Vol. 169, No. 1, 1990 May 31, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 253-259
CORRELATION OF PROTEIN KINASE C TRANSLOCATION, P-GLYCOPROTEIN PHOSPHORYLATION AND REDUCED DRUG ACCUMULATION IN MULTIDRUG RESISTANT HUMAN KB CELLS Timothy
C. Chambers,
Ira
Chalikonda,
and Gabriel
Eilon
Department of Biological Chemistry Merck Sharp & Dohme Research Laboratories West Point, PA 19486 Received
April
12,
1990
SUMMARY: Treatment of drug-resistant human KB carcinoma cells (KB-Vl) with 0.2 PM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of 13H]vinblastine in KB-Vl cells was 0.8 -t 0.1 pmol/mg/30 min in the absence, and 1.9 +- 0.2 pmol/mg/30 min in the presence, of 20 PM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase G mediated phosphorylation stimulates the drug transport activity of P-glycoprotein. 01990 Academic Press, Inc.
The development limiting
factor
resistance
of in
involves
encodes
a transmembrane
protein
has
anticancer
a broad drugs
and
important. phosphorylation
treatment
the
amplification protein
In
cells
lines,
[see
Ca++/phospholipid-dependent
catalyzed
the (1,2)
human by
enzyme
One
family
This
in
of
reviews].
In
phosphorylation to
KB carcinoma
activated
which
efflux
recent
likely
kinase
of
P-glycoprotein.
for
is
protein
mechanism
multigene
undergoes
modification
a significant
energy-dependent
P-glycoprotein
multidrug-resistant is
rn&
KDa termed for
is
tumors.
the
150-170
specificity
resistant
agents
malignant of
post-translational the
chemotherapeutic of
of
substrate
cell
this
to
the
from
multidrug-resistant (3-7),
resistance
be
functionally
cell C cell
line, (PW
KB-Vl, (7),
membranes
a by
Abbreviations - PKC, protein kinase C; PM&, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; DMSO, PBS, phosphate buffered saline; dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl) dimethyl-ammonioll-propanesulfonate. Do06291x/90 253
All
$1.50
Copyright 0 1990 by Academic Press. Inc. rights of reproduction in any form reserved.
Vol.
169, No. 1, 1990
BIOCHEMICAL
diacylglycerol
and
using
cells
KB-VI
verapamil-induced in
tumor-promoting indicated
human
observations
suggest
may modulate
its
effects here
of that
drug
phorbol the
the
activity
esters phorbol
accumulation
breast
cancer
(7),
cells
possibility
transport
ester
phorbol that
[3H]vinblastine
drug-resistant
AND BIOPHYSICAL
that properties.
P-glycoprotein
(8).
Preliminary
studies
ester
treatment
reduced
similar
to earlier
Taken
(9).
phosphorylation Based
on phosphorylation of
RESEARCH COMMUNICATIONS
and is
together, of
on kinetic drug
stimulated
results
P-glycoprotein studies
accumulation, by
these
PKC
of
the
we show catalyzed
phosphorylation.
MATERIALS
AND METHODS
Materials - [32P]orthophosphate (8 mCi/ml), [gamma-32P]ATP (3000 Ci/mmol) obtained from Amersham and C219 and [3H]vinblastine (23 Ci/mmol) were monoclonal antibody, which recognizes P-glycoprotein (lo), was from Centocor. Cell Culture - Drug-sensitive KB-3 and drug-resistant KB-Vl cells were maintained in DMEM (4.5 g/l glucose) containing 10% fetal bovine serum and 10 ml-l Hepes, pH 7.3. KB-Vl cells were grown in the presence of 1 pg/ml vinblastine. For metabolic labeling with [32P], cells in 160 cm2 flasks were incubated for 4 hr in 10 ml of phosphate-free medium containing 0.8 mCi of [32P]orthophosphate. Prenaration of Extracts and PKC Determination - Near-confluent cultures of cells in 160 cm2 flasks were treated for various times with freshly prepared PMA dissolved in DMSO; controls received DMSO alone (0.1% v/v final concentration). Cells were gently scraped into ice-cold PBS, sedimented, and pellets suspended in 2 ml of buffer A (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM 2-mercaptoethanol, 1 mM EGTA, 1 mM EDTA and 1 mM PMSF). Cells were 15 min), debris removed by disrupted by nitrogen cavitation (400 p.s.i., centrifugation at 1,000 x g for 10 min, and a cytosolic fraction obtained by The membrane pellet was suspended in centrifugation at 100,000 x g for 1 hr. 1 ml of buffer A containing 1% w/v Triton X-100 and after 15 min on ice clarified by centrifugation (100,000 x g, 30 min). Samples were loaded