Photoinduced Inactivation of Protein Kinase C by Dequalinium Identifies the RACK-1-binding Domain as a Recognition Site*

Susan A. RotenbergDagger and Xiao-guang Sun

From the Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York 11367

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 PKCalpha , 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 (alpha  or beta ), with IC50 = 7-18 µM. UV/DECA treatment of synthetic peptides modeled after the RACK-1-binding site in the C2 region of PKCbeta 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 PKCalpha bound well to RACK-1, whereas UV/DECA-inactivated PKCalpha 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 PKCalpha translocation. Overall, this work identifies DECA as a tool that prevents PKC translocation by inhibiting formation of the PKC·RACK-1 complex.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 PKCbeta 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


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.   Structure of DECA.

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 PKCbeta that lie immediately C-terminal to the phorbol ester-binding domain (6). When bound to the C2 region of PKCalpha , 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 PKCbeta 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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Recombinant human PKCalpha and PKCbeta 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). [gamma -32P]ATP was purchased from NEN Life Science Products. Antiserum for PKCalpha 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 [gamma -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 [gamma -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 [gamma -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 lambda 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 PKCalpha (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 PKCalpha 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 PKCalpha 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 PKCalpha antiserum to demonstrate the position of native PKCalpha (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 PKCalpha (Santa Cruz Biotechnology).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Mode of Dequalinium-mediated Inhibition of PKCalpha with Respect to RACK-1-- The binding interactions of recombinant RACK-1 and DECA with purified human PKCalpha were first explored by classical kinetics. For these studies, conditions of reversible binding of DECA were employed. Pilot experiments established that measurements of PKCalpha 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).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Dixon plot of DECA-mediated inhibition of human PKCalpha as a function of RACK-1 concentration. The concentration dependence of DECA-mediated inhibition was measured with the following fixed concentrations of RACK-1: 0 (black-square), 100 (black-triangle), and 175 (bullet ) nM. Data points are each the average of triplicate measurements that were within 10% error. The plot is representative of three independent experiments.

Photoinduced Inactivation of PKC-- The following experiment demonstrates that irradiation with 365-nm light causes irreversible inhibition of PKCalpha 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.

Our findings indicate that a 5-min UV irradiation (365 nm) of mixtures of PKCalpha and DECA produced an inactive PKC·DECA covalent complex that persisted despite the dilution step. As shown in Fig. 3, UV irradiation of PKCalpha and DECA alone produced a dose-dependent decrease in the recoverable kinase activity as the DECA concentration was increased; with 20 µM DECA, only 5% of the total PKCalpha activity remained. The IC50 for UV-induced inactivation by DECA is 12 ± 5 µM (average of four independent experiments) and was consistent with Ki = 11.5 ± 5 µM for human PKCalpha (Fig. 2) and with IC50 = 11 µM previously obtained for recombinant rat PKCbeta 1 (10), the latter two values having been measured under conditions of reversible binding (i.e. no UV light). With recombinant human PKCbeta 1, UV-induced inactivation by DECA was again observed in the same concentration range, exhibiting IC50 = 14 µM ± 4, the average of four independent measurements (data not shown).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Photoinduced inactivation of human PKCalpha by DECA. PKCalpha was treated with DECA in the presence (black-square) and absence (square ) of UV light. The data points reflect the means of triplicate measurements that were within 10% error. The plot is representative of four independent experiments.

In control experiments that omitted UV irradiation, reversible binding of DECA to PKCalpha was verified by assay of PKCalpha activity incubated with increasing concentrations of DECA. It can be seen in Fig. 3 that, in the absence of UV irradiation, incubation of PKC with up to 20 µM DECA produced 80-100% of the total activity following a 24-fold dilution into the assay medium. Losses of enzymatic activity attributable to UV light alone were usually within 5-20% of the control PKC activity. Western blot analysis of the irradiated enzyme showed no degradation of pure PKCalpha protein following UV irradiation for 5 min (data not shown).

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 PKCbeta (Fig. 4A). The PKCbeta peptide sequences are closely aligned with the corresponding sequences of PKCalpha , 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).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Photolabeling of a C2 domain peptide. A, shown are the synthetic peptides that define the RACK-1-binding domain. Peptide sequences, modeled on the sequence of rat PKCbeta , are shown beneath the corresponding sequence of human PKCalpha ; differences between the two isoform sequences are underlined. B, peptide beta C2-4 or a control peptide was UV-irradiated for 45 min with DECA, followed by characterization of the products by HPLC. Peak detection was carried out at 333 nm, the lambda max of DECA. Chromatograms for peptide beta C2-4 (lower trace) and a control peptide (upper trace) are shown. AU, absorbance units.

