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J. Biol. Chem., Vol. 279, Issue 50, 52714-52725, December 10, 2004
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(PKC) from 14-3-3
-BINDING SITES*
¶
From the
Department of Biochemistry and Division of Biology, Kansas State University, Manhattan, Kansas 66506
Received for publication, March 18, 2004 , and in revised form, September 29, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PKC)to the membrane and the phosphorylation of Cx43 by PKC and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565–572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160–1168). Gap junction activity in lens epithelial cells is regulated by PKC-mediated phosphorylation of Cx43. PKC activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKC-C1B domain that are responsible for its interaction with 14-3-3
. Two sites, C1B1 (residues 101–112) and C1B5 (residues 141–151), are located within the C1 domain of PKC. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3, resulting in the release of endogenous cellular PKC from 14-3-3, in vivo or in vitro, in activation of PKC enzyme activity, phosphorylation of PKC, in the subsequent translocation of PKC to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 µM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKC are regulated by its binding to 14-3-3 at the C1B domain of PKC. Synthetic peptides corresponding to these regions of PKC successfully competed for the binding of 14-3-3 to endogenous PKC, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, , , 
, 
, 
, and 
) have been identified (13, 14). 14-3-3 proteins are most abundant in the cytoplasm, and all actions of 14-3-3 proteins involve binding to target proteins.
Binding of 14-3-3 to its partners depends on the phosphorylation of specific Ser or Thr residues in the recognition domains. By using peptides derived from Raf-1, Muslin et al. (15) identified the optimal motif for association of target proteins with 14-3-3 proteins as RSXpSXP, where pS represents phosphorylated Ser and X represents any amino acid. Yaffe et al. (16) and Rittinger et al. (17) reported that there are two preferred 14-3-3-binding motifs, RSXpSXP and RXXXpSXP. Most of the 14-3-3 partners identified to date contain one of these motifs. PKC has two potential 14-3-3-binding motifs within the C1B domain.
PKC plays an integral part in the cell signaling machinery. PKC comprises a family of serine/threonine kinases, which contain at least 11 isoforms and can be found in most cell types (18). PKCs phosphorylate growth factor receptors, ion channels, structural proteins, and gap junction connexin proteins (19). Classical PKCs (including 
, , 1, and 2) translocate to membranes after both diacylglycerol (DAG) and calcium binding to C1 and C2 domains, respectively (18).
For activation to occur, the PKCs are often covalently modified by phosphorylation on serine and threonine residues. This is thought to induce a conformational change in the PKC protein, resulting in an enhanced interaction with membranes (21). Several signaling proteins use two membrane-targeting modules to reversibly regulate their activation state and cellular distribution (21, 22). Each module of the classical PKC binds membranes with low affinity, with tight membrane binding achieved when both domains are membrane-bound. When the affinity of one module for membranes is dependent on stimulus-dependent changes in membrane composition (generation of lipid secondary messengers) or protein structure (phosphorylation), the interaction with the membrane can be reversibly regulated (21). PKC has served as a model for the reversible regulation of membrane location by the concerted action of two membrane-targeting modules. PKC has two C1 domain repeats, C1A and C1B. However, unlike the classical PKC isoform, both the C1A and C1B domain have high affinity for DAG, even at low Ca2+ levels (23). This enzyme can be activated by very low DAG levels (2). In addition, the C1 domain of PKC has been hypothesized to act as an oxidative stress switch through modifications of the Cys residues without a DAG or Ca2+ signal (48). We have reported recently that PKC from whole lens is activated by exogenous hydrogen peroxide (20). This activation could be regulated by the interaction of the PKC with membrane docking proteins such as 14-3-3.
The C1 domain of PKC is a Cys-rich region from 36 to 151 of PKC residues. It is present as a tandem repeat, designated C1A and C1B (24). The NMR structure of the C1B domain of PKC reveals a globular domain with two pulled -sheets forming the ligand binding pocket (25). Two Zn2+ atoms are coordinated by His and Cys residues at opposite ends of the primary sequence, helping to stabilize the domain. Several reports (26, 27) suggest that docking of PKC to 14-3-3 involves the C1B domain of PKC.
