[CANCER RESEARCH 48, 1910-1919, April 1, 1988)
Phorbol Ester-mediated
Association of Protein Kinase C to the Nuclear Fraction in
NIH 3T3 Cells
Thomas P. Thomas, Harvinder S. Talwar, and Wayne B. Anderson1
Division of Cancer Biology and Diagnosis, National Cancer Institute, NIH, Bethesda, Maryland 20892
ABSTRACT
Treatment of intact NIH 3T3 cells with 12-0-tetradecanoylphorbol13-acetate (TPA) causes a rapid redistribution (stabilization) of protein
kinase C to the participate fraction. Part of the enzyme activity stabilized
to the membrane fraction in response to TPA can be recovered associated
with nuclear-cytoskeletal
components. An apparently pure nuclear frac
tion prepared from NIH 3T3 cells was found to contain 25-30% of the
total membrane-associated
protein kinase C activity when isolated in the
presence of Ca2*. In untreated control cells, most of this activity found
with the nuclear fraction can be extracted by chelators. Phorbol ester
(TPA) treatment of NIH 3T3 cells induces the tight association of protein
kinase C to the nucleus; this tightly bound activity is not dissociable by
chelators and can be recovered only by solubilization with detergent.
Nuclei purified from untreated human promyelocytic leukemic III.-60
cells contain higher amounts of chelator-stable, detergent-extractable
protein kinase C activity compared with control NIH 3T3 cells. However,
TPA treatment of HL-60 cells does not enhance the amount of protein
kinase C found tightly associated with the nuclear fraction. Immunohistochemical studies with poh donai antibodies directed against protein
kinase C further indicate that TPA treatment of NIH 3T3 cells does
significantly enhance the amount of protein kinase C found tightly
associated with the nucleus and cytoskeleton, whereas exposure of HL60 cells to TPA does not appreciably alter the amount of protein kinase
C observed to be associated with the nuclear fraction. The TPA-mediated
association (activation) of protein kinase C to the nuclear and cytoskeletal
fractions with NIH 3T3 cells is further supported by the enhanced
phosphorylation of specific endogenous proteins noted when purified
nuclei and cytoskeletal preparations are incubated with |-y--"P|ATP.
These results suggest that tumor promoters may induce association
(activation) of protein kinase C with different subcellular components to
alter the availability of endogenous substrates. This may result in differ
ential responses by different cell types during exposure to tumor pro
moters.
INTRODUCTION
In resting unstimulated cell populations in culture, protein
kinase C activity is found predominantly in the cytosolic frac
tion or loosely associated with membranes. Treatment of resting
target cells with certain tumor promoters and hormones can
stimulate protein kinase C either directly (in the case of tumor
promoters) or through increased diacylglycerol production (in
the case of certain hormones shown to enhance phosphatidylinositol turnover) (1); this activation of protein kinase C occurs
concomitantly with stabilization (chelator-stable association) of
the enzyme to the paniculate fraction (2). Thus, the biologically
active phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (3) and hormones, such as interleukin 2 (4),
interleukin 3 (5), the a-agonist phenylephrine (6), gonadotropin-releasing hormone (7), thyrotropin-releasing hormone (8,
9), and adrenocorticotropin (10), have all been shown to alter
the subcellular distribution of protein kinase C to the membrane
fraction. Most of this stimulated protein kinase C activity
Received 10/31/86; revised 5/4/87. 11/23/87; accepted 1/6/88.
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.
1To whom requests for reprints should be addressed, at NIH, Building 36,
Room 1D22, Bethesda, MD 20892.
appears to be stabilized with the plasma membrane (3).
Yet, in vitro and in vivo studies suggest that proteins derived
from subcellular fractions other than plasma membrane, espe
cially from the nucleus and cytoskeleton, might serve as impor
tant substrates for protein kinase C. Nuclear proteins, such as
histone HI (11) and DNA methyltransferase (12) and topoisomerase II (13), and cytoskeletal related proteins, such as
vinculin (14, 15), filamin (15), microtubule-associated proteins
(16, 17), talin (18), and troponin (19), and other membrane
skeletal-associated proteins (20, 21) have all been tentatively
identified as possible substrates for phosphorylation by protein
kinase C. Although protein kinase C has been implicated in the
induction of proteins, such as ornithine decarboxylase (22) and
histidine decarboxylase (23) and probably c-myc (24, 25), c-fos
(25, 26), and plasminogen activator (27), the mode of signal
transmission of protein kinase C activation to alter gene expres
sion is unknown.
Thus, it is of importance to determine whether the phorbol
ester tumor promoter TPA2 might induce association (stabili
zation) of protein kinase C to subcellular fractions other than
plasma membrane. By immunohistochemical localization stud
ies, Girard et al. (28) have shown that, in rat brain, protein
kinase C can be found localized to the periphery of nuclei or to
axons. In this communication we have assessed whether TPA
can mediate association of protein kinase C to the nuclearcytoskeletal fraction and have determined differences in this
response to TPA with different cell types.