Irradiation of 100 nmol of each peptide for 45 min in the presence of 12 nmol of DECA was carried out. Separation of the unreacted material from the product(s) was performed by HPLC with a gradient of water and acetonitrile, with detection at 333 nm (lambda max of free DECA). Of the three peptides tested, only beta C2-4 (amino acids 218-226) yielded a detectable signal at 333 nm, indicative of a DECA-peptide adduct (Fig. 4B, lower trace). While DECA itself typically eluted at 45% acetonitrile, this new peak eluted at 17% acetonitrile. This peak was not detected in the untreated peptide, peptide irradiated alone, or peptide mixed with DECA but not irradiated (data not shown). When a scrambled control peptide of beta C2-4 (Fig. 4A) was UV-irradiated with DECA under identical conditions, no major peaks at 17% acetonitrile were detected (Fig. 4B, upper trace). This finding implied a sequence-specific interaction of DECA with beta C2-4.

Sequence analysis (Table I) of the DECA-beta C2-4 adduct demonstrated that the modification had occurred at the tryptophan residue of beta C2-4, which corresponds to Trp223 of PKC. These results suggest that Trp223 is a site in the intact enzyme that can be covalently modified by DECA during PKC inactivation. It is of interest to note that this amino acid is strictly conserved in the C2 domains of the conventional PKC isoforms (alpha , beta 1/beta 2, and gamma ) (21).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Sequence analysis of unmodified and DECA-modified beta C2-4
Shown are the results from Edman degradation of the synthetic peptide beta C2-4 (Fig. 4A) that had been treated with or without UV/DECA and isolated by HPLC (Fig. 4B). See "Experimental Procedures" for details.

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 PKCalpha (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 PKCalpha was subjected to UV treatment with or without DECA, prior to the overlay step. For these experiments, DECA-inactivated PKCalpha was irreversibly inhibited by 70-90% as compared with the UV-treated control enzyme. As shown in Fig. 5A, UV-treated control PKCalpha was observed to bind well to endogenous RACK-1 (lane 2), whereas UV/DECA-inactivated PKCalpha displayed significantly reduced binding to RACK-1 (lane 3). These results demonstrate that, coincident with its inactivation by DECA, PKCalpha 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 PKCalpha .


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 5.   Overlay assays with DECA-inactivated human PKCalpha . The amount of inactivated or control PKCalpha bound to immobilized native RACK-1 was analyzed immunochemically. In A, RACK-1 (5 µg) was immunoprecipitated from human breast cells and immobilized on nitrocellulose, and in B, crude lysate (60 µg) from the same cell line was immobilized on nitrocellulose. For both A and B: lane 1, RACK-1 protein detected with anti-RACK-1 antisera; lane 2, RACK-1 overlaid with UV-treated control PKCalpha , followed by detection with anti-PKCalpha antisera; lane 3, RACK-1 overlaid with PKCalpha inactivated by UV/DECA treatment, followed by detection with anti-PKCalpha antisera. The UV/DECA-treated enzyme had been inactivated by >80% as compared with the PKC activity for the UV-irradiated control sample. For B: lane 4, immobilized cell lysate control (without an overlay) detected directly with anti-PKCalpha antisera. Results are representative of three independent experiments.

Additional high affinity interactions with human PKCalpha were observed with immobilized whole lysates of MDA-MB-231 cells (Fig. 5B, lane 2) that were also eliminated when the enzyme was inactivated by UV/DECA treatment (lane 3). This result suggests that, in addition to RACK-1, UV/DECA treatment blocks the ability of PKCalpha to bind other PKC-binding proteins as well, possibly localized in different subcellular compartments (reviewed in Ref. 22).

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, PKCalpha 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 PKCeta , PKCiota , 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 PKCalpha 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 PKCalpha 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.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 6.   DECA inhibits phorbol ester-induced translocation of PKCalpha in human breast adenocarcinoma cells. Cells were pretreated with 10 µM DECA or Me2SO for 30 min and irradiated with UV light (5 min) as indicated, followed by incubation with 1 µM TPA or Me2SO for 1 h. Western blot analysis was conducted for particulate (lanes 1-5) and soluble (lanes 6-10) fractions isolated from cells treated under the following conditions: Me2SO (control; lanes 1 and 6), Me2SO + TPA only (lanes 2 and 7), UV/Me2SO + TPA (lanes 3 and 8), DECA + TPA (lanes 4 and 9), and UV/DECA + TPA (lanes 5 and 10). A representative experiment is shown.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 PKCalpha activity (Fig. 3) with concomitant loss of high affinity binding by PKCalpha 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 PKCalpha .

