaCl/1% Triton X-100/0.5% deoxycholate/0.1 % sodium
±29
StationaryNo
8.2 ±0.5+Fresh
144 ±28
dodecyl sulfato/1 mg/ml BSA/10 Mg/ml aprotinin/1 HIMphenylmeth1Mean ±SD of triplicate determinations.
ylsulfonyl fluoride. The cell extracts were spun at 12,000 x g for 15
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REGULATION OF PROTEIN KINASE C LEVELS
«- 30
'O
I
IDO»
.A
25
75
È
20
¡
,5
50
>-
IO
>
5
<
u
-o-o'
IO
UJ
2O
30
30 O
IO
FRACTION NUMBER
Fig. 1. Protein kinase C activity in growing and stationary cells. Triton X-100
extracts from growing (A) or stationary (H) Swiss 3T3 cells were fractionated on
DEAE-cellulose as described. Fractions were assayed in the presence (•)or
absence (O) of phosphatidylserine and PDBu; calcium was included in all assays.
Protein kinase activity is expressed as cpm incorporated into histone/10 min/SOfil aliquot. In this experiment, when activity was corrected for the amount of
protein loaded (350 Mg for growing cells and 500 fig for stationary cells), the
specific activity was 755 and 640 cpm/min/Mg protein loaded for growing and
stationary cells, respectively.
-
20
S
75
E
(-
50
o-
25
z
it
0
24
48
72
96
TIME OF PDBu PRETREATMENT (hours)
Fig. 3. Loss of protein kinase C activity after long-term PDBu treatment.
Growing (A) or stationary (H) cultures of Swiss 3T3 cells were treated with 200
n,MPDBu for the times indicated. Protein kinase C activity was measured after
fractional ion of Triton X-100 cell lysates on DEAE-cellulose as described in
"Materials and Methods," and expressed as a percentage of that in parallel
dimethyl sulfoxide-treated cultures. Points shown are pooled from several inde
pendent experiments in which the dimethyl sulfoxide-treated growing or station
ary cultures typically yielded approximately 300 to 500 cpm of "P incorporated
into histone/min/Mg of protein loaded onto the columns. The curves shown were
calculated using a nonlinear regression analysis program.
100
75
-
100
5O
0.02
0.2
2.0
20
PDBu (nM)
200
2000
Fig. 2. Dose response for inhibition of EGF binding by PDBu in growing and
stationary Swiss 3T3 cells. Growing (•)or stationary (O) cells were treated for
10 min with the indicated doses of PDBu prior to the addition of '"I EGF as
described in "Materials and Methods." Specific binding to untreated cells was
4410 ±230 cpm/35-mm well and 702 ±28 cpm/IX mm well for growing and
stationary cultures, respectively. The EGF binding detected after cells were treated
with 2000 nM PDBu (250 ±116 cpm in growing cultures and 134 ±14 cpm in
stationary cultures) represents low affinity binding and was subtracted prior to
calculating the percentage of inhibition. The 50% effective dose values calculated
using a nonlinear regression analysis program were 5.9 ±1.3 nM and 7.0 ±0.7
nM for growing and stationary cells, respectively.
a population of high affinity receptors has been shown to reflect
activation of protein kinase C by tumor promoters (cf. Refs. 5
and 6). When growing and stationary cells were exposed to
PDBu, the dose-response curves for inhibition of EGF binding
were similar (Fig. 2). The 50% effective dose for this effect was
approximately 6 nM in both cases. These results indicate that
the short-term response of Swiss 3T3 cells to an exogenous
activator of protein kinase C is independent of growth state.
In contrast, when cells were treated for prolonged periods of
time with PDBu, a growth state-dependent loss of protein
kinase C activity was observed. In growing cells (Fig. 3/i), PDBu
(200 HM)caused a rapid loss of approximately 75% of the total
protein kinase C during the initial 24 h of treatment. Continued
treatment did not further reduce protein kinase C below ap
proximately 25% of control levels. In stationary cells (Fig. 35),
a similar rapid loss of activity (f....= approximately 6 h) was
observed, but the amount of protein kinase C continued to
decrease with time to a significantly lower level than in the
growing cells.