There are two possible 14-3-3 consensus binding regions in the C1B of PKC for 14-3-3 binding, residues 101–112 and 141–151. In order to identify which 14-3-3 isoform interacts with PKC, we first examined the interaction between PKC and 14-3-3 isoforms, , , and . PKC is usually cytoplasmic and inactive and translocates to membranes upon binding of lipids and Ca2+ (28, 29). But as reported previously (2, 23), PKC will translocate with only a DAG signal. PKCs can bind to a scaffold protein, such as 14-3-3, which is thought to bind in the cytoplasm and can either activate or inhibit an individual PKC isoform (26).
Previously in our laboratory, we have shown that IGF-I or LEDGF decreases gap junction activity through the activation of PKC, causing a PKC-mediated phosphorylation of connexin 43 (Cx43), and then a decrease in gap junction activity (1, 2). PKC-mediated phosphorylation of Cx43 and subsequent decrease of gap junction activity could be regulated through the release of PKC from 14-3-3, followed by the activation and translocation of PKC to the membrane, and then by subsequent PKC-catalyzed phosphorylation of Cx43. In this study, we have investigated the interaction of PKC with 14-3-3 and have identified the residues on PKC which interact with 14-3-3. We report the competition of endogenous PKC binding to 14-3-3 in live cells or in vitro by C1 domain peptides. This resulted in the endogenous PKC translocation to the plasma membrane, in the phosphorylation Cx43, and in subsequent decreases in gap junction activity and gap junction plaque formation in vivo.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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were purchased from BD Biosciences. Polyclonal antibodies against 14-3-3, -, and -, Raf-1, and protein A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit antibody against phosphoserine was purchased from Zymed Laboratories Inc.. Anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase and PepTag nonradioactive protein kinase C assay system was purchased from Promega (Madison, WI). Dulbecco's modified Eagle's medium (low glucose), trypsin-EDTA, gentamicin, and penicillin/streptomycin were purchased from Invitrogen. Fetal bovine serum was purchased from Atlanta Biologicals (Norcross, GA). LEDGF was from Dr. Toshimichi Shinohara (Omaha, NE). GST-14-3-3 clone was generously provided by Dr. M. B. Yaffe (Massachusetts Institute of Technology). PKC clone was kindly provided by Dr. Peggy Zelenka (National Eye Institute, Bethesda, MD). IGF-I and protease inhibitor mixture were purchased from Sigma. Lucifer yellow and rhodamine dextran were purchased from Molecular Probes (Eugene, OR). Cells and Cell Cultures—N/N1003A rabbit lens epithelial cells were cultured in 75-cm2 flasks (Midwest Scientific) in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 50 µg/ml gentamicin. The cells were grown at 37 °C in an atmosphere of 90% air and 10% CO2 and used for experiments when they reached 90% confluency.
Endogenous DAG Assay—Cells were cultured in 75-cm2 flasks and dosed at 10 ng/ml LEDGF for various times. Sample preparation and radioenzymatic assays were performed according to the manufacturer's instructions for the sn-1,2-diacylglycerol assay reagents system (Amersham Biosciences). Total lipids were extracted with chloroform/methanol and used as the substrates for DAG kinase. Phosphatidic acid (PA). which was labeled by 32P. was produced by the reaction. The product mixtures were separated by thin layer chromatography, and zones corresponding to 32P-PA were visualized by autoradiography of the dried plates overnight. The spots containing 32P-PA were quantitated by scintillation counting. Endogenous DAG levels were calculated from DAG standard curves.
