From the Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York 11367
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ABSTRACT |
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1,1-Decamethylenebis-4-aminoquinaldinium
diiodide (DECA; dequalinium) is an anti-tumor agent and protein kinase
C (PKC) inhibitor whose mechanism of action with PKC is unknown. This
study reports that with human PKC
, DECA exhibited competitive
inhibition (Ki = 11.5 ± 5 µM)
with respect to RACK-1 (receptor for activated C kinase-1), an adaptor protein
that has been proposed to bind activated PKC following translocation
(Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol.
Chem. 270, 24180-24187). When exposed to UV light, DECA
covalently modified and irreversibly inhibited PKC (
or
), with
IC50 = 7-18 µM. UV/DECA treatment of
synthetic peptides modeled after the RACK-1-binding site in the C2
region of PKC
induced modification of
Ser218-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr226, but
not of a control peptide. This modification occurred at a tryptophan
residue (Trp223) that is conserved in all conventional PKC
isoforms. In overlay assays with native RACK-1 that had been
immobilized on nitrocellulose, UV-treated control PKC
bound well to
RACK-1, whereas UV/DECA-inactivated PKC
had reduced binding
activity. The significance of these findings is shown with
adenocarcinoma cells, which, when pretreated with 10 µM
DECA and UV light, exhibited diminished
12-O-tetradecanoylphorbol-13-acetate-induced PKC
translocation. Overall, this work identifies DECA as a tool that
prevents PKC translocation by inhibiting formation of the PKC·RACK-1
complex.
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INTRODUCTION |
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Protein kinase C (PKC),1 a monomeric Ca2+- and phospholipid-dependent serine/threonine protein kinase, plays a critical role in growth factor-activated signaling and malignant transformation (1, 2). A current focus of investigation is to identify a specific inhibitor of PKC that is targeted to a structural element of the protein that influences PKC behavior in cells. Because the regulatory domain of PKC distinguishes it from other protein kinases, it is this segment of PKC, contained in the N-terminal portion of the protein, that attracts the greatest interest. This domain houses the binding regions for PKC-activating ligands such as phosphatidylserine (PS), diacylglycerol (DAG), and phorbol esters (C1 region) as well as for Ca2+ (C2 region).
1,1-Decamethylenebis-4-aminoquinaldinium diiodide (DECA;
dequalinium) (Fig. 1) is a
dicationic lipophilic PKC inhibitor whose mechanism of action remains
unknown. Identified as a potent anti-tumor agent in several animal
models (8) and as a drug that is selectively accumulated by cancer
cells (9), DECA inhibits PKC
1 activity in vitro and in
murine embryo fibroblasts at low micromolar concentrations (10).
Observations that associate DECA action with both the catalytic and
regulatory domains of PKC (10) relegate this compound to a general
category of PKC inhibitor defined as "mixed-type" (11). However,
previous findings have shown that, in its interaction with the
regulatory domain, DECA is not competitive with PS (10) and phorbol
ester or
Ca2+.2
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A candidate DECA recognition site in the regulatory domain with
functional significance is the RACK-1-binding site. Identified as the
receptor for activated C
kinase, RACK-1 is a PKC-binding protein found in the
Triton-X-insoluble material of the particulate fraction (3-7). It is
believed to recognize specific sites in the C2 region of PKC that
lie immediately C-terminal to the phorbol ester-binding domain (6).
When bound to the C2 region of PKC
, this adaptor protein has been
proposed to stabilize the activated form of the enzyme, but acts as
neither substrate nor inhibitor. Synthetic peptides modeled after the
RACK-1-binding domain in the C2 domain of PKC
were observed to block
the association of PKC with RACK-1 in vitro and to inhibit
PKC translocation in cardiac myocytes and Xenopus oocytes
(6).
The photochemically induced behavior of DECA makes possible a novel approach to elucidate the inhibitory mechanism of this interesting compound. Irradiation by long-wave UV light (365 nm) in the presence of a target protein can cause covalent modification of that protein, as originally described with the mitochondrial F1-ATPase (12). The present study harnesses the photolyzable behavior of DECA to show that the RACK-1-binding domain of PKC is a recognition site for DECA.