In order to quantify the statistical significance of these results,
we fit the two sets of data by biphasic (growing cells) and simple
(stationary cells) exponential decay models. We used a nonlin
ear regression program which provides best-fit estimates of the
initial activity, the rate of decay, and (in the case of the biphasic
model) residual activity. This program also defines the signifi
cance of the model fit, which depends on the statistical errors
in the data and on the number of free parameters in each model.
Our analysis shows that, under growing conditions, the hypoth
esis of a simple exponential decay model can be rejected (the
X2per degree of freedom for this model exceeds 4.3); that this
model adequately fits the data for the stationary conditions (x2
per degree of freedom = approximately 1.6); and that the
biphasic exponential decay model does adequately describe the
data for the growing conditions (x2 per degree of freedom =
approximately 1.4). Furthermore, comparison of the decay rates
of the two models shows that they are not statistically different
(i.e., they lie within 2.5 a of one another and, thus, do not differ
at the 99% confidence level). Instead, the difference between
the data can be ascribed to a difference in the residual activity;
under growing conditions, the model fits demonstrate a statis
tically significant nonzero residual activity, whereas under sta
tionary conditions, the data are consistent with decay to zero
activity. Thus, growing cells maintain a minimal level of protein
kinase C even after extensive treatment with PDBu.
For both growth states, the loss of protein kinase C activity
following long-term PDBu treatment could be attributed to a
loss of the corresponding protein. When [3sS]methionine-labeled extracts of Swiss 3T3 cells were immunoprecipitated with
rabbit polyclonal antiserum prepared against porcine protein
kinase C (18), two molecular weight bands of approximately
87,000 and 58,000 were detected that were not present in
samples treated with preimmune antiserum (Fig. 4/1). Of these
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REGULATION OF PROTEIN KINASE C LEVELS
^^^^^_
-
Table 3 Effect of PDBu treatment on protein kinase C activity in ras-transformed
NIH 3T3 cells
Cells were treated for 96 h with 200 n\i PDBu in dimethyl sulfoxide or
dimethyl sulfoxide alone in the presence or absence of 10% calf serum. After
DEAE-cellulose chromatography of cell extracts, fractions containing protein
kinase C activity were pooled, and the total activity and protein in the peak were
determined.
97K
87K -,
Growth
conditionPlus
68K
serum
43K
Fig. 4. Use of antiserum to detect protein kinase C in Swiss 3T3 cells before
and after down modulation with POBu.. I, immunoprecipitation of protein kinase
C from untreated Swiss 3T3 cells. Protein kinase C was immunoprecipitated from
>extracts of ["SJmethionine-labeled cells as described in "Materials and Methods."
Lane I, 2 u\ of noninmmune serum; Lane 2, 1 f<lof antiserum to porcine protein
kinase C; Lane 3, 2 M'of antiserum to porcine protein kinase C; Lane 4, 4 n\ of
antiserum to porcine protein kinase C. Molecular weights of protein standards
are indicated. II. immunoblotting of cell lysates from stationary (Lanes 1 to 3) or
growing (Lanes 4 io 6) Swiss 3T3 cells treated for 120 h with dimethyl sulfoxide
(Lanes I, 2, 4, 5) or 200 n\i PDBu (Lanes 3. 6). Cell extracts were analyzed as
described in "Materials and Methods." Lanes 1 to 3, samples containing 45, 22.5,
and 27 pg of protein, respectively, Lanes 4 to 6, samples containing 250, 125,
and 120 jig of protein, respectively. No bands were detected in the nonimmune
control.
Table 2 Effect of PDBu treatment on protein kinase C activity
in transformed cells
Cells were treated with 200 n\i PDBu in dimethyl sulfoxide or dimethyl
sulfoxide alone for the indicated times. Protein kinase C activity was measured
after fractionating cell lysates on DEAE-cellulose as described in "Materials and
Methods." Total activity in the column eluate was determined and expressed per
UKprotein loaded onto the column.
Cell line
Treatment
Protein kinase C activity
(cpm MP incorporated/
min/Mgcellular protein)
sulfoxide, 72 h
100(19.5)
PDBu, 72 h
441 (100)
Dimethyl sulfoxide, 72 h
54(12.2)
PDBu, 72 h
296 (100)
A431Dimethyl Dimethyl sulfoxide, 48 h
PDBu, 48 h514(100)"
0(0)
* Numbers in parentheses, amount of kinase activity, shown as percentage,
relative to dimethyl sulfoxide-treated controls.