onto 0.3 ml columns of DEAE-Sephacel equilibrated with buffer B (buffer A without sucrose), and, after washing with 5 ml buffer B, PKC was completely eluted with 2 ml 0.1 M NaCl in buffer B. PKC was assayed as described previously (11) by measuring incorporation of 32P from [gamma-32P]ATP to histone III-S in the presence of 0.5 mM CaC12, 50 pg/ml phosphatidyl-L-serine and 5 pg/ml diolein. Blanks, the values of which were subtracted, contained 1 mM EGTA instead of the cofactors. Protein was determined by the method of Bradford (12) using bovine serum albumin as a standard, P-glvconrotein Phosnhorvlation - 32P-labeled KB-Vl cells were treated with PMA for various periods, washed with ice-cold PBS, and membrane vesicles prepared as described previously (7). P-glycoprotein was solubilized by adding the detergent CHAPS to 1% w/v final concentration, as described (13). Immunoprecipitation with C219 monoclonal antibody and preparation of samples for SDS gel electrophoresis, performed using 6% acrylamide gels by the method of Laemmli (14), were carried out as detailed previously (7). Gels were stained with silver nitrate using reagents from BioRad Laboratories and quantitation of P-glycoprotein was performed by densitometric scanning of the gel. Radioactivity of excised bands was determined by scintillation counting. Drug Accumulation Assays - KB-Vl cells in drug-free medium were plated at 3 x 10s cells/well in 24-well Costar plates. PMA (0.2 pm> was added to 254
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
appropriate wells. The next day, PMA was added to additional wells for various periods up to 2.5 hr. Then, 30 min prior to processing, the medium of each well was removed and replaced with 0.5 ml DMRM containing 50 mM Hepes, pH 7.35, 0.1 pCi [3H]vinblastine, and, in some experiments, 20 PM verapamil. After 30 min incubation, the plates were washed three times in ice-cold PBS, the cells were detached with trypsin, and radioactivity determined (15). NaOH, 0.5 ml of 0.5 M, was added instead of trypsin to control groups of cells from each plate to determine protein content. RESULTS The kinetics Figure and
1.
of
The
relatively
translocation this
of
activity
to near
unchanged
for
cells
to
PMA
after of
relatively
was near
resulted
in
Membrane
PKC was decreased,
but
was still
elevated
relative
lines
of
PKC
(16,17).
PMA up to
to
1 PM,
PMA-induced
to
unexpected
results
in Further
confirmed down-regulation
2.5 the
the
30 min
membrane.
basal
4-fold.
cytosolic to
level
PKC maximum
(Fig.
long-term
complete
in During
3- to of
PKC (Table
1).
exposure
depletion,
experimentation, that
in
or using
KB-Vl I).
In
cells contrast,
Time (hi) Time-course of PMA-induced PKC translocatian in KB-Vl cells. Fieure 1. Cultures of cells were treated with 0.2 pM PMA for the times indicated, fractions prepaied and partially purified by cytosolic and membrane DEAE-Sephacel chromatography, and PKC activity determined, as described in "Materials and Methods."
255
in
resulted
relative
since
shown
for hr,
a return
level.
were
are
increased
basal
cell
resistant
to
PKC to activity
cells
stimulation
up
cytosolic
24 hr
numerous
of
after
treatment
PKC specific
PMA for
in KB-Vl
maximal
of
50% of
24 hr PMA treatment
down-regulation, concentrations
PKC translocation
periods
approximately
obtained,
The results to
which
membrane-associated
Exposure
levels
response,
of
time,
PMA-induced
is
Vol.
169, No. 1, 1990
Table
I.
Duration (hr)
BIOCHEMICAL
Effect
given
continuous characteristic
100 11 11 10
are averages
exposure
of
Figure
2.
4-fold
increase
increasing
times
since
membrane
treated
with
PMA-induced effects phosphate
of
exposure with
PKC
activity
PMA for
24 hr,
was
P-glycoprotein
phosphorylation
Accumulation KB-Vl as
cells
with
variation
ratio
in absolute
a rrlag't
period
amount
of
of drug
[3H]vinblastine preincubation utilized levels The
verapamil,
results,
of values
30 min,
of
PMA for an
accumulation presented
of
Figure
inverse the
3A.
drug
Figure
cells of
increase
in
to
the
increase
in
of
preincubation
time
of 40%
and
are
values,
since
exposure
of
cells
relative
to
control
examined show
24 hr.