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant human PKC and PKC
1 were
purchased from PanVera Corp. (Madison, WI); dioleoylphosphatidylserine
and DAG were obtained from Avanti Polar Lipids (Alabaster, AL); and TPA
was purchased from LC Services (Woburn, MA). [
-32P]ATP
was purchased from NEN Life Science Products. Antiserum for PKC
was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse IgM
anti-RACK-1 monoclonal antiserum was obtained from Transduction
Laboratories (Lexington, KY). Conjugated and unconjugated rabbit
anti-mouse IgM antisera were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The modified pseudosubstrate peptide RFARKGSLRQKNV (Ser25 peptide), which represents
amino acids 19-31 of PKC, was synthesized by N. Pileggi (Protein Core
Facility, Columbia University). Peptide sequence analysis of the
DECA-peptide adduct was carried out by Dr. J. Farmar (New York Blood
Center, New York).
Synthesis of DECA-- Dequalinium diiodide was synthesized by an established method (10, 13). Elemental analysis was as follows. Calculated: 50.74% C, 5.63% H, 7.89% N; found: 50.63% C, 5.57% H, 7.62% N (Desert Analytics, Tucson, AZ). It should be noted that commercially available dequalinium salts contain a major impurity.
Assay of PKC--
PKC activity was taken as the difference in
32Pi transferred from
[-32P]ATP to the modified pseudosubstrate peptide
RFARKGSLRQKNV in the presence and absence of lipid activators (20 µg
of PS and 1 µg of DAG) as described previously (10). Assays were
conducted in triplicate, and the results were averaged. Values were
typically within 10% error. Curve fitting was carried out with
Cricketgraph® software.
UV-induced Inactivation of PKC by DECA--
The UV light-induced
photolysis of DECA was carried out with a Blak-Ray 365-nm ultraviolet
lamp (Model UVL-56) that was positioned 2.3 ± 0.1 cm above a
porcelain welled plate seated in an ice bath. From this distance, the
lamp delivered an intensity of 2700 microwatts/cm2. Into
each well was placed the medium (50 µl) to be irradiated: PKC in
buffer (20 mM Tris, pH 7.4, 2 mM EDTA, and 2 mM EGTA) plus DECA at the specified concentration or 4%
(v/v) Me2SO for controls. Control experiments demonstrated
that this concentration of Me2SO did not affect PKC
activity. Following 5 min of irradiation, an aliquot of the enzyme was
diluted into the assay medium as described above but without
[-32P]ATP, producing a 24-fold dilution of the
irradiated enzyme. Assay tubes were equilibrated in a 30 °C water
bath for 5 min prior to initiation of the phosphotransferase reaction
by addition of [
-32P]ATP.
Photolabeling of C2 Domain Peptides by
Dequalinium--
Synthetic peptides (1 mM) modeled after a
C2 domain segment were UV-irradiated with 120 µM DECA for
45 min, and the product(s) were analyzed by HPLC on a Waters Delta-Pak
C18 reverse-phase column (2 × 150 mm; 300 Å) at a
flow rate of 0.4 ml/min. Peptides were eluted with a binary gradient of
0-30% solvent B in 60 min, where solvent A was 6 mM HCl
in water and solvent B was 6 mM HCl in acetonitrile (14).
Detection of the DECA-peptide adduct was carried out at 333 nm, the
max of DECA. Peptide sequence analysis was carried out
on an Applied Biosystems Model 477A sequenator.
Isolation of RACK-1 Protein-- Recombinant RACK-1 protein was expressed as a fusion protein with maltose-binding protein tagged with a FLAG sequence (DTKDDDDK). The fusion protein was isolated by affinity chromatography on a 0.5-ml amylose column and cleaved from its fusion partner as described previously (6). RACK-1 was purified from the cleavage products by buffer replacement (to remove the eluant), followed by affinity chromatography on amylose; purified RACK-1 protein was recovered in the flow-through material. The isolation of recombinant RACK-1 as a 35-kDa protein was confirmed by Western blotting with anti-RACK monoclonal antiserum and a rabbit anti-mouse IgM-horseradish peroxidase conjugate. Protein content was determined by Bio-Rad protein reagent with bovine serum albumin as a standard.