MATERIALS AND METHODS
Materials. Diolein, phosphatidylserine, PMSF, 3,3'-diaminobenzidine, and histone HI (type III-S, lysine-rich histone) were purchased
from Sigma Chemical Co. (St. Louis, MO). Leupeptin was from Boehringer Mannheim (Mannheim, West Germany) and [-/-32P]ATP was
from ICN (Irvine, CA). The biotin-avidin-peroxidase-based immunostaining kit (Vectastain) was from Vector Laboratories (Burlingame,
CA). Hexylene glycol was obtained from Fluka AG (Buchs, Switzer
land). Rabbit anti-mouse brain protein kinase C antisera (29) was
generously provided by Drs. A. Jeng and P. Blumberg (NCI, NIH),
while rabbit anti-pig brain protein kinase C antisera (28, 30) was kindly
supplied by Drs. P. Girard and J. F. Kuo (Emory University, Atlanta,
GA).
Cell Culture. Human promyelocytic leukemic HL-60 cells (obtained
from Dr. T. Breitman, NCI, NIH) were grown in suspension in RPMI
1640 (Gibco Laboratories, Grand Island, NY) supplemented with 10%
fetal bovine serum, antibiotics (SO units/ml of penicillin and 50 ug/ml
of streptomycin), and 4 m\i glutamine in a 5% CO- humidified atmos
phere. Mouse NIH 3T3 fibroblasts (generously provided by Dr. R.
Bassin, NCI, NIH) were grown attached to culture dishes at 37°Cin a
5% humidified atmosphere in Dulbecco-Vogt-modified Eagle's medium
(Gibco) containing 10% fetal calf serum, 4 HIMglutamine, 50 units/ml
penicillin, and 50 ¿ig/mlstreptomycin.
Preparation of Nuclear-Cytoskeletal Components. Confluent NIH
3T3 cells (~20 x IO6 cells) were incubated for 20 min in the presence
and absence of 0.1 n\i TPA. Cells attached to the culture dish then
2The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;
PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; NP-40,
Nonidet P-40; NCI, National Cancer Institute; EGTA, ethylene glycol bis(/3aminoethyl ether)-A',W,A'',A"-tetraacetic acid.
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TUMOR PROMOTER-INDUCED
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
were rapidly washed with warm (30°C)PBS and extracted for 3 min at
saturated lithium carbonate; (0 incubate in distilled water for 1 min;
30°Cwith gentle shaking in a buffer containing 0. l M piperazine-A^W(u) incubate in 70% ethanol for 2 min; (v) incubate in absolute ethanol
bis(2-ethanesulfonic acid) (pH 6.9), 5 IHMMgCl2, 0.2 mM EGTA, 4 M for 2 min; (w) incubate in xylene for 2 min and mount under a coverslip
with Permount. The slides were photographed with a Carl Zeiss phoglycerol, 100 MMleupeptin, and 0.05% Triton X-100 (see Ref. 31). The
solubilized fraction was quickly removed to leave nuclear-cytoskeletal
tomicroscope at X135.
In Vitro Phosphorylation of Endogenous Proteins. The attached nu
structures attached to the substratum. The attached nuclei and filamen
clear-cytoskeletal fraction was washed with buffer B and then scraped
tous cytoskeletal proteins were washed twice with cold PBS and twice
with chelator-containing buffer A [20 mM Tris-HCl (pH 7.5), 2 IHM from the culture dish into buffer B. Chelator-washed, nuclear-cytoskel
EDTA, 0.5 mM EGTA, 2 HIM PMSF, 0.25 M sucrose, and 25 MM etal fraction or purified nuclei prepared from control and TPA-treated
leupeptin]. To separate nuclei from the cytoskeletal structures, the
cells (100 Mgprotein used per experiment) were incubated in a final
reaction volume of 250 M' with 10 MM[>-32P]ATP (2000 cpm/pmol),
attached components were rapidly scraped into 5 ml of a buffer con
taining 10 HIMTris-HCl (pH 8), 3 mM CaCl2, 0.25 M sucrose, 2 mM
750 MMCaCl2, 10 mM MgCl2, 25 MMleupeptin, and 20 mM Tris-HCl,
PMSF, 1% NP-40, and 100 MMleupeptin; vortexed; and centrifuged
pH 7.5, in the presence and absence of 24 Mgphosphatidylserine and
immediately at 1000 x g for 4 min at 4"C. The supernatant (designated
1.6 Mg diolein for 3 min at 30"C. The phosphorylation reaction was
as the cytoskeletal fraction) was retained and incubated at 4"C with
terminated by the addition of 1.5 ml ice-cold acetone:NH3 (5.3:0.3).
rotation for 60 min to allow complete detergent (NP-40) solubilization
The precipitated protein fractions were collected by centrifugation in a
of cytoskeleton-associated protein kinase C. The nuclear pellet was
microfuge; the pellet was dissolved in sodium dodecyl sulfate sample
resuspended in buffer B (buffer A with 50 MMleupeptin and without
buffer and boiled for 3 min; and phosphorylated proteins were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%
sucrose) containing 1% NP-40 and incubated with rotation for 60 min
at 4°C.The solubilized cytoskeletal (adjusted to 3.5 mM EGTA) and
acrylamide) and detected by autoradiography.
nuclear preparations were fractionated by DEAE cellulose chromatography, and protein kinase C was measured as described elsewhere (2).