3T3ANN-1
NIH
species, the A/, 87,000 protein was the only band that comigrated with purified rat brain protein kinase C and was also
immunoprecipitated by polyclonal antiserum prepared against
a peptide (19) from protein kinase C (data not shown). To
determine the effect of down modulation, growing and station
ary cultures of Swiss 3T3 cells were treated with 100 ng/ml
PDBu for 120 h and then analyzed by immunoblotting with the
polyclonal antiserum against protein kinase C (Fig. 45). In both
stationary and growing cells, the level of M, 87,000 protein
detected was reduced under these down modulation conditions
by over 84%. Similar results were obtained with antiserum
against the protein kinase C peptide (data not shown). Thus,
the loss of protein kinase C activity as detected by in vitro
kinase assays correlates with degradation of a corresponding
M, 87,000 protein.
To determine whether cells which display perturbations in
growth control are subject to PDBu-induced loss of protein
kinase C, we measured protein kinase C activity in growtharrested transformed cells before and after tumor promoter
treatment. The v-oA/-transformed cell line, ANN-1, and the
parent NIH 3T3 line were growth arrested by serum starvation
as described in "Materials and Methods." After treatment with
PDBu for 72 h under these conditions, protein kinase C activity
was reduced to a comparable extent in both the transformed
and nontransformed NIH 3T3 lines (Table 2). Human epider-
kinase C activity
(cpm "P incorporated/
Mgprotein/min)5779
(100)*
sulfoxide
PDBu
3794 (58.7)
No serumTreatmentDimethyl
Dimethyl sulfoxide
6036 (104)
PDBuProtein
1582(27.4)
* Numbers in parentheses, amount of kinase activity, shown as percentage,
relative to that in serum-fed, dimethyl sulfoxide-treated cells.
mal carcinoma cells (A431), which growth arrest in response
to tumor promoters, had no detectable protein kinase C activity
after treatment for 48 h with PDBu, even in the presence of
serum. Thus, the response of growth-arrested transformed cells
to prolonged tumor promoter treatment results in a loss of
protein kinase C activity similar to that of nontransformed
cells.
In transformed cells, as in nontransformed cells, the extent
of loss of protein kinase C activity after prolonged PDBu
treatment can be influenced by growth conditions. For example,
analysis of a ra.v-transformcd NIH 3T3 cell line revealed no
difference in the levels of protein kinase C in growing and
growth-arrested cultures (Table 3). When growing cells were
treated with PDBu for 96 h, protein kinase C activity was
reduced by 41 %. Growth-arrested cells treated for a comparable
period showed a greater reduction (approximately 75%) in
protein kinase C activity. In these transformed cells, the amount
of C kinase activity remaining after treatment of growing cells
was significantly higher than that seen previously in the nontransformed cells. These results indicate that the down modu
lation of protein kinase C levels is similar, but not identical, in
transformed and nontransformed cells.
The in vivo activity of the residual protein kinase C in downmodulated cells, when monitored by the ability to inhibit EGF
binding in Swiss 3T3 cells, was also subject to growth state
regulation. To measure inhibition of EGF binding in downmodulated cells, cultures were treated with PDBu (200 nivi)
continuously for up to 96 h. After washing for 2 h to remove
PDBu and allow the EGF binding to recover to control (un
treated) levels, PDBu was then added back, and EGF binding
was measured. Pretreatment of growing cells with PDBu did
not affect the extent of inhibition of EGF binding, while pre
treatment of stationary cells resulted in a dramatic attenuation
of the ability of PDBu to cause inhibition of EGF binding (Fig.
5). The results after 48 h of PDBu treatment suggest that, in
stationary cells, the remaining protein kinase C activity (10 to
25%) is insufficient to cause inhibition of EGF binding, whereas
in growing cells, the remaining activity (~25% of original levels)
may cause total inhibition of EGF binding. In cells which have
not been down modulated, PDBu binding studies demonstrate
that all the protein kinase C must be activated to elicit a similar
response (Ref. 11; Footnote 4). The fact that PDBu mediates
complete inhibition of EGF binding in down-modulated grow
ing cells, which contain only a small fraction of the original
protein kinase activity, suggests that prolonged tumor promoter
exposure can sensitize growing but not stationary cells to acti
vators of protein kinase C.