(15),
that
to the
some
Following
was observed
60% after
of
expressed
experiments.
transport
3B, 256
In
the
control
time
LSH]vinblastine, in
phosphatase
1).
The results
and decreased of
with
PMA,
relationship
decreased
hr,
a
time-course.
in different
2.5
to
phosphorylation
similar
was observed
was
in
shown
observation),
(Fig.
to
versus
inhibitor of
in
and
are
protein
of
response
a magnitude
shown
to
level
as a function
accumulated
in
declined
(unpublished
high
a similar
a clear
resulted
corresponded
levels
cells
PMA-treated
accumulation with
of
PM PMA is
PMA
and
constant
Thus ) in
[SH]vinblastine
0.2
percentage
a
the
of
these
a relatively
and follows
1 hr
may be due
relatively
is
to
phosphorylation after
This
from
maintained.
PKC activity,
cells
Phosphorylation
PMA.
was
100 342 192 133
I).
content.
membranes
P-glycoprotein
membrane
KB-3
observed
to
100 56 74 59
experiments.
P-glycoprotein
were
in
associated
independent
of PKC (Table
of
Maximum
of three
100 293 0 0
drug-sensitive
depletion
The kinetics
and
PKC Activitv (% untreated) KB-3 KB-Vl Cvtosol Membrane Cvtosol Membrane
0 0.2 0.2 1.0
Values
RESEARCH COMMUNICATIONS
of shortand long-term exposure to PMA on activity distribution of PKC in KE cells
PMA Treatment Concentration (l&t)
0 0.5 24 24
activity
AND BIOPHYSICAL
between to
after We next
induce effect
PMA.
higher of
verapamil-induced
PMA.
Vol.
169, No. 1, 1990
BIOCHEMICAL
a --Fe-%
02
AND BIOPHYSICAL
--+-k-e4
--i-e-e‘l
03
Time (hr)
RESEARCH COMMUNICATIONS
Time (hr)
Time (hr)
Figure 2. Time-course of P-glycoprotein PMA-induced phosphorylation. 32P-labeled cells were treated with 0.2 PM PMA for the times indicated, membrane vesicles prepared, P-glycoprotein immunoprecipitated, and samples analyzed by 6% acrylamide SDS gel electrophoresis, silver staining and autoradiography. Quantitation of P-glycoprotein phosphorylation was based on comparison of radioactivity and amount of protein as described in "Materials and Methods." The inset shows typical results obtained after 0, 1 and 24 hr treatment with PMA. Fieure 3. Effect of PMA on [3H]vinblastine accumulation. KB-Vl cells ware preincubated with 0.2 pM PMA for the times indicated and 0.1 &i [sH]vinblastine added alone (A) or with 20 PM verapamil (B). After an additional 30 min, cells were washed and radioactivity and protein determined, as described in "Materials and Methods." In (A), 100% - 0.785 pmol/mg; in (B), 100% = 1.895 pmol/mg. Results are expressed for each time point as [mean (PMA)/mean (control)] x 100, n-4. The magnitude of the error bar at each time point was calculated from J[S,s + S,*), where S, - S.E. (control) and S, = S.E. (PMA), n-4.
[3H]vinblastine
accumulation
time-dependent
manner,
no
was
lag
period
shorter
times
verapamil
did
accumulation contrast
exposure.
not
affect
and It
effect
the on drug
significantly
the
should
of
showed at
of
be noted
that
3A.
that
the
maximal of
analog
0.1
the
in
this
a
case,
pronounced conditions
at used,
(7). effect
- 0.5
phorbol
in
more
under
P-glycoprotein
PMA treatment
However,
PMA was
of
concentrations
inactive
by
Fig.
effect
phosphorylation
curves
PMA,
reduced
to results
observed,
was observed to
significant
similar
of
Dose-response
was
FM.
of
PMA on
Additionally,
12,13-diacetate
drug in
had
no
accumulation. DISCUSSION
To examine cells
by
PKC
the (7),
role
of P-glycoprotein
the
effect
of
phosphorylation,
PMA on 257
[3H]vinblastine
catalyzed accumulation
in KB-VI was
Vol.
169, No. 1, 1990 Here
examined. relevant
we show
measure
of
by preincubation
membrane
significantly
the
are
related
indicate
was
of
maximal
a non-catalytic
after
level
a more
active
basal
thus
the
of
drugs
appears
Whereas
stimulation
not multiple
conformation
of
PMA,
and
then
Also,
ability by appeared
phosphorylation
of
While
on activity,
sites.
in
stimulated
reduced.
protein,
the
transport
the
account
PMA
resulting
be of
[aH]-
conditions,
Thus,
into
the
reduced
PKC,
to
to
take
at
same
P-glycoprotein.