RESULTS
Isolation and Purification of Nuclei. NIH 3T3 cells (control and TPA
treated) were detached from the culture dish with 0.1% trypsin, and
Effect of TPA Treatment of Intact Cells on Protein Kinase C
fetal calf serum (10% final) was immediately added to the trypsinized
Activity Associated with the Nuclear-Cytoskeletal Fraction. As
cell suspension. NIH 3T3 and HL-60 cell suspensions were centrifuged
has been reported for numerous other cell types, exposure of
at 150 x g for 2 min, and the cell pellets were washed once by
resuspension in PBS. Cell pellets (-15 x IO6cells) were chilled to 4°C intact NIH 3T3 fibroblasts and human promyelocytic leukemia
TPA for 20 min caused a redistribution
on ice and resuspended in a buffer containing 50 mM piperazine-A^TV"- HL-60 cells to 0.1 /¿M
of
protein
kinase
C
activity
from the cytosolic to the paniculate
bis(2-ethanesulfonic acid) (pH 6.5), 1 mM CaCl2,0.5 M hexylene glycol,
fraction
(Fig.
1).
Since
the
enzyme
complex formed with TPA
and 50 MMleupeptin and incubated on ice for 10 min to allow the cells
requires higher salt concentrations for elution from DEAEto swell (see Ref. 32). The swollen cells were disrupted by 30 strokes
cellulose (2), the complete recovery of detergent-solubilized
with a Dounce homogenizer (B pestle). Crude nuclei were isolated
membrane-associated protein kinase C was obtained with elu
immediately by centrifugation at 1,000 x g for 3 min. This nuclear
pellet was resuspended in 0.5 ml nuclear suspension buffer [50 mM
tion of the DEAE-cellulose column with 0.1 M NaCl. Under
Tris-HCl (pH 7.5), 0.3 M sucrose, 4 HIMMnCl2, 25 mM KC1, 0.1 mM
these conditions, total protein kinase C activity (soluble plus
EDTA, 1 mM 2-mercaptoethanol, 1 mM PMSF, and 50 MMleupeptin]paniculate) remained unchanged with TPA treatment of NIH
by gentle agitation with a Pasteur pipet and mixed with 4 ml of this
3T3 cells for 20 min. However, after exposure of HL-60 cells
same nuclear suspension buffer containing 2 M sucrose and 0.1 mM
to TPA for 20 min, the activity gained in the membrane fraction
PMSF. This nuclear suspension was centrifuged at 25,000 x g for 30
accounted for only ~30% of the activity lost from the cytosol.
min. The supernatant was removed, and the purified nuclear pellet was
rinsed three times with the nuclear suspension buffer. The purified
nuclei then were resuspended in 0.5 ml PBS for cytospin onto slides or
3T3+
resuspended in 5 ml of buffer B for measurement of protein kinase C
PA1^i-+TPA1HL-60
lNIH
14,000P
>_
activity. The purity of the nuclear preparation was determined by
electron microscopy and was routinely monitored by phase contrast
12,000ÌJjì
1
microscopy after staining with méthylène
blue.
Immunohistochemical Localization Studies. Nuclei purified from NIH
1txoo°||
3T3 and HL-60 cells as described were attached to polylysine-coated
glass slides by cytocentrifugation (33). The attached nuclei, or the
80002
nuclear-cytoskeletal fraction prepared as described, were fixed by in
eK
cubation at room temperature for 30 min with 4% formaldehyde-0.1%
6000-i
Sglutaraldehyde in 50 mM Tris-HCl, pH 7.5. The fixed fractions then
were rinsed and/or incubated successively with: (a) PBS (rinse three
1
4000
times); (¿>)
0.2 M lysine; (c) PBS (rinse three times); (d) incubate with
—0
<
TPAi
1% horse serum in 50 mM Tris-HCl, pH 7.5, for 20 min; (e) incubate
2000n. nñ
in goat serum diluted 50 times in 50 mM Tris-HCl, pH 7.5-150 mM
NaCl buffer for 20 min; (/ ) incubate with rabbit anti-protein kinase C
(1:500 dilution) or rabbit preimmune serum (1:500 dilution) for 60 min
Cytosol Membrane
Cytosol Membrane
at room temperature and then overnight at 4°C;(g) rinse with 50 mM
Fig. 1. Phorbol ester (TPA)-mediated association of protein kinase C to the
Tris-HCl, pH 7.5-150 mM NaCl buffer; (h) incubate with 1% horse
crude paniculate fraction in NIH 3T3 and HL-60 cells. Cells were incubated in
serum in 50 mM Tris-HCl, pH 7.5, for 10 min; (/') incubate with
nWi +TPA
i_ 1IIj
II-+
the presence and absence of 0.1 JIMTPA for 20 min. After treatment, cells were
biotinylated goat anti-rabbit IgG (1:500 dilution) for 30 min in a
harvested, subcellular fractions were prepared, and protein kinase C activity was
determined in cytosolic and NP-40 detergent-solubilized paniculate preparations
humidified box; (j) repeat steps g and h; (k) incubate with 0.3% H2O2
fractionated by DEAE-cellulose chromatography as described elsewhere (2). Pro
in methanol for 30 min; (/) incubate with 1% horse serum in 50 mM
tein kinase C activity was determined by subtracting the amount of 3JP incorpo
Tris-HCl, pH 7.5, for 20 min; (m) incubate with avidin-biotin complex
ration into histone noted in the absence of added phospholipids from the amount
of "P incorporation noted in the presence of phospholipids. Total protein kinase
(Vectastain) for 60 min in a humidified box; (/;) repeat steps g and It
C activity is expressed as the pmol "P incorporated per 3 min per total volume
and strain with diaminobenzidine (0.05%) for 3 min; (o) incubate with
of each cytosolic and membrane preparation prepared from 15 x 10* cells. The
PBS for 10 min; (p) incubate with hematoxylin for 1 min; (q) rinse
data represent means of triplicate determinations from two separate experiments
with NIH 3T3 cells and three independent experiments with HL-60 cells. Bars.
slide by dipping (20 times) into 4% acetic acid; (r) rinse slide by dipping
SD.