Native RACK-1 protein was immunoprecipitated from whole cell lysates obtained from human breast adenocarcinoma MDA-MB-231 cells by an established method (15). Mouse anti-RACK-1 monoclonal antiserum was used as primary antibody, followed by rabbit anti-mouse IgM as secondary antibody. The immunocomplexes were collected with protein A-agarose beads (Vector Labs, Inc.), and the immunoprecipitated protein was prepared for SDS-polyacrylamide gel electrophoresis.Overlay Assay--
Immunoprecipitated native human RACK-1 (5 µg) or whole cell lysates (60 µg) were subjected to 9%
SDS-polyacrylamide gel electrophoresis, followed by electrophoretic
transfer to a nitrocellulose filter by an established method (16). The
nitrocellulose filter was blocked overnight at 4 °C in a 5%
powdered milk suspension containing 0.1% Tween 20 and 0.01% sodium
azide. The overlay assay utilized individual lanes, either by excising
them as nitrocellulose strips or by sequestering the lanes of an intact
filter in a Deca-Probe (Pharmacia Biotech Inc.). Purified recombinant
human PKC (0.27 µg) was UV-irradiated for 5 min in the presence of
1 mM DECA or 10% (v/v) Me2SO as a vehicle
control. Following irradiation, each PKC
sample was tested for
enzymatic activity in triplicate and for high affinity binding to
immobilized native RACK-1. For the binding assay, each lane was treated
with the PKC sample (3.5 nM) in Ca2+, DAG, and
PS and incubated with gentle rocking for 30 min at room temperature as
described previously (6). Lanes that were used in the overlay step were
washed and detected with PKC
antisera. Additional lanes that
excluded the overlay step were analyzed immunochemically with either
anti-RACK-1 antisera to demonstrate the RACK-1 signal at 33 kDa or with
PKC
antiserum to demonstrate the position of native PKC
(77 kDa)
in cell lysates.
Cell Culture and Translocation Assay--
MDA-MB-231 cells were
cultured to 60-70% confluence on 150-mm plates in complete medium
(Iscove's modified Dulbecco's medium with L-glutamine,
10% fetal bovine serum, and 1% penicillin/streptomycin). In
serum-free medium, plated cells were treated with 10 µM
DECA (or Me2SO) for 30 min at room temperature and then, as
indicated, subjected to 5 min of irradiation using a 365-nm UV lamp
(American Ultraviolet Co., Murray Hill, NJ) at a distance that
delivered 2000 microwatts/cm2. Cells were washed with
phosphate-buffered saline and replaced with complete medium containing
TPA (1 µM) or 0.1% (v/v) Me2SO for 1 h
at room temperature. Cells were washed with phosphate-buffered saline
and harvested, and cell pellets were lysed in 200 µl of homogenization buffer (20 mM Tris, pH 7.4, 250 mM sucrose, 2 mM EDTA, 10 mM
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/liter leupeptin, 10 µg/liter soybean trypsin inhibitor, and 10 µg/ml calpain inhibitor II) by Dounce homogenization (40 strokes),
followed by sonication (3 × 3 s). The homogenate was centrifuged at 200 × g for 10 min to remove unlysed
cells, and the resulting supernatant was centrifuged at 100,000 × g for 1 h. The supernatant (soluble fraction) was
analyzed for protein content and prepared for electrophoresis. The
pellet (particulate fraction) was resuspended in buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 5 mM
EGTA, and 5 mM 2-mercaptoethanol plus protease inhibitors) containing 0.1% Triton X-100, placed on ice for 30 min with periodic Vortex mixing, and prepared for electrophoresis. Soluble (10 µg/lane) and particulate (5 µg/lane) fractions were analyzed by Western blotting and immunochemically stained for PKC (Santa Cruz
Biotechnology).
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RESULTS |
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Mode of Dequalinium-mediated Inhibition of PKC with Respect to
RACK-1--
The binding interactions of recombinant RACK-1 and DECA
with purified human PKC
were first explored by classical kinetics. For these studies, conditions of reversible binding of DECA were employed. Pilot experiments established that measurements of PKC
catalytic activity lay on the initial velocity component of the reaction curve. The Dixon plot (17) shown in Fig.
2 depicts concentration-dependent inhibition by DECA conducted in the
presence of fixed nanomolar concentrations of recombinant RACK-1. The
results reveal that DECA-mediated inhibition is competitive with
respect to RACK-1. The Ki value for inhibition was
11.5 ± 5 µM (obtained by averaging three
independent experiments).
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Photoinduced Inactivation of PKC--
The following experiment
demonstrates that irradiation with 365-nm light causes irreversible
inhibition of PKC activity by DECA, as revealed by dilution of the
enzyme-DECA complex into the assay medium. Dilution would be expected
to dissociate a reversibly bound complex such that full enzymatic
activity is recovered. However, if PKC is irreversibly inhibited, the
dilution step should be ineffective.