1 hour,
does
significantly
activation, of
and biologically
and verapamil-induced
of phosphorylation
phosphorylation
promote
is
Under
exposure
effect
phosphorylation
differential
and
time
an accurate
Both
decreased.
by PKC.
to
P-glycoprotein
PMA.
transport
catalyzed
RESEARCH COMMUNICATIONS
P-glycoprotein,
phosphorylation to
phosphorylation
accumulation,
with
association,
P-glycoprotein
AND BIOPHYSICAL
of
cells
increased
directly
drug
activity
accumulation
induces
net
that
the
of
vinblastine
of
BIOCHEMICAL
this
may
measurement
the
possibility
of
phosphorylation
not
of
readily
may
reversed
by
dephosphorylation. To
further
define
P-glycoprotein studies
function,
utilizing
where
PKC is
since
KB-VI This
1).
was not to
the
it
role
will
depleted
by
are
prolonged
PKC was relatively property
of
KB-Vl
of
PKC-catalyzed
in
cells,
inhibit
progress. of
This
is
cells
in
this
in
phosphorylation,
and
to
but thus
cell
approach,
PMA, was
precluded
down-regulation
unusual
phenomenon
resistance
phosphorylation
An alternative
to PMA-induced
which
cells.
drug
to
exposure
resistant
by KB-3
development
of
be necessary
PKC inhibitors
exhibited
further
the
not
unique
appears line,
(Table
to
and
(16,17), be related
hence,
merits
investigation. Evidence
processes
for
has
been
neurotransmitters, glucose
and
proteins
cases
(19)
and
role
documented.
ions
such
as Ca for
phosphorylation Na+/H+
antiporter
of PKC in numerous
PKC activation
hormones,
responsible
some
a regulatory
enzymes ++
, H+,
some of h (20).
and other Na+,
these &
is
associated
agents,
K+ and processes
Cl-
258
to
with
(8,18).
release transport
The
been
e.g.,
our
and transport
and with
have
demonstrated, However,
secretion
knowledge,
of
transporter
identified glucose
of
and
in
transporter there
are
no
Vol.
169, No. 1, 1990
reports
in which
catalyzed here
a change
in
phosphorylation
suggest
multidrug with
BIOCHEMICAL
a key
of role
transporter,
other
transporters
for
AND BIOPHYSICAL
transport
activity
specific PKC in
RESEARCH COMMUNICATIONS
has
been
correlated
transporters.
The
results
phosphorylation
and
regulation
a protein
which
shares
(21,22;
see also
23).
significant
with
PKC
presented of
structural
the
homology
ACKNOWLEDGMENTS The providing typing
authors the
the
are cell
grateful lines
used
to
Drs.
M. Gottesman
in
this
study
and
and to
I.
Dorothy
Pastan
for
Bonenberger
kindly for
manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. ia. 19. 20. 21. 22. 23.
Juranka, P.F., Zastawny, R.L., and Ling,V. (1989) FASEB J. 2, 2583-2592. van der Bliek, A.M. and Borst, P. (1989) Adv. Cancer Res. 2, 165-203. Garman, D., Albers, L., and Center, M.S. (1983) Biochem, Pharmacol. 32, 3633-3637. Roy, S.N., and Horwitz, S.B. (1985) Cancer Res. 45, 3856-3863. Hamada, H., Hagiwara, K-I., Nakajima, T., and Tsuruo, T. (1987) Cancer Res. 47, 2860-2865. Richert, N.D., Aldwin, L., Nitecki, D., Gottesman, M.M., and Pastan, I. (1988) Biochemistry 21, 7607-7613. Chambers, T.C., McAvoy, E.M., Jacobs, J.W., and Eilon, G. (1990) J. Biol. Chem. In press. Nishizuka, Y. (1986) Science 233, 305-312. Fine, R.L., Patel, J., and Chabner, B.A. (1988) Proc. Natl. Acad. Sci. U.S.A. S5, 582-586. Kartner, N., Everndelle-Porelle, D., Bradley, G., and Ling, V. (1985) Nature 316, 820-823. Kikkawa, V., Minakuchi, R., Takai, Y., and Nishizuka, Y. (1983) Methods Enzymol. 99, 288-298. Bradford, M. (1976) Anal. Biochem. 