(10 times) into distilled water; (s) rinse by dipping (20 times) into
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TUMOR PROMOTER-INDUCED
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
Concomitant with this apparent loss in HI -60 protein kinase
C activity, a higher background activity (phosphotransferase
activity measured in the absence of added phospholipids) was
observed in the membrane fraction of treated HL-60 cells. This
observed increase in phospholipid-independent
phosphotrans
ferase activity conceivably could be due to increased formation
of protein kinase M, the M, 50,000 fragment of protein kinase
C (34), and may account, in part, for the loss in total protein
kinase C activity noted with TPA treatment of HL-60 cells.
Alternatively, in HL-60 cells, a complex of protein kinase C
with TPA, calcium, and phospholipids might be formed that is
stable to detergent solubilization. Such a complex conceivably
could be recovered from the DEAE-cellulose column in an
active form that would not exhibit further stimulation with the
addition of phospholipids. Further studies are required to re
solve these possibilities.
To further characterize possible activation and localization
of protein kinase C in subcellular fractions other than plasma
membrane in response to tumor promoters, phospholipid-dependent phosphotransferase activity was determined in isolated
nuclear and cytoskeletal fractions. A nuclear-cytoskeletal frac
tion was prepared from control and TPA-treated NIH 3T3 cells
by rapid extraction of the cells with mild detergent as described
in "Materials and Methods." Under the conditions used, the
the majority of purified nuclei (Fig. 2A). Further, a pronounced
condensation of the nuclear matrix and its associated chromâtin
was a common feature of the purified nuclei. Surrounding the
condensed portion of the nuclear matrix, ribonucleoprotein
particles and strands of what appear to be extruded DNA can
be seen at higher magnifications (Fig. 2B). As indicated by
arrows, nuclear pores are visible on the outer portion of the
membrane surrounding the condensed matrix. These character
izations show that the preparations contain intact nuclei and
nuclei with broken nuclear envelopes, with minimal contami
nation by other subcellular fragments and organelles.
As given in Table 1, treatment of NIH 3T3 cells with TPA
for 20 min caused a significant activation of protein kinase C
at the nucleus as indicated by the increase (~ 12-fold) in chelator-stable, detergent-solubilized, phospholipid-dependent phos
photransferase activity found associated with the purified nu
clear fraction. This activity stabilized to the nuclei constituted
25-30% of the total protein kinase C activity found associated
with the total 100,000 x g crude particulate fraction. Further,
TPA treatment of NIH 3T3 cells was found to enhance chelatorstable, detergent-soluble activity associated with the nuclear
fraction, even when the nuclei were prepared in the continuous
presence of chelators (2 HIMEDTA and l IHMEGTA) through
out the isolation procedure. However, the purity and integrity
plasma membrane was solubili/ed, leaving nuclei and associated
of the nuclear fraction is improved when isolated in the presence
cytoskeletal fragments attached to the culture dish as monitored
of calcium; therefore calcium was routinely present during
by phase contrast microscopy (31). This nuclear-cytoskeletal
purification of nuclei. In contrast, although HL-60 cells exhib
ited a higher level of tightly associated detergent-solubilized
preparation was washed with buffer containing EGTA to re
move any protein kinase C loosely associated through Ca2+- protein kinase C activity initially associated with the nuclear
fraction, treatment of HL-60 cells with TPA did not result in a
mediated binding to those subcellular fractions. The washed
significant increase in nuclear-associated protein kinase C ac
nuclear-cytoskeletal preparation then was extracted with deter
gent to solubilize any chelator-stable, tightly associated protein
tivity (Table 1).
kinase C. Tightly associated, detergent-soluble protein kinase
Immunohistochemical Evidence for TPA-induced Association
C activity was found to be markedly increased in both the of Protein Kinase C to the Nuclear and Cytoskeletal Fractions.
To verify that the increase in phospholipid-dependent phospho
nuclear and cytoskeletal components of this particulate prepa
ration in response to TPA treatment of intact NIH 3T3 cells. transferase activity measured in the nuclear and cytoskeletal
fractions in response to TPA treatment represents an increase
There was an increase in protein kinase C activity of 2731 pmol
32P incorporated per 3 min in the nuclei and 3097 pmol 32P in enzyme protein, immunohistochemical studies were carried
incorporated per 3 min in the cytoskeletal fraction upon treat
out with polyclonal antibody preparations raised against puri
ment of confluent NIH 3T3 cells (20 x 10" cells) with 0.1 iiM fied protein kinase C. As shown in Fig. 3, TPA treatment of
TPA for 20 min. This activity gained in the nuclear-cytoskeletal
intact NIH 3T3 cells caused a pronounced increase in the
fraction after exposure to TPA accounted for about 40% of the amount of ¡mmunoreactive protein kinase C found associated
total chelator-stable, membrane-associated protein kinase C with the nuclear-cytoskeletal fraction. In further agreement with
activity. Attempts to prepare a similar nuclear-cytoskeletal
the phosphotransferase activity measurements, highly purified
fraction from HL-60 cells growing in suspension were not
nuclei from TPA-treated NIH 3T3 cells also exhibit a signifi
successful.