PKC Enzyme Activity—Activation of the PKC isoform was determined by measurement of enzyme activity. Cells were grown ± LEDGF for 10, 20, or 30 min. For peptide studies, cells were grown ± 100 µM peptides for 2 h prior to the addition of LEDGF. PKC activity was determined by use of a PepTag assay kit. Equal protein from whole cell extracts was immunoprecipitated with PKC antisera at 4 °C for 4 h as described previously (2). Immunoprecipitated PKC-agarose bead complexes were incubated with the PKC reaction mixture according to the manufacturer's instructions for the PepTag assay kit. Reactions were stopped, and fluorescent PepTag peptides (PKC reaction products) were resolved by agarose gel electrophoresis and visualized under UV light. The phosphorylated peptide bands were excised and measured as described at 570 nm (2).
Synthetic Peptides—Two possible sites of the C1 domain of PKC, residues 101–112 and 141–151, were synthesized by Sigma Genosys. Synthetic peptide C1B1 sequence is HKFRLHSYSSPT; C1B5 sequence is RCVRSVPSLCG, and nonspecific peptide is SFGKCHLYPKV.
Measurement of Uptake of Synthetic Peptides Using HPLC and Mass Spectrometry—7.2 x 106 cells were cultured in 75-cm2 flasks and treated with 100 µM of C1B5 (equivalent to 11.76 µg per flask) for 2 h. 5 µl of 250 µl of whole cell extract of treated cells was dissolved in 295 µl of 0.1% (v/v) trifluoroacetic acid in water and then used for HPLC purification and quantification, using a 1.6 mm x 25 cm C18 reverse phase column (Vydac, Torrance, CA) with a 5–60% (v/v) linear gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid, over a period of 60 min. Identification was confirmed by MALDI-TOF mass spectrometry. The amount of uptake of peptides by the cells was determined from the standard curve of C1B5 peptide alone, using the identical HPLC gradient.
Western Blot Analyses—Cell lysates were prepared by lysing cells in ice-cold lysis buffer consisting of 20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.5% (v/v) Triton X-100, 25 µg/ml aprotinin, and 25 µg/ml leupeptin. 20 µg of total protein were boiled and resolved on 12% SDS-PAGE minigels under reducing conditions. Proteins were electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat dried milk and probed with diluted antibodies overnight as indicated. Bands were revealed by chemiluminescence reaction (ECL, Pierce). Blots were routinely stripped in a denaturing buffer (0.5 M Tris-HCl (pH 8.6), 10% SDS, 1% -mercaptoethanol) and reprobed with antibodies for loading control. In this and subsequent experiments Western blots are quantified by image analyses using Un-scan-it gel software(Orem, UT).
Co-immunoprecipitation—Cells were cultured to 90% confluency in 75-cm2 tissue culture flasks and then treated with 10 ng/ml LEDGF for 30 min (1). The cells were harvested with 1 ml of ice-cold cell lysis buffer, containing 20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.5% (v/v) Triton X-100, 25 µg/ml aprotinin, and 25 µg/ml leupeptin. The cells were homogenized and centrifuged at 13,000 rpm (20,000 x g) for 30 min at 4 °C. 500 µg of total soluble proteins (cell lysate) from treated or control cell cultures were used for co-immunoprecipitation. Lysates were first pre-cleared by treatment with 10 µl of protein A/G-agarose for 1 h at 4 °C. After centrifugation at 2,000 rpm, the supernatant was collected, and anti-mouse PKC was added to cell lysates to a final concentration of 5 µg/ml of antisera and then incubated overnight at 4 °C with constant mixing. 20 µl of protein A/G-agarose beads were added to the mixture, and the mixture was further incubated for another 2 h on ice. After centrifugation, supernatants were discarded, and the precipitates were washed three times with phosphate-buffered saline (PBS). Loading buffer was added to the washed precipitates, which were subjected to Western blot analyses with various antibodies as described.
In Vitro Competition Assay—GST-14-3-3 was expressed in Escherichia coli and purified using anti-GST-agarose beads (Santa Cruz Biotechnology). In vitro transcribed/translated PKC was made by using TNT-coupled reticulocyte systems (Promega). GST-14-3-3-agarose beads were washed and incubated with 5 µl of in vitro transcribed/translated PKC for 1 h. Various concentrations of C1B5 were added to the complex for an additional 1 h. The complex was then washed and separated by 12% SDS-PAGE as described above.