2, 248-254. Hamada, H., and Tsuruo, T. (1988) J. Biol. Chem. 263, 1454-1458. Laemmli, U.K. (1970) Nature 227, 680-685. Fojo, A., Akiyana, S-I., Gottesman, M.M., and Pastan, I. (1988) Cancer Res. 45, 3002-3007. Kikkawa, U., Kishimoto, A., and Nishizuka, Y. (1989) Ann. Rev. Biochem. 5tJ, 31-44. (1986) In Phosphoinositides and Niedel, J.E., and Blackshear, P.J. Receptor Mechanisms (J. Putney, Jr., Ed.), pp. 47-88. Alan R. Liss, NY. Shearman, M. S., Sekiguchi, K., and Nishizuka, Y. (1989) Pharmacol. Rev. 4J, 211-237. Witters, L. A., Vater, C. A., and Lienhard, G. E. (1985) Nature, 315, 777-778. Sardet, C., Counillon, L., Franchi, A., and Pouyssegur, J. (1990) Science, 247, 723-725. Gros, P., Croop, J., and Housman, D. (1986) Cell 47, 371-380. Chen, C-J., Chin, J.E., Ueda, K., Clark, D.P., Pastan, I., Gottesman, M. M ., and Roninson, I.B. (1986) Cell 47, 381-389. Riordan, J. R., et al. (1989) Science 245, 1066-1073. 259
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 253-259
CORRELATION OF PROTEIN KINASE C TRANSLOCATION, P-GLYCOPROTEIN PHOSPHORYLATION AND REDUCED DRUG ACCUMULATION IN MULTIDRUG RESISTANT HUMAN KB CELLS Timothy
C. Chambers,
Ira
Chalikonda,
and Gabriel
Eilon
Department of Biological Chemistry Merck Sharp & Dohme Research Laboratories West Point, PA 19486 Received
April
12,
1990
SUMMARY: Treatment of drug-resistant human KB carcinoma cells (KB-Vl) with 0.2 PM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of 13H]vinblastine in KB-Vl cells was 0.8 -t 0.1 pmol/mg/30 min in the absence, and 1.9 +- 0.2 pmol/mg/30 min in the presence, of 20 PM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase G mediated phosphorylation stimulates the drug transport activity of P-glycoprotein. 01990 Academic Press, Inc.
The development limiting
factor
resistance
of in
involves
encodes
a transmembrane
protein
has
anticancer
a broad drugs
and
important. phosphorylation
treatment
the
amplification protein
In
cells
lines,
[see
Ca++/phospholipid-dependent
catalyzed
the (1,2)
human by
enzyme
One
family
This
in
of
reviews].
In
phosphorylation to
KB carcinoma
activated
which
efflux
recent
likely
kinase
of
P-glycoprotein.
for
is
protein
mechanism
multigene
undergoes
modification
a significant
energy-dependent
P-glycoprotein
multidrug-resistant is
rn&
KDa termed for
is
tumors.
the
150-170
specificity
resistant
agents
malignant of
post-translational the
chemotherapeutic of
of
substrate
cell
this
to
the
from
multidrug-resistant (3-7),
resistance
be
functionally
cell C cell
line, (PW
KB-Vl, (7),
membranes
a by
Abbreviations - PKC, protein kinase C; PM&, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; DMSO, PBS, phosphate buffered saline; dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl) dimethyl-ammonioll-propanesulfonate. Do06291x/90 253
All
$1.50
Copyright 0 1990 by Academic Press. Inc. rights of reproduction in any form reserved.
Vol.
169, No. 1, 1990
BIOCHEMICAL
diacylglycerol
and
using
cells
KB-VI
verapamil-induced in
tumor-promoting indicated
human
observations
suggest
may modulate
its
effects here
of that
drug
phorbol the
the
activity
esters phorbol
accumulation
breast
cancer
(7),
cells
possibility
transport
ester
phorbol that
[3H]vinblastine
drug-resistant
AND BIOPHYSICAL
that properties.
P-glycoprotein
(8).
Preliminary
studies
ester
treatment
reduced
similar
to earlier
Taken
(9).
phosphorylation Based
on phosphorylation of
RESEARCH COMMUNICATIONS
and is
together, of
on kinetic drug
stimulated
results
P-glycoprotein studies
accumulation, by
these
PKC
of
the
we show catalyzed
phosphorylation.