cant increase in immunoreactive protein kinase C (Fig. 4). That
To further define changes (activation) of protein kinase C at this increase in immunoreactive enzyme in the nuclear and
the nucleus in response to tumor promoter, it was necessary to cytoskeletal fractions is mediated by TPA is indicated by the
obtain highly purified nuclei free of possible contamination by control panels where little staining is noted with nuclei and
fragments of plasma membrane. Highly purified nuclei were cytoskeleton prepared from untreated NIH 3T3 cells incubated
prepared from control and TPA-treated NIH 3T3 and HL-60
with anti-protein kinase C antisera. In contrast, nuclei purified
cells as described in "Materials and Methods." Measurement
from untreated and TPA-treated HL-60 cells exhibit a similar
of adenylate cyclase activity was used to monitor the purified
intensity of immunostaining (Fig. 5). This is in agreement with
nuclear preparation for possible contamination by fragments of the phosphotransferase activity measurements which indicate
little effect of TPA treatment of HL-60 cells to increase the
plasma membrane. Adenylate cyclase activity found in the
nuclear fraction prepared from NIH 3T3 and HL-60 cells never
amount of protein kinase C found associated with the nuclear
accounted for more than I and 3%, respectively, of the total
fraction.
adenylate cyclase activity present in the crude particulate frac
That the positive immunohistochemical staining noted at the
nucleus of TPA-treated 3T3 cells is due to an increase in protein
tion. The purified nuclei were routinely monitored for purity
by phase contrast microscopy after staining with méthylène kinase C enzyme is further established by the Western blot
blue. In addition, the purity of the nuclear preparation was analysis of proteins extracted from purified nuclear prepara
verified by electron microscopic examination. Careful exami
tions (Fig. 6). The antibody preparation specifically recognizes
nation of the purified nuclei reveals that the outer leaflet of the
a MT 80,000 band corresponding to protein kinase C, which is
nuclear envelope had been sheared away from the nucleus in markedly enhanced in nuclei prepared from 3T3 cells exposed
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TUMOR PROMOTER-INDUCED
- £f-.r
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
.-
••'iV^"*""
•
.•"•
jfc
•
.If•-•'
' H.T-. ;
T
Fig. 2. Electron micrograph of purified nuclei from NIH 3T3 cells. Nuclei were purified from NIH 3T3 cells as described in "Materials and Methods." The
purified nuclei were fixed with 4% paraformaldehyde/0.25% glutaraldehyde for l h followed by exposure to 2% osmium tetroxide in PBS for an additional I li. The
fixed nuclear pellet was then dislodged from the centrifuge tube, dehydrated, and embedded in plastic for subsequent electron microscopic evaluation. Ultrathin
sections (800-1,000 A) were cut from portions of the embedded nuclear pellet. The sections were stained with uranyl acetate and lead citrate prior to examination in
a Phillips 400 electron microscope. Photographs of these sections were taken at magnifications varying from 2,150 (A) to 10,000 (B) diameters. Arrows, nuclear pores
on the outer portion of the membrane surrounding the condensed matrix. The authors gratefully acknowledge the electron microscopic evaluation of these preparations
by Dr. G. H. Smith, NCI, NIH.
Table I Effect of TPA treatment of intact NIH 3T3 and HL-60 cells on the
amount of protein kinase C activity found associated with nuclei purified in the
presence or absence of calcium chelators
Cells were incubated in the presence and absence of 0.1 »IM
TPA for 20 min,
and then nuclei were isolated and purified in the presence and absence of chelators
as described in "Materials and Methods." The purified nuclei first were extracted
with chelator-containing buffer B for 30 min at 41 to obtain the chelatorextractable kinase activity. The chelator-extracted nuclei were harvested by centrifugation and solubilized in buffer B containing I % NP 40 to obtain detergentsolubilized kinase activity. Protein kinase C activity was determined in the
chelator-extracted and detergent-solubilized fractions following DEAE-cellulose
chromatography as described elsewhere (2).
Protein kinase C activity*
(pinol 32P incorporated/3 min)
to TPA. Other protein bands recognized by the antisera prep
aration present a similar pattern with nuclei prepared from
control and TPA-treated NIH 3T3 cells. The recognition of
similar multiple bands common to nuclei from treated and
untreated cells may reflect some nonspecific interaction by the
antisera used, or it may indicate detection of different forms of
protein kinase C as suggested by Girard et al. (30). Nonetheless,
only the TPA-mediated specific association of the M, 80,000
protein correlates with the observed increase in phospholipiddependent protein kinase C activity and the increase in immunohistochemical staining of nuclei in response to TPA treat
ment of NIH 3T3 cells. To further establish that the antibody
Addition of cheChelator-extracted
preparation is recognizing protein kinase C, the antisera was
solubilizedUntreated384
rfurinoCell
lators
preadsorbed with purified protein kinase C prior to use in
Untreated3,260
typeNIH
Western blot analysis and immunohistochemical studies. This
3T3HL-60subfractionation"
preadsorbed blocked antisera did not recognize either the M,
+
3121,337
158784
2,870738
80,000 or 50,000 proteins and did not show enhanced immunostaining with nuclei prepared from NIH 3T3 cells treated with
-1238+TPA52427417839Detergent568+TPA4,609
612
TPA (results not shown).