4 Unpublished results.
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REGULATION OF PROTEIN KINASE C LEVELS
100«
75
feg
50
2
t *
5i —
I
25
24
TIME
48
72
OF PDBu PRETREATMENT
96
(hours)
Fig. 5. Inhibition of EGF binding after long-term POBu treatment. Growing
(•)or stationary (O) Swiss 3T3 cells were pretreated with 200 nM PDBu for the
times indicated. After washing to remove PDBu, I25I-EGF was added with or
without fresh 200 nM PDBu. The extent of inhibition of EGF binding after
rechallenge with PDBu is expressed as a percentage of the maximal inhibition
measured in cells which were pretreated with dimethyl sulfoxide only. In growing
cells which were pretreated with dimethyl sulfoxide only, PDBu caused a reduction
in EGF binding from 15,363 ±339 cpm bound/35-mm well to 1,080 ±164 cpm
bound. In stationary cells, EGF binding was reduced from 1,171 ±92 cpm/18mni well to 452 ±68 cpm. Nonspecific binding measured in the presence of I
fig/ml EGF was 635 ±72 cpm/well and 170 ±37 cpm/well for growing and
stationary cultures, respectively. The data for growing and stationary cells were
obtained in independent experiments.
DISCUSSION
We have determined the effect of cell growth state on protein
kinase C activity in both normal and transformed cells. Two
approaches were used to assess protein kinase C activity: assay
of the enzyme in detergent extracts of cells; and inhibition of
EGF binding in response to PDBu treatment. In untreated
Swiss 3T3 cells, total levels of protein kinase C activity and the
extent of PDBu-induced inhibtion of EGF binding were inde
pendent of growth state. In contrast, long-term treatment with
this tumor promoter resulted in more complete down modula
tion of protein kinase C in stationary than in growing cultures.
A similar type of control was observed in transformed cells.
Further, the biological response to activators of protein kinase
C was also subject to growth state regulation, since PDBu
stimulation resulted in inhibition of EGF binding in growing,
but not stationary, cells after down modulation. These results
suggest that, for certain biologically relevant targets, desensitization of growing cells to tumor promoters effectively does not
occur despite loss of protein kinase C activity.
The two approaches used to assess protein kinase C provide
a reasonable measure of the level of active enzyme. Direct assay
of the enzyme following detergent extraction and removal of
inhibitors by DEAE-cellulose fractionation allowed us to quantitate the total amount of potentially active enzyme in the cell.
To measure the activation of protein kinase C in intact cells,
we assayed inhibition of high affinity EGF binding. Although
there is evidence for the existence of multiple pathways by
which the EGF receptor can be modulated (13, 26) changes in
EGF binding remain valuable in vivo indicators of protein
kinase C activity in response to tumor promoters.
The observed decrease in protein kinase C activity appears
to result from degradation of the enzyme. The loss of phorbol
ester binding sites, as well as the decrease in protein kinase C
activity that we have observed after tumor promoter treatment,
has been demonstrated previously for quiescent 3T3 cells (11,
20, 21). It doesn't appear that an inhibitor of the enzyme is
responsible for the decrease in activity, since this was not
detected by mixing experiments (Ref. 20; Footnote 4). Results
based upon antiserum to protein kinase C indicate that the loss
of enzyme activity following PDBu treatment correlates with
loss of detectable protein as assessed by immunoblotting tech
niques (21). We have observed a similar loss in protein kinase
C in both growing and stationary cell cultures by immunoblot
ting under our down modulation conditions. The level of pro
tein kinase C detected in cells is a function of the rate of
synthesis as well as degradation of the enzyme, and either or
both of these parameters might by influenced differentially by
growth state. The fact that the initial rate of decay of protein
kinase C activity after PDBu treatment is similar for cells in
both growth conditions suggests that the rate of synthesis rather
than the rate of degradation is primarily responsible for the
observed difference in residual enzyme activity.
Whatever growth state-dependent mechanism is controlling
the extent of down modulation of protein kinase C in Swiss
3T3 cells appears to be operative in transformed cells as well.