MATERIALS
AND METHODS
Materials - [32P]orthophosphate (8 mCi/ml), [gamma-32P]ATP (3000 Ci/mmol) obtained from Amersham and C219 and [3H]vinblastine (23 Ci/mmol) were monoclonal antibody, which recognizes P-glycoprotein (lo), was from Centocor. Cell Culture - Drug-sensitive KB-3 and drug-resistant KB-Vl cells were maintained in DMEM (4.5 g/l glucose) containing 10% fetal bovine serum and 10 ml-l Hepes, pH 7.3. KB-Vl cells were grown in the presence of 1 pg/ml vinblastine. For metabolic labeling with [32P], cells in 160 cm2 flasks were incubated for 4 hr in 10 ml of phosphate-free medium containing 0.8 mCi of [32P]orthophosphate. Prenaration of Extracts and PKC Determination - Near-confluent cultures of cells in 160 cm2 flasks were treated for various times with freshly prepared PMA dissolved in DMSO; controls received DMSO alone (0.1% v/v final concentration). Cells were gently scraped into ice-cold PBS, sedimented, and pellets suspended in 2 ml of buffer A (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM 2-mercaptoethanol, 1 mM EGTA, 1 mM EDTA and 1 mM PMSF). Cells were 15 min), debris removed by disrupted by nitrogen cavitation (400 p.s.i., centrifugation at 1,000 x g for 10 min, and a cytosolic fraction obtained by The membrane pellet was suspended in centrifugation at 100,000 x g for 1 hr. 1 ml of buffer A containing 1% w/v Triton X-100 and after 15 min on ice clarified by centrifugation (100,000 x g, 30 min). Samples were loaded onto 0.3 ml columns of DEAE-Sephacel equilibrated with buffer B (buffer A without sucrose), and, after washing with 5 ml buffer B, PKC was completely eluted with 2 ml 0.1 M NaCl in buffer B. PKC was assayed as described previously (11) by measuring incorporation of 32P from [gamma-32P]ATP to histone III-S in the presence of 0.5 mM CaC12, 50 pg/ml phosphatidyl-L-serine and 5 pg/ml diolein. Blanks, the values of which were subtracted, contained 1 mM EGTA instead of the cofactors. Protein was determined by the method of Bradford (12) using bovine serum albumin as a standard, P-glvconrotein Phosnhorvlation - 32P-labeled KB-Vl cells were treated with PMA for various periods, washed with ice-cold PBS, and membrane vesicles prepared as described previously (7). P-glycoprotein was solubilized by adding the detergent CHAPS to 1% w/v final concentration, as described (13). Immunoprecipitation with C219 monoclonal antibody and preparation of samples for SDS gel electrophoresis, performed using 6% acrylamide gels by the method of Laemmli (14), were carried out as detailed previously (7). Gels were stained with silver nitrate using reagents from BioRad Laboratories and quantitation of P-glycoprotein was performed by densitometric scanning of the gel. Radioactivity of excised bands was determined by scintillation counting. Drug Accumulation Assays - KB-Vl cells in drug-free medium were plated at 3 x 10s cells/well in 24-well Costar plates. PMA (0.2 pm> was added to 254
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
appropriate wells. The next day, PMA was added to additional wells for various periods up to 2.5 hr. Then, 30 min prior to processing, the medium of each well was removed and replaced with 0.5 ml DMRM containing 50 mM Hepes, pH 7.35, 0.1 pCi [3H]vinblastine, and, in some experiments, 20 PM verapamil. After 30 min incubation, the plates were washed three times in ice-cold PBS, the cells were detached with trypsin, and radioactivity determined (15). NaOH, 0.5 ml of 0.5 M, was added instead of trypsin to control groups of cells from each plate to determine protein content. RESULTS The kinetics Figure and
1.
of
The
relatively
translocation this
of
activity
to near
unchanged
for
cells
to
PMA
after of
relatively
was near
resulted
in
Membrane
PKC was decreased,
but
was still
elevated
relative
lines
of
PKC
(16,17).
PMA up to
to
1 PM,
PMA-induced
to
unexpected
results
in Further
confirmed down-regulation
2.5 the
the
30 min
membrane.
basal
4-fold.
cytosolic to
level
PKC maximum
(Fig.
long-term
complete
in During
3- to of
PKC (Table
1).
exposure
depletion,
experimentation, that
in
or using
KB-Vl I).
In
cells contrast,
Time (hi) Time-course of PMA-induced PKC translocatian in KB-Vl cells. Fieure 1. Cultures of cells were treated with 0.2 pM PMA for the times indicated, fractions prepaied and partially purified by cytosolic and membrane DEAE-Sephacel chromatography, and PKC activity determined, as described in "Materials and Methods."