" Chelators (Final concentration, 2 ni\i EDTA and l IHMEGTA) were included
Effect of TPA Treatment of Intact Cells on in Vitro Phosphoin the buffers used during cell homogenization and nuclei purification where
rylation of Endogenous Proteins Present in the Nuclear and
indicated by +.
* The activity given is for the nuclear fraction prepared from 15 x 10* cells.
Cytoskeletal Fraction. To further assess possible changes in
The total protein kinase C activity (cytosol + crude paniculate) in 15 x 10' NIH
protein kinase C activity in the nuclear and cytoskeletal frac
3T3 cells was 15.2 nmol 3:P incorporated/3 min, and the total activity redistrib
tions in response to TPA treatment of intact cells, in vitro
uted to the 100,000 x g crude paniculate fraction with the standard TPA
treatment of NIH 3T3 cells was 13.5 nmol "P incorporated/3 min. The total
phosphorylation studies were carried out to determine differ
protein kinase C activity determined in 15 x 10' HL-60 cells was 4.58 nmol 3:P
ences in the phosphorylation pattern of endogenous proteins
incorporated/3 min, and total activity redistributed to the crude paniculate
fraction in response to TPA was 1.18 nmol "P incorporated/3 min.
present in these subcellular fractions. Nuclei purified from
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TUMOR PROMOTER-INDUCED
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
Fig. 3. Iminumili istochcinical localization of protein kinase C in the nuclear-cytoskeletal fraction of NIH 3T3 cells. Confluent NIH 3T3 cells growing in Titer Tek
wells were incubated in the presence (right) and absence (left) of 0.1 nM TPA for 20 min. Cells were then rapidly (3 min) extracted with 0.05% Triton X 100 to
remove the plasma membrane and cytosolic fraction to leave the nuclear-cytoskeletal components attached to the substratum. The nuclear-cytoskeleton preparations
were fixed and analyzed for protein kinase C by immunohistochemical staining as described in "Materials and Methods." Rabbit anti-rat brain protein kinase C
(kindly provided by Drs. A. Jeng and P. Blumberg) (1:1000 dilution) was used with Vectastain kit components of biotinylated second antibody (goat anti-rabbit IgG)
and avidin-biotin complex. Diaminobenzidine was used as the coloring agent. The nuclear-cytoskeletal fraction prepared from control (untreated) NIH 3T3 cells
exhibited negligible staining with both preimmune rabbit serum and with rabbit anti-protein kinase C antisera.
control and TPA-treated NIH 3T3 and HL-60 cells were incu
bated with [7-32P]ATP in the presence and absence of phosphatidylserine plus diolein as described in "Materials and Methods"
(Fig. 7). Incubation of nuclei purified from TPA-treated NIH
3T3 cells with [7-32P]ATP showed enhanced phosphorylation
of Afr 24,000 and 31,000 protein bands when compared with
the phosphorylation pattern of control nuclei. In addition, there
was a less pronounced increase in the phosphorylation of Mr
18,000 and 19,000 nuclear proteins. Addition of phospholipid
and diolein to the control nuclear preparation does stimulate
the phosphorylation of the above mentioned M, 18,000,19,000,
24,000, and 31,000 bands (predominantly the M, 18,000 and
M, 19,000 bands). This indicates the presence of some unsiim
ulated protein kinase C associated with the nuclear fraction as
previously suggested by the activity measurements presented in
Table 1. Incubation of [7-32P]ATP with a nuclear-cytoskeletal
preparation from NIH 3T3 cells treated with TPA resulted in
the specific phosphorylation of proteins with apparent molec
ular weights of 25,000, 41,000, 59,000, and 71,000 (Fig. 8).
This is in addition to the enhanced phosphorylation of M,
18,000, 19,000, 24,000, and 31,000 proteins apparently con
tributed by the nuclei present in this preparation (see Fig. 7).
The M, 25,000, 41,000, 59,000, and 71,000 bands apparently
represent cytoskeletal proteins specifically phosphorylated with
TPA activation of protein kinase C at the cytoskeleton. These
observations lend additional support to the above results, which
indicate that TPA treatment of NIH 3T3 cells results in in
creased protein kinase C activity associated with the nuclear
fraction.
There appears to be a general enhancement in the phospho
rylation of all bands noted with nuclei prepared from HL-60
cells exposed to TPA (Fig. 7). This suggests that TPA-induced
phosphorylation of specific proteins is negligible in nuclei pre
pared from TPA-treated HL-60 cells. Furthermore, the addi
tion of phospholipids to the phosphorylation reaction contain
ing nuclei from either control or TPA-treated HL-60 cells had
little effect on the phosphorylation of specific HL-60 nuclear
proteins.
DISCUSSION
Biologically active phorbol esters bind to and stimulate pro
tein kinase C, and this enzyme appears to serve as the primary
cellular phorbol ester receptor or target to mediate the effects
1914
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TUMOR PROMOTER-INDUCED NUCLEAR ASSOCIATION OF PROTEIN KINASE C
Fig. 4. Immunohistochemical localization of protein kinase C in nuclei purified from NIH 3T3. Nuclei were purified from NIH 3T3 cells incubated in the presence
(right) and absence (left) of 0.1 fiM TPA for 20 min as given in "Materials and Methods." The nuclear preparations were fixed and analyzed for the presence of protein
kinase C by immunohistochemical staining as described in "Materials and Methods" and in the legend to Fig. 2.