Translocation—Cells were treated with 100 µM of peptides for 2 h prior to addition of LEDGF. As a negative control, cells were treated with a nonspecific peptide at the same dose and time. Cells were harvested in 50 mM Tris (pH 7.5) and 20 mM MgCl2 (2). The cell lysates were centrifuged for 1 h at 35,000 rpm (100,000 x g) at 4 °C. The sample was separated into supernatant (cytosolic) and pellet (membrane) fractions and analyzed by Western blot as described previously above. The separated proteins were transferred to nitrocellulose membranes and probed with anti-PKC antibodies (1:1000) or other antisera as indicated.
Gap Junction Activity—Scrape loading/dye transfer assay (SL/DT) is a common technique to measure gap junction activity. The SL/DT assay provides a rapid and simple measurement of the gap junction communication between cultured cells in a monolayer, and it is based on introducing a transient tear in the plasma membrane without affecting cell viability or colony-forming ability. Lucifer Yellow, which is an intensely fluorescent 4-aminophthalimide, is used as the tracer dye in the SL/DT assay. It does not diffuse through intact plasma membranes, and its low molecular weight (Mr 457.2) permits its transmission from one cell to another presumably through patent gap junctions. Rhodamine dextran is a high molecular weight polymer (Mr 10,000), and it cannot diffuse through intact plasma membranes nor cross the gap junctions; therefore, it is used as a control to differentiate dye transfer from diffusion because of cell damage (30). Cells were then fixed with 2.5% (w/v) paraformaldehyde, and the numbers of cells transferring dye are counted using a standard fluorescent or laser-scanning confocal microscope.
Cells were grown to 90% confluency on glass coverslips and washed three times with PBS. Then 2.5 ml of 1% (w/v) lucifer yellow and 0.75% (w/v) rhodamine dextran (control for dye transfer) in PBS were added at the center of the coverslip. Using a razor blade, two cuts crossing each other were made on the coverslip, passing through the dye in the center of the coverslip. These cells were incubated with the dye for 1 min, and then the cells were washed three times with PBS. This was followed by incubating the cells in tissue culture medium for 10 min at room temperature, allowing dye to transfer from cell to cell. The cells were then washed three times with PBS and fixed with 2.5% paraformaldehyde in PBS for 30 min at room temperature. Dye transfer was evaluated by examining the cells under a fluorescent microscope. For quantitation, the extent of dye transfer was estimated by counting the number of fluorescent-labeled cells, lucifer yellow and rhodamine dextran, in the microscopic field under x20 magnification in the center intersection of the cut (31). Each treatment was repeated three times, and each slide was counted three times. Results are expressed as the average number of cells that allows dye transfer from cell to cell in 10 min.
Gap Junction Plaques—Another method of measuring gap junction activity is by measurements of plaques, large cluster of up to 10,000 gap junction hexamer channels (connexons). Plaques form more functional channels and can be viewed by confocal microscopy of the cell surface. Cells were treated with 100 µM peptides for 2 h prior to LEDGF exposure at various times. The cells were then fixed for 10 min with 2.5% (w/v) paraformaldehyde in PBS. This was followed by incubation with 1 ml of 0.15% (v/v) Triton X-100 for 20 min and blocking of the nonspecific sites with 3% (w/v) bovine serum albumin in PBS for 2 h at room temperature. Anti-Cx43 monoclonal antibodies were diluted in blocking buffer (3% bovine serum albumin) and then added to the fixed cells and incubated for 18 h at 4 °C. The working dilution of the primary anti-Cx43 (host-rabbit) was 1:250. The secondary antisera, Alexa Fluor 568 (Molecular Probes) which is goat anti-rabbit and has an excitation/emission wavelength of 578/603, was used with the working concentration of 5 µg/ml in blocking buffer. The cells were mounted on slides with 1% (v/v) glycerol in PBS. Slides were examined using a laser-scanning confocal microscope (Nikon C1), focusing on the top of the plasma membrane. The number of plaques per cell was determined under x100 objective lens.