Transformed cells in a growth-arrested state do respond to
long-term PDBu treatment with loss of a significant fraction of
protein kinase C. In addition, raj-transformed cells growing in
serum have higher levels of protein kinase C after PDBu treat
ment than serum-starved cells. However, the regulation of C
kinase levels in transformed cells does differ from that in
nontransformed cells in several respects. The ras-transformed
cells lose a smaller fraction of C kinase activity following longterm PDBu treatment in both serum-starved and serum-fed
states. Further, in untreated src-, abl-, and mi-transformed NIH
3T3 cell lines, there is approximately one half the cytosolic
protein kinase C activity per mg of protein relative to the parent
line (Ref. 9; Footnote 4). Thus, the transformed cell lines appear
to possess similar, but distinct, mechanisms for control of
cellular protein kinase C.
The amount of protein kinase C apparently required to inhibit
EGF binding differs in PDBu-pretreated cells relative to control
cells, suggesting that extensive pretreatment of cells with tumor
promoters could alter cell properties other than protein kinase
C levels. In untreated Swiss 3T3 cells, maximal inhibition of
EGF binding requires maximal mobilization of cellular C kinase
as monitored by cell surface binding of tumor promoters (Ref.
11; Footnote 4). However, we show that, in growing cells which
have been pretreated with PDBu, maximal inhibition of EGF
binding can be attained with only a fraction (less than 25%) of
the protein kinase C. Similar results have been reported for two
other systems, where loss of approximately 80% of the cell
surface PDBu binding sites had no effect on the ability of tumor
promoters to inhibit EGF binding in pituitary cells (22) or
suppress globin gene expression and differentiation in 1IL 60
cells (23). One explanation proposed for these results is that
heterogenous populations of protein kinase C exist which are
subject to differential loss upon treatment with tumor pro
moters (22). Our observation that stationary down-modulated
cells do show an attenuated response to PDBu argues against
heterogeneous enzyme populations in these cells.
An alternative possibility is that extensive pretreatment of
cells with tumor promoters could alter secondary signaling
systems. In Swiss 3T3 cells, the loss of EGF binding in response
to tumor promoter stimulation appears to be protein kinase C
dependent (11, 26). However, analysis of the sites of phosphorylation of the EGF receptor after tumor promoter treatment
In vivo suggests that other kinases may be activated either
independently or as a result of protein kinase C stimulation (7,
13,24). Thus, pretreatment of cells with tumor promoters could
affect other EGF receptor modulators, or even differential
populations of the EGF receptor itself, and render them more
sensitive to tumor promoter stimulation.
1085
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REGULATION
OF PROTEIN
Our results illustrate the important point that tumor pro
moter-induced loss of protein kinase C activity is a complex
process that depends on factors such as growth state, tumor
promoter dose, and cell type. Under our conditions, cells must
be growth arrested in order for total loss of the C kinase activity
to occur. The >90% reduction in C kinase activity that we have
observed for growth-arrested cells is not limited to confluent
cultures, since subconfluent BALB/c 3T3 cells which were
growth arrested by serum starvation behave in a similar manner
(Footnote 4). The inability of PDBu pretreatment to cause
complete loss of protein kinase C in growing cells cannot be
ascribed to a serum factor, since A431 cells which were growth
arrested in the presence of PDBu and serum still lost all
detectable kinase C activity. In addition to growth state, loss of
C kinase depends on the type and dose of tumor promoter, and
the half-life for this process ranges from hours to days for
different treatment conditions (20, 21). Some cell lines are
partially or totally resistant to loss of PDBu binding sites and
presumably protein kinase C activity, after tumor promoter
treatment (22, 25); in these cases, cell type as well as growth
state may be a controlling factor. Our results indicate that it is
difficult, if not impossible, to eliminate protein kinase C from
growing cells by down-modulation, and that, in growing cells,
low levels of the enzyme may have significant biological activity.
ACKNOWLEDGMENTS
We would like to thank Dr. M. Böigerof the University of Southern
California for statistical data analysis programs; Dr. P. Girard, Dr. J.
F. Kuo, and Dr. P. Parker for kindly providing antibody; B. Friedman
for helpful comments; and E. Fahci for assistance in preparing the
manuscript.
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Growth State-dependent Regulation of Protein Kinase C in
Normal and Transformed Murine Cells
Patricia G. McCaffrey and Marsha Rich Rosner
Cancer Res 1987;47:1081-1086.
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