Evidence of covalent modification by DECA of the RACK-1-binding domain in PKC was presented with the synthetic peptide beta 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 beta 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 beta 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 PKCbeta 1 (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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Rotenberg, S. A., and Weinstein, I. B. (1991) in Biochemical and Molecular Aspects of Selected Cancers (Pretlow, T. G., II, and Pretlow, T. P., eds), Vol. I, pp. 25-73, Academic Press, Orlando, FL
  2. Newton, A. C. (1995) J. Biol. Chem. 270, 28495-28498[Free Full Text]
  3. Mochly-Rosen, D., Khaner, H., and Lopez, J. (1991) Proc. Natl. Acad Sci. U. S. A. 88, 3997-4000[Abstract]
  4. Mochly-Rosen, D., Miller, K. G., Scheller, R. H., Khaner, H., Lopez, J., Smith, B. L. (1992) Biochemistry 31, 8120-8124[Medline] [Order article via Infotrieve]
  5. Smith, B. L., and Mochly-Rosen, D. (1992) Biochem. Biophys. Res. Commun. 188, 1235-1240[Medline] [Order article via Infotrieve]
  6. Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187[Abstract/Free Full Text]
  7. Ron, D., Chen, C.-H., Caldwell, J., Jamieson, L., Orr, E., and Mochly-Rosen, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 839-843[Abstract]
  8. Weiss, M. J., Wong, J. R., Ha, C. S., Bleday, R., Salem, R. R., Steele, G. D., Chen, L. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5444-5448[Abstract]
  9. Chen, L. B. (1988) Annu. Rev. Cell Biol. 4, 155-181[CrossRef]
  10. Rotenberg, S. A., Smiley, S., Ueffing, M., Krauss, R. S., Chen, L. B., Weinstein, I. B. (1990) Cancer Res. 50, 677-685[Abstract]
  11. Nakadate, T., Jeng, A. Y., and Blumberg, P. M. (1988) Biochem. Pharmacol. 37, 1541-1545[Medline] [Order article via Infotrieve]
  12. Zhuo, S., and Allison, W. S. (1988) Biochem. Biophys. Res. Commun. 152, 968-972[Medline] [Order article via Infotrieve]
  13. Taylor, E. P. (1951) J. Chem. Soc. (Lond.) 1150-1157
  14. Young, P. M., and Wheat, T. E. (1990) Pept. Res. 3, 287-292[Medline] [Order article via Infotrieve]
  15. Rosenberg, I. M. (1996) Protein Analysis and Purification, p. 38, Birkhaeuser Press, Inc., Boston
  16. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
  17. Segel, I. H. (1975) Enzyme Kinetics, p. 110, John Wiley & Sons, Inc., New York
  18. Quest, A. F. G., Bardes, E. S. G., and Bell, R. M. (1994) J. Biol. Chem. 269, 2961-2970[Abstract/Free Full Text]
  19. Shao, X., Davletov, B. A., Sutton, R. B., Sudhof, T. C., Rizo, J. (1996) Science 273, 248-251[Abstract]
  20. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Sudhof, T. C., Sprang, S. R. (1995) Cell 80, 929-938[Medline] [Order article via Infotrieve]
  21. Parker, P. J. (1992) in Protein Kinase C, Current Concepts and Future Perspectives (Lester, D. S., and Epand, R. M., eds), pp. 3-24, Ellis Horwood Ltd., West Sussex, United Kingdom
  22. Faux, M. C., and Scott, J. D. (1996) Trends Biochem. Sci. 21, 312-315[CrossRef][Medline] [Order article via Infotrieve]
  23. Zhuo, S., Paik, S. R., Register, J. A., Allison, W. S. (1993) Biochemistry 32, 2219-2227[Medline] [Order article via Infotrieve]
  24. Orr, J. W., and Newton, A. C. (1994) J. Biol. Chem. 269, 8383-8387[Abstract/Free Full Text]
  25. Emma, D., Gamboa, G., Liao, S., Berman, M., DiSaia, P., and Manetta, A. (1993) Gynecol. Oncol. 50, 38-44[CrossRef][Medline] [Order article via Infotrieve]
  26. Helige, C., Smolle, J., Zellnig, G., Fink-Puches, R., Kerl, H., and Tritthart, H. A. (1993) Eur. J. Cancer 29A, 124-128
  27. Lester, D. S. (1992) in Protein Kinase C, Current Concepts and Future Perspectives (Lester, D. S., and Epand, R. M., eds), pp. 80-101, Ellis Horwood Ltd., West Sussex, United Kingdom
  28. Bazzi, M. D., and Nelsestuen, G. L. (1989) Biochemistry 28, 9317-9323[Medline] [Order article via Infotrieve]
  29. Mochly-Rosen, D. (1995) Science 268, 247-251[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.