Statistical Analysis—The level of significance (see * in figure legends) was considered at p < 0.05 using Student's t test analysis. All data are presented as mean ± S.E. of at least three independent experiments from different batches of cultures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Enzyme Activity, and PKC Autophosphorylation—We have shown previously that PKC is a major regulator of gap junction assembly/disassembly when both PKC and - are activated in lens epithelial cells (34) and that LEDGF can cause the translocation of PKC, not PKC, from the cytosol to the membrane. Additionally, the activation of PKC by LEDGF induced increases in DAG can cause a decrease in gap junction activity through the phosphorylation of Cx43 (1). A schematic diagram of PKC activation is shown in Fig. 10.
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200 pmol/mg of fresh tissue, a 2-fold increase when compared with basal levels of 100 pmol/mg.
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is quite sensitive to small changes in the natural activator, DAG, because of the presence of two tandem zinc-binding motifs in the C1 domain which bind DAG at very low DAG levels (1–6 nM) (33). Because LEDGF can increase DAG levels, does the elevation of DAG in the presence of LEDGF increase PKC enzyme activity as well? Cells were treated with 10 ng/ml of LEDGF for 10, 20, or 30 min. The results show a consistent 2-fold increase in PKC enzyme activity similar to a 2-fold increase of DAG levels compared with basal levels (Fig. 1B) within a similar time range. We also examined the effects of an increase in concentration of LEDGF. 20 ng/ml LEDGF for 10 min can increase PKC enzyme activity by 2.75-fold compared with control (Fig. 1C).
PKC activation is often accompanied by phosphorylation of the specific PKC on serine residues (2). To demonstrate PKC, phosphorylation cells were treated with 0, 10, or 20 ng/ml of LEDGF for 10 min. Co-immunoprecipitation was performed. The results show a 1.75- and 2.0-fold increase in PKC phosphorylation in the presence of 10 and 20 ng/ml LEDGF compared with control, respectively (Fig. 1D). Thus, production of DAG, activation of PKC enzyme activity, and PKC phosphorylation occur within a similar time and dose range after LEDGF addition to the cells.
14-3-3 Protein Co-immunoprecipitates with PKC, and This Is Reversed after LEDGF or IGF-1 Treatment—In order to identify which 14-3-3 isoform interacts with PKC, co-immunoprecipitation studies were done. Previously we have shown that growth factors such as IGF-I or LEDGF can induce PKC translocation to membranes, interactions of the PKC with the gap junction protein, connexin 43, and this results in the inhibition of gap junction activity in lens epithelial cells (1, 2). 14-3-3 has been reported to interact with PKCs and can either cause the inhibition or activation of PKC enzyme activity (26). Here we identify the 14-3-3 isoform that interacts with PKC in cells. Cells were treated with growth factors, IGF-I or LEDGF, at optimal times of 15 min and concentrations of 10 ng/ml LEDGF or 25 ng/ml IGF-I (1, 2). PKC was immunoprecipitated from cells by using protein A/G PLUS-agarose beads in the presence of anti-PKC antibodies (Fig. 2). The nitrocellulose membranes were probed with anti-14-3-3 (, , or ) polyclonal antibodies. Western blots from cells, which were treated with 10 ng/ml LEDGF for 15 min or 25 ng/ml IGF-I for 15 min, showed a decrease in co-immunoprecipitation of PKC and 14-3-3 compared with control without treatment (Fig. 2A). The results suggest that LEDGF or IGF-I treatment resulted in a release of PKC from 14-3-3. There were no significant changes of interaction between PKC and 14-3-3 and 14-3-3 under these same treatments (Fig. 2A). The time course for growth factor effects on binding of PKC to 14-3-3 was examined. As a negative control, cells were treated with GST alone because the recombinant LEDGF is fused with GST. GST was used at 10 ng/ml for 15 min. The results show that after a 20-min incubation of cells with LEDGF, there is a decreased interaction of PKC with 14-3-3 (Fig. 2A); however, IGF-I decreased this interaction of PKC with 14-3-3 after only a 5-min incubation (Fig. 2B). The graphs in Fig. 2B are the total average pixel intensity of three experiments. To date, it has not been reported that PKC directly interacts with 14-3-3 or is released from 14-3-3 upon treatment with growth factors. Our results clearly demonstrate that both LEDGF and IGF-I can induce the release of endogenous PKC from 14-3-3. Because 14-3-3 was released from PKC after LEDGF or IGF-I treatment, further studies concentrated on the interaction of these two proteins.