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Photolabeling of Synthetic C2 Domain Peptides--
In view of
results that indicate that DECA competes with RACK-1 for the
RACK-1-binding domain, it was next considered that synthetic peptides
previously shown to define the RACK-1-binding domain of PKC (6) may be
covalently modified by DECA when irradiated with UV light. For these
experiments, highly purified peptides were employed whose sequences had
been modeled after the corresponding amino acid sequences of the C2
domain of PKC (Fig. 4A).
The PKC
peptide sequences are closely aligned with the corresponding
sequences of PKC
, although minor deviations are evident, as shown by
the underlined residues. The segment of the C2 domain
described by these sequences lies C-terminal to the phorbol
ester-binding domain (amino acids 102-144) (18) and within the general
region that has been ascribed to Ca2+ binding (amino acids
187-249) (19, 20).
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Overlay Experiments--
The ability of the DECA-inactivated
enzyme to bind RACK-1 in an overlay assay was addressed. Under
conditions that are optimal for RACK-1 binding (3, 7), samples of
recombinant human PKC (in the presence of PS/Ca2+/DAG)
were incubated with strips of nitrocellulose representing individual
lanes of a membrane onto which native human RACK-1 (immunoprecipitated
from MDA-MB-231 cells) had been immobilized by electrophoretic
transfer, as described under "Experimental Procedures." Purified
recombinant human PKC
was subjected to UV treatment with or without
DECA, prior to the overlay step. For these experiments,
DECA-inactivated PKC
was irreversibly inhibited by 70-90% as
compared with the UV-treated control enzyme. As shown in Fig.
5A, UV-treated control PKC
was observed to bind well to endogenous RACK-1 (lane 2),
whereas UV/DECA-inactivated PKC
displayed significantly reduced
binding to RACK-1 (lane 3). These results demonstrate that,
coincident with its inactivation by DECA, PKC
is rendered unable to
bind RACK-1. This finding is consistent with the idea that DECA and
RACK-1 recognize the same binding site in PKC
.
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DECA Reduces Translocation of PKC in Human Breast Adenocarcinoma
Cells--
Because activated PKC is thought to bind RACK-1 as a
consequence of translocation (6), the ability of DECA to intervene in
phorbol ester-induced translocation of PKC was tested in human breast
adenocarcinoma cells (MDA-MB-231). In these cells, PKC is the most
abundant isoform as well as the only isoform with a C2 domain. (The
other isoforms found by Western blotting in these cells were PKC
,
PKC
, and PKCµ (data not shown).) Following pretreatment with DECA
(10 µM) for 30 min and subsequent UV irradiation for 5 min, the cells were stimulated with the tumor-promoting phorbol ester
TPA for 1 h to induce translocation of cytosolic PKC into cellular
membranes. The Western blot shown in Fig.
6 indicates that preincubation with
UV/DECA followed by TPA treatment produced a marked retention of PKC
in the soluble fraction (lane 10) as compared with no
retention when UV treatment alone preceded TPA addition (lane
8). Similarly, a decrease in TPA-induced PKC
recruitment to the
particulate fraction was evident with UV/DECA treatment (Fig. 6,
lane 5) as compared with UV treatment alone prior to TPA
addition (lane 3). In both particulate and soluble fractions, analogous effects of DECA alone (without UV light) were
observed, but were less pronounced than the effects achieved by a
combination of UV light and DECA (Fig. 6, compare lanes 4 and 5 or lanes 9 and 10). UV
treatment alone had no independent effect on TPA-induced PKC
translocation in either fraction (Fig. 6, compare lanes 2 and 3 or lanes 7 and 8). These
findings demonstrate that, at low micromolar doses, UV light
potentiates the effect of DECA in suppressing PKC translocation.
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DISCUSSION |
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In this work, we demonstrate that, under reversible binding
conditions, DECA and RACK-1 display competitive binding kinetics. When
irradiated with UV light (365 nm), DECA causes irreversible inhibition
of recombinant human PKC activity (Fig. 3) with concomitant loss of
high affinity binding by PKC
to immobilized RACK-1 (Fig. 5A) and other unidentified proteins (Fig. 5B).