of such tumor promoters (35, 36). This activation of protein
kinase C has been shown to result in a redistribution of protein
kinase C activity from the soluble to the particulate fraction,
predominantly by enhancing association with the plasma mem
brane (37). In the present study we have investigated whether
TPA might stabilize the association (activation) of protein
kinase C to subcellular membrane structures other than plasma
membrane. Treatment of intact NIH 3T3 cells with TPA is
shown to promote an increase in chelator-stable association of
protein kinase C activity with nuclei and cytoskeleton, in addi
tion to the plasma membrane. This is demonstrated by direct
measurement of calcium, phospholipid-dependent
phosphotransferase activity found in chelator-stable, detergent-solubilized extracts of purified nuclei and isolated cytoskeletal com
ponents (Table 1). That this increase in protein kinase C activity
is due to increased stabilization of enzyme protein with the
nuclear and cytoskeletal fractions is further indicated by im
munohistochemical localization studies (Figs. 3 and 4).
This TPA-mediated association of protein kinase C to the
nuclear fraction is not observed following exposure of HL-60
leukemic cells to TPA (Table 1; Fig. 5). HL-60 cells exhibit a
higher basal level of protein kinase C activity associated with
the nuclear fraction than observed with NIH 3T3 cells, but this
is not further increased by TPA treatment. Rather, TPA-induced binding of protein kinase C to the particulate fraction of
HL-60 cells is recovered associated with the plasma membraneenriched fractions.3 This is in agreement with immunocytochemical localization studies with intact HL-60 cells, which
demonstrate that exposure of these cells to TPA caused translocation of protein kinase C to the plasma membrane (38).
Previous studies have shown that growing cell populations
exhibit higher levels of particulate protein kinase C than noted
under conditions of growth arrest (37). Since HL-60 cells are
growing immortal cells, it was conceivable that the higher basal
levels of protein kinase C activity found in the nuclei of HL-60
cells might be a result of the proliferative state of these cells.
However, both growing and growth-arrested NIH 3T3 cell
populations exhibit similar amounts of nuclear protein kinase
C activity, and both cell populations exhibit similar increases
in nuclear protein kinase C activity upon treatment with TPA.3
The ratio of protein kinase C found in the cytosol and
particulate fractions appears to be regulated, in part, by calcium
(37, 39). With a reconstitution system of partially purified rat
brain protein kinase C and a crude membrane fraction isolated
from NIH 3T3 cells, it has been shown that the tight association
(chelator insoluble) of protein kinase C to the membranes
requires the simultaneous presence of TPA, Ca2+, enzyme, and
phosphatidylserine (40). Conceivably, protein kinase C nor3T. P. Thomas and W. B. Anderson, unpublished results.
1915
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TUMOR PROMOTER-INDUCED
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
'
• I
Fig. 5. Immunohistochemical localization of protein kinase C in nuclei punii«!from HL-60 cells. Nuclei were purified from HL-60 cells incubated in the presence
(right) and absence (left) of O.I I¡M
TPA for 20 min as described in "Materials and Methods." The nuclear preparations were fixed and analyzed for protein kinase C
as described in 'Materials and Methods" and in the legend to Fig. 2.
NIH 3T3 cells.3 Furthermore, under conditions where chelator
tnally is present in cells in weak calcium-dependent association
was continually present during cell disruption and preparation
(chelator sensitive) with membranes, as evidenced by the pres
ence of high amounts of chelator-extractable protein kinase C of purified nuclei and nuclear-cytoskeletal components (com
plete absence of Ca2+), TPA treatment of NIH 3T3 cells en
activity found with purified nuclei (Table 1). TPA intercalation
at the membrane might facilitate a high-affinity (chelator-stahanced protein kinase C activity associated with these subcel
ble) interaction between protein kinase C and the membrane.
lular fractions. That protein kinase C is present at the nucleus
With such a mechanism, it would be necessary for TPA to is also supported by subcellular distribution studies of phorbol
ester binding activity with mouse brain (45), epidermis (46),
rapidly traverse the cell membrane and cytosol to stabilize
and liver (47), which demonstrate the presence of specific, highprotein kinase C association with the nucleus. In fact, fluores
affinity, saturable receptors for phorbol esters in the nuclear
cent derivatives of TPA have been shown to be rapidly distrib
uted throughout the cytoplasm within minutes after addition to fraction. Although Nishizuka (1) has reported that immunocyintact cells (41). Yet, it is not apparent why TPA does not
tochemical studies with monoclonal antibodies directed against
stabilize protein kinase C binding to nuclei in HL-60 cells.
protein kinase C indicate little, if any, protein kinase C in the
Perhaps other, as yet unidentified, regulatory factors are deci
nucleus, immunohistochemical localization studies by Girard
sive in directing and determining the sites of protein kinase C et al. (28) indicate that some protein kinase C is found at the
binding to paniculate fractions. An interesting possibility is nucleus in rat brain. As the present results suggest, protein
that different forms (i.e., different gene products or modified
kinase C association with the nucleus may be dependent on cell
enzyme protein) of protein kinase C (42-44) might exhibit
type and also on the activated state of the enzyme.
altered preference for association with different subcellular
An increase in protein kinase activity associated with nuclear
membranous structures.