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Cause Release of Endogenous PKC from Endogenous 14-3-3—To identify the interaction sites on PKC for 14-3-3, we identified two potential binding peptides corresponding to putative interaction sites for PKC to 14-3-3 (34, 35). PKC is a classical PKC that has two cysteine-rich regions within the C1-DAG-binding domain. As discussed above these regions both have high affinity for DAG. Because the C1 domain peptides do not have PKC catalytic activity alone, we hypothesized that the presence of excessive C1 domain peptides would compete for endogenous PKC binding to endogenous 14-3-3, resulting in the displacement of the endogenous PKC from the 14-3-3 as measurable by a reduced co-immunoprecipitation reaction. This could result in enhanced interaction of the PKC with membranes. The sequence of PKC reveals two possible 14-3-3-binding consensus sites at residues 101–112 and 141–151 (Fig. 3A). We examined the association between endogenous PKC and endogenous 14-3-3 in the presence of these peptides in vivo. Cells were treated with 100 µM of C1B1 or C1B5 peptides for 2 h (see Fig. 3A for sequences). Whole cell extracts were immunoprecipitated with anti-PKC antibody overnight, followed by immunoblot analysis with anti-14-3-3 antibody (Fig. 3B). Endogenous PKC was released from 14-3-3 in the presence of C1B1 or C1B5 peptides at 100 µM; however, although a greater effect was observed in the presence of both C1B1 and C1B5 peptides together (100 µM each) (Fig. 3B), this was not statistically significant from individual peptides. The results demonstrate that 100 µM peptides for 2 h can compete endogenous PKC from binding to endogenous 14-3-3. 14-3-3 was also co-immunoprecipitated with Raf-1, and its association was not affected by the presence of C1B1 or C1B5 peptides (Fig. 3C). The same blot was re-probed with anti-PKC antibodies, showing that C1B1 or C1B5 peptides could compete with endogenous PKC for the binding of 14-3-3 (Fig. 3C, bottom). These results suggest that PKC binds to 14-3-3 at the C1B1 and/or C1B5 region within the C1B domain of PKC.
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17% of the C1B5 peptide was internalized (results not shown). This suggests that the cellular concentration after treatment for 2 h with 100 µM peptide was 4.72 nmol per cell.
C1 Domain Peptides Cause a Translocation of Endogenous PKC to Membrane Fractions—Because the C1B peptides can compete with endogenous PKC for 14-3-3-binding sites, we determined the effects on the cellular location of endogenous PKC. Endogenous PKC once released from 14-3-3 should translocate from the cytosol to the membrane in the presence of C1B1 and/or C1B5. This could result in enhanced interactions with membrane proteins such as Cx43. Peptides were incubated with cells for 2 h at 100 µM prior to separation of these cells into the supernatant and pellet (membrane) fractions. The results show that C1B1 and/or C1B5 peptides can increase PKC translocation from the cytosol (supernatant) to membrane fractions (pellet) by 50–76% (Fig. 4). 100 µM of C1B1 and/or C1B5 peptides is sufficient to translocate PKC to the membrane. The total amount of 14-3-3 was not affected in whole cell extracts during this time. (Note: 14-3-3 proteins are always cytosolic.) Quantitation of three independent experiments from different batches of cell cultures is graphed as total average pixel intensity in Fig. 4 (bottom).