255
in
resulted
relative
since
shown
for hr,
a return
level.
were
are
increased
basal
cell
resistant
to
PKC to activity
cells
stimulation
up
cytosolic
24 hr
numerous
of
after
treatment
PKC specific
PMA for
in KB-Vl
maximal
of
50% of
24 hr PMA treatment
down-regulation, concentrations
PKC translocation
periods
approximately
obtained,
The results to
which
membrane-associated
Exposure
levels
response,
of
time,
PMA-induced
is
Vol.
169, No. 1, 1990
Table
I.
Duration (hr)
BIOCHEMICAL
Effect
given
continuous characteristic
100 11 11 10
are averages
exposure
of
Figure
2.
4-fold
increase
increasing
times
since
membrane
treated
with
PMA-induced effects phosphate
of
exposure with
PKC
activity
PMA for
24 hr,
was
P-glycoprotein
phosphorylation
Accumulation KB-Vl as
cells
with
variation
ratio
in absolute
a rrlag't
period
amount
of
of drug
[3H]vinblastine preincubation utilized levels The
verapamil,
results,
of values
30 min,
of
PMA for an
accumulation presented
of
Figure
inverse the
3A.
drug
Figure
cells of
increase
in
to
the
increase
in
of
preincubation
time
of 40%
and
are
values,
since
exposure
of
cells
relative
to
control
examined show
24 hr.
(15),
that
to the
some
Following
was observed
60% after
of
expressed
experiments.
transport
3B, 256
In
the
control
time
LSH]vinblastine, in
phosphatase
1).
The results
and decreased of
with
PMA,
relationship
decreased
hr,
a
time-course.
in different
2.5
to
phosphorylation
similar
was observed
was
in
shown
observation),
(Fig.
to
versus
inhibitor of
in
and
are
protein
of
response
a magnitude
shown
to
level
as a function
accumulated
in
declined
(unpublished
high
a similar
a clear
resulted
corresponded
levels
cells
PMA-treated
accumulation with
of
PM PMA is
PMA
and
constant
Thus ) in
[SH]vinblastine
0.2
percentage
a
the
of
these
a relatively
and follows
1 hr
may be due
relatively
is
to
phosphorylation after
This
from
maintained.
PKC activity,
cells
Phosphorylation
PMA.
was
100 342 192 133
I).
content.
membranes
P-glycoprotein
membrane
KB-3
observed
to
100 56 74 59
experiments.
P-glycoprotein
were
in
associated
independent
of PKC (Table
of
Maximum
of three
100 293 0 0
drug-sensitive
depletion
The kinetics
and
PKC Activitv (% untreated) KB-3 KB-Vl Cvtosol Membrane Cvtosol Membrane
0 0.2 0.2 1.0
Values
RESEARCH COMMUNICATIONS
of shortand long-term exposure to PMA on activity distribution of PKC in KE cells
PMA Treatment Concentration (l&t)
0 0.5 24 24
activity
AND BIOPHYSICAL
between to
after We next
induce effect
PMA.
higher of
verapamil-induced
PMA.
Vol.
169, No. 1, 1990
BIOCHEMICAL
a --Fe-%
02
AND BIOPHYSICAL
--+-k-e4
--i-e-e‘l
03
Time (hr)
RESEARCH COMMUNICATIONS
Time (hr)
Time (hr)
Figure 2. Time-course of P-glycoprotein PMA-induced phosphorylation. 32P-labeled cells were treated with 0.2 PM PMA for the times indicated, membrane vesicles prepared, P-glycoprotein immunoprecipitated, and samples analyzed by 6% acrylamide SDS gel electrophoresis, silver staining and autoradiography. Quantitation of P-glycoprotein phosphorylation was based on comparison of radioactivity and amount of protein as described in "Materials and Methods." The inset shows typical results obtained after 0, 1 and 24 hr treatment with PMA. Fieure 3. Effect of PMA on [3H]vinblastine accumulation. KB-Vl cells ware preincubated with 0.2 pM PMA for the times indicated and 0.1 &i [sH]vinblastine added alone (A) or with 20 PM verapamil (B). After an additional 30 min, cells were washed and radioactivity and protein determined, as described in "Materials and Methods." In (A), 100% - 0.785 pmol/mg; in (B), 100% = 1.895 pmol/mg. Results are expressed for each time point as [mean (PMA)/mean (control)] x 100, n-4. The magnitude of the error bar at each time point was calculated from J[S,s + S,*), where S, - S.E. (control) and S, = S.E. (PMA), n-4.