The biological significance of PKC inactivation by DECA was observed in
cells whose treatment with UV/DECA significantly diminished TPA-induced translocation of PKC
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Evidence of covalent modification by DECA of the RACK-1-binding domain
in PKC was presented with the synthetic peptide C2-4 (amino acids
218-226). This peptide is one of three peptides known to define the
RACK-1-binding site (Fig. 4A) and has been shown to
interfere with PKC/RACK-1 binding interactions in cardiac myocytes and
Xenopus (6). The modification of a tryptophan residue in
C2-4 by DECA is consistent with an earlier study with mitochondrial F1-ATPase in which a phenylalanine residue was derivatized
by this compound (23). The preferential modification of
C2-4 as compared with the scrambled control peptide underscores the sequence specificity of the modification event and argues against random reactivity with tryptophan residues in the holoenzyme.
Previous work with the catalytic fragment of PKC showed that DECA causes inhibition of phosphotransferase activity with the same potency as with the intact enzyme (10), signifying that a critical target site is located in the catalytic domain. Neither Mg2+·ATP nor peptide substrate is competitive with DECA, however (10).2
Other work from this laboratory has shown that DECA is ineffective as an inhibitor up to 1 mM with other protein kinases such as the cAMP-dependent protein kinase catalytic subunit, the calmodulin-dependent myosin light chain kinase, and pp60src.3 The lack of effect with cAMP-dependent protein kinase is particularly compelling since the catalytic domains of PKC and cAMP-dependent protein kinase exhibit almost 50% sequence homology and are believed to have very similar three-dimensional structures (24).
The specificity of DECA for PKC may be explained in part by coincident
binding interactions with both the catalytic domain (unidentified) and
the regulatory domain (at the RACK-1-binding site). Inhibition of PKC
by DECA involving the two domains could be due to either one or two
molecules of DECA binding per molecule of PKC. A two-molecule binding
model depicts the binding of one DECA molecule to the RACK-1-binding
domain, which occurs in parallel with the binding of a second molecule
of DECA to its target site in the catalytic domain. In a one-molecule
binding model, the aminoquinaldinium moieties of DECA bind
simultaneously to the RACK-1-binding domain and catalytic domain of the
intact enzyme. Thus, the bipartite structure of DECA (Fig. 1)
participates in a unique two-point contact such that the regulatory and
catalytic domains are tethered. This idea is lent support by the
structure-activity relationships of DECA analogues and their inhibition
of PKC1 (10), from which it is known that the 10-carbon distance
that exists between the aminoquinaldinium moieties is a critical
determinant of the inhibitory potency of DECA, thus implicating both
ends of the molecule in DECA-mediated inhibition. A one-molecule
binding model is also strengthened by the present study, which showed that nanomolar concentrations of RACK-1 (C2 domain interaction) were
sufficient to diminish the DECA-mediated inhibition of PKC activity
(catalytic domain interaction) (Fig. 2).
An important finding of this work is that interaction of DECA with the RACK-1-binding domain of PKC produces significant inhibition of translocation. This result may explain the ability of DECA to delay the morphological response by fibroblasts treated with phorbol ester (10), to act as an anti-tumor agent (8, 25), and to inhibit cell motility and invasion (26). That DECA did not entirely abolish TPA-induced translocation but merely suppressed it may be explained by the artificially strong stimulus provided by TPA (27) that also produces direct interactions of PKC with membrane lipids (28). In view of the proposal that RACK-1 binds activated PKC (29), the present study suggests that DECA impedes this association (as well as other unidentified high affinity protein/protein interactions), thereby blocking downstream PKC-directed signaling events.
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ACKNOWLEDGEMENTS |
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We thank Dr. Daria Mochly-Rosen (Stanford University Medical Center) for generously providing the C2 domain peptides and the bacterial RACK-1 expression system used in this study. Stimulating discussions with Prof. Corinne A. Michels, Prof. William S. Allison, and Dr. Susan Jaken are gratefully acknowledged. Technical assistance was provided by Regina Sullivan.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA60618 and by the Gustavus and Louise Pfeiffer Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, Queens College, City University of New York, 65-30 Kissena Blvd., Flushing, NY 11367. Tel.: 718-997-4133; Fax: 718-997-5531; E-mail: rotenber{at}qcvaxa.acc.qc.edu.
1
The abbreviations used are: PKC, protein kinase
C; PS, phosphatidylserine; DAG, diacylglycerol; DECA,
1,1-decamethylenebis-4-aminoquinaldinium diiodide; TPA,
12-O-tetradecanoylphorbol-13-acetate;
Me2SO, dimethyl sulfoxide; HPLC, high pressure liquid
chromatography.
2 S. A. Rotenberg, unpublished results.
3 A. Francis-Smith, S. A. Rotenberg, unpublished results.
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REFERENCES |
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