and cytoskeletal components in response to TPA is further
That this TPA-mediated localization of protein kinase C to indicated by results of in vitro phosphorylation studies carried
the nucleus is taking place within the intact cell and not after
out with subcellular components isolated from control and
cell disruption is indicated by the observation that inclusion of TPA-treated NIH 3T3 cells. Incubation of nuclei isolated from
TPA in the lysis buffer during cell disruption did not result in NIH 3T3 cells pretreated with TPA with [7-32P]ATP shows
increased binding of protein kinase C to the nuclear fraction of enhanced phosphorylation of M, 18,000, 19,000, 24,000, and
1916
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TUMOR PROMOTER-INDUCED
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
PKC CON +TPA
31,000 protein bands. Similar phosphorylation studies with a
nuclear-cytoskeletal preparation from NIH 3T3 cells treated
with TPA indicate enhanced phosphorylation of M, 25,000,
41,000, 59,000 and 71,000 cytoskeletal proteins. In contrast,
little change in the pattern of phosphorylation was observed
with nuclei prepared from control and TPA-treated HL-60
cells. The addition of phosphatidylserine and diacylglycerol to
these isolated fractions did not significantly enhance the phos
phorylation of endogenous proteins. This probably indicates
that the kinase tightly associated to the particulate preparation
already is in an activated state and does not require additional
stimulation. As this suggests, phosphorylation of endogenous
nuclear and cytoskeletal proteins would presumably take place
within the intact cell with TPA activation of the kinase at these
subcellular sites. Thus, the pattern of phosphorylation noted
with the in vitro studies presented might give only a partial
indication of possible endogenous substrates because phospho
rylation of endogenous protein within unlabeled intact cell
might prevent the incorporation of additional amounts of 32P
from [7-32P]ATP exogenously added to the isolated nuclei.
—43K
—26K
—18K
Fig. 6. Western blot analysis of immunoreactive proteins extracted from nuclei
purified from control (CON) and TPA-treated NIH 3T3 cells. Nuclei were
prepared as described in "Materials and Methods." Chelator-washed nuclei were
solubilized in sodium dodecyl sulfate sample buffer and electrophoresed in 10%
polyacrylamide gel; the protein bands were then transferred electrophoretically
to nitrocellulose membrane. The membrane was incubated (3 h at 37°C)with 3%
bovine serum albumin in Tris/NaCl/NP-40 buffer [20 mw Tris-HCl (pH 7.5),
150 min NaCl, and 0.1 % NP-40] to block nonspecific binding sites. The membrane
then was incubated overnight at 4°Cwith protein kinase C (PKC) antiserum
diluted 1:500 in the Tris/NaCl/NP-40 buffer containing 1% bovine serum albu
min. The membrane exposed to antiserum then was washed with Tris/NaCl/NP40 buffer, incubated with 125I-labeled protein A, and washed again. The labeled
proteins were detected by autoradiography. The mono Q-purified protein kinase
C used as marker in Lane 1 was generously provided by Dr. Peter Blumberg
(NCI, NIH). Ordinate, molecular weight in thousands (K).
Std.
Phorbol ester tumor promoters have been shown to markedly
alter the regulation of cellular growth (see Refs. 48 and 49). In
many cell systems, TPA can act, usually in concert with other
growth factors, to stimulate proliferation (48-51). On the other
hand, TPA acts to inhibit the growth of HL-60 leukemic cells
and induces differentiation into macrophage-like cells (52).
Feuerstein et al. (53) have reported that the differential phos
phorylation of specific proteins may account, at least in part,
for the differential effect of TPA to act as an inhibitor versus a
stimulator of cell growth. Not only does TPA, apparently acting
through protein kinase C, exhibit both positive and negative
effects on cell growth depending on cell type, but also activation
of protein kinase C can have both positive and negative effects
within a given cell. Activation (stabilization) of protein kinase
C at the plasma membrane is intimately related to negative
feedback control mechanisms such as the down-regulation of
cell surface receptors and inhibition of phosphatidylinositol
turnover (1, 37). Positive effects of protein kinase C activation,
such as altered gene expression and enhanced cell proliferation,
NIH 3T3
Control +TPA
Control
HL-60
+TPA
Std.
200KFig. 7. Effect of TPA treatment of NIH
3T3 and HL-60 cells on in vitro phosphoryla
tion of endogenous proteins present in the
purified nuclear fraction. Purified nuclei pre
pared from untreated and TPA-treated (0.1 JIM
TPA for 20 min) NIH 3T3 and HL-60 cells
were washed with chelator-containing buffer.
The chelator-washed nuclei were resuspended
and incubated in a phosphorylation reaction
mixture containing [T-32P]ATP at 30'C for 3
min as described in "Materials and Method."
The phosphorylated nuclei were boiled in so
dium dodecyl sulfate sample buffer and sub
jected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 32Pphosphoryl
ated proteins were determined by autoradi
ographic analysis. PS, phosphatidylserine;
Stcl.. standard; ordinales, molecular weight in
thousands (A).
- 200K
97K68K43K-
-97K
-68K
* r *
H it
26K-
18K
14K
-31K
-43K
-24K
- 19K
- 18K
-26K
-18K
-14K
PS+DIOL
-r
1917
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TUMOR PROMOTER-INDUCED
Std.
Control
NUCLEAR ASSOCIATION OF PROTEIN KINASE C
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ACKNOWLEDGMENTS
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200K —
— 59K(C)
1918
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Phorbol Ester-mediated Association of Protein Kinase C to the
Nuclear Fraction in NIH 3T3 Cells
Thomas P. Thomas, Harvinder S. Talwar and Wayne B. Anderson
Cancer Res 1988;48:1910-1919.
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