[3H]vinblastine
accumulation
time-dependent
manner,
no
was
lag
period
shorter
times
verapamil
did
accumulation contrast
exposure.
not
affect
and It
effect
the on drug
significantly
the
should
of
showed at
of
be noted
that
3A.
that
the
maximal of
analog
0.1
the
in
this
a
case,
pronounced conditions
at used,
(7). effect
- 0.5
phorbol
in
more
under
P-glycoprotein
PMA treatment
However,
PMA was
of
concentrations
inactive
by
Fig.
effect
phosphorylation
curves
PMA,
reduced
to results
observed,
was observed to
significant
similar
of
Dose-response
was
FM.
of
PMA on
Additionally,
12,13-diacetate
drug in
had
no
accumulation. DISCUSSION
To examine cells
by
PKC
the (7),
role
of P-glycoprotein
the
effect
of
phosphorylation,
PMA on 257
[3H]vinblastine
catalyzed accumulation
in KB-VI was
Vol.
169, No. 1, 1990 Here
examined. relevant
we show
measure
of
by preincubation
membrane
significantly
the
are
related
indicate
was
of
maximal
a non-catalytic
after
level
a more
active
basal
thus
the
of
drugs
appears
Whereas
stimulation
not multiple
conformation
of
PMA,
and
then
Also,
ability by appeared
phosphorylation
of
While
on activity,
sites.
in
stimulated
reduced.
protein,
the
transport
the
account
PMA
resulting
be of
[aH]-
conditions,
Thus,
into
the
reduced
PKC,
to
to
take
at
same
P-glycoprotein.
1 hour,
does
significantly
activation, of
and biologically
and verapamil-induced
of phosphorylation
phosphorylation
promote
is
Under
exposure
effect
phosphorylation
differential
and
time
an accurate
Both
decreased.
by PKC.
to
P-glycoprotein
PMA.
transport
catalyzed
RESEARCH COMMUNICATIONS
P-glycoprotein,
phosphorylation to
phosphorylation
accumulation,
with
association,
P-glycoprotein
AND BIOPHYSICAL
of
cells
increased
directly
drug
activity
accumulation
induces
net
that
the
of
vinblastine
of
BIOCHEMICAL
this
may
measurement
the
possibility
of
phosphorylation
not
of
readily
may
reversed
by
dephosphorylation. To
further
define
P-glycoprotein studies
function,
utilizing
where
PKC is
since
KB-VI This
1).
was not to
the
it
role
will
depleted
by
are
prolonged
PKC was relatively property
of
KB-Vl
of
PKC-catalyzed
in
cells,
inhibit
progress. of
This
is
cells
in
this
in
phosphorylation,
and
to
but thus
cell
approach,
PMA, was
precluded
down-regulation
unusual
phenomenon
resistance
phosphorylation
An alternative
to PMA-induced
which
cells.
drug
to
exposure
resistant
by KB-3
development
of
be necessary
PKC inhibitors
exhibited
further
the
not
unique
appears line,
(Table
to
and
(16,17), be related
hence,
merits
investigation. Evidence
processes
for
has
been
neurotransmitters, glucose
and
proteins
cases
(19)
and
role
documented.
ions
such
as Ca for
phosphorylation Na+/H+
antiporter
of PKC in numerous
PKC activation
hormones,
responsible
some
a regulatory
enzymes ++
, H+,
some of h (20).
and other Na+,
these &
is
associated
agents,
K+ and processes
Cl-
258
to
with
(8,18).
release transport
The
been
e.g.,
our
and transport
and with
have
demonstrated, However,
secretion
knowledge,
of
transporter
identified glucose
of
and
in
transporter there
are
no
Vol.
169, No. 1, 1990
reports
in which
catalyzed here
a change
in
phosphorylation
suggest
multidrug with
BIOCHEMICAL
a key
of role
transporter,
other
transporters
for
AND BIOPHYSICAL
transport
activity
specific PKC in
RESEARCH COMMUNICATIONS
has
been
correlated
transporters.
The
results
phosphorylation
and
regulation
a protein
which
shares
(21,22;
see also
23).
significant
with
PKC
presented of
structural
the
homology
ACKNOWLEDGMENTS The providing typing
authors the
the
are cell
grateful lines
used
to
Drs.
M. Gottesman
in
this
study
and
and to
I.
Dorothy
Pastan
for
Bonenberger
kindly for
manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. ia. 19. 20. 21. 22. 23.
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