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to PKC. GST-14-3-3 was expressed in E. coli and purified by using anti-GST-agarose beads. GST-14-3-3 was incubated with in vitro transcribed/translated PKC with various concentrations of C1B1 peptides for 1 h. The results show that 10 µM of C1B5 peptide can compete 49% of PKC from GST-14-3-3 compared with control without addition of C1B5 peptides (Fig. 5).
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Enzyme Activity—To examine the effect of C1B1 or C1B5 peptides on PKC enzyme activity, we also tested peptide stimulation of PKC enzyme activity after treatment of cells with 100 µM of each peptide. Endogenous PKC was immunoprecipitated, and its enzyme activity was measured by use of the PKC peptide substrate assay (2). The enzyme activity was normalized by calibration of the relative level of phosphorylated substrates to the relative amount of PKC in the immunoprecipitate, as determined by Western blotting, and was expressed as percentage of nontreated specific PKC activity (Fig. 6). The results demonstrate that treatment of cells with 100 µM C1B1 or C1B5 peptides significantly increased PKC activity in cells. Cells treated with both C1B1 and C1B5 did not show further increased PKC activity compared with treatment with C1B1 or C1B5 alone. Thus, either C1B1 or C1B5 peptides can cause an increase in PKC enzyme activity and are not additive for enzyme activation (Fig. 6) or for effects on co-immunoprecipitation (Fig. 3) of a peptide substrate. However, the C1B5 peptide does show more effect on PKC translocation (Fig. 4), but the values are not statistically significant.
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, and This Results in an Increase in Phosphorylation of Cx43 on Ser-368 —PKC interacts with Cx43 and subsequently phosphorylates Cx43 on Ser-368 of the C terminus (1, 2, 34). We examined the effects of peptide treatment of cells on the phosphorylation of Cx43 on Ser-368. Cells were treated with 100 µM of C1B1, C1B5, or nonspecific peptides for 2 h. Western blots demonstrated that C1B1 or C1B5 peptide treatment can increase the phosphorylation of Cx43 on Ser-368 (Fig. 7, top band). The average total pixel intensity of three experiments shows that 100 µM C1B1 or C1B5 peptides can increase phosphorylation of Cx43 on Ser-368 by 3- or 2-fold, respectively (Fig. 7, bottom band). However, the nonspecific peptide has no effect on Cx43 phosphorylation. Cx43 levels did not change during this time.
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by LEDGF or IGF-I decreases the gap junction dye transfer activity through the phosphorylation of Cx43 in cells. Since C1B1 and/or C1B5 peptide treatment resulted in the release of PKC from 14-3-3, does the presence of these peptides also cause a decrease in gap junction activity? Regulation of gap junction activity was measured as dye transfer in cells in the presence and/or absence of peptides (Fig. 8). Cells were treated with 100 µM of C1B1, C1B5, or both peptides for 2 h. The results show that C1B1 or C1B5 treatment can decrease gap junction activity in vivo compared with control cells without peptide or with a nonspecific peptide (Fig. 8). Quantitation of six different experiments are graphed as the average number of cells taking up the dye in the microscopic field under x20 magnification in the center intersection of the cut (Fig. 8, bottom). Rhodamine dextran has been subtracted for these results.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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has a unique activation mechanism for a classical PKC. It appears that both the C1A and C1B domains are exposed and that this PKC isoform can be activated without increased Ca2+ (23). Growth factors such as LEDGF or IGF-I can cause the translocation of PKC in cells (1, 2). LEDGF, a lens and retina growth factor (36), can increase the endogenous DAG level, PKC enzyme activity, and phosphorylation level of PKC (Fig. 1). DAG binds specifically to a hydrophobic groove in the tandem C1 domains, C1A and C1B, with equal affinity for PKC, resulting in an increased translocation to membranes (23). Immunoprecipitation assays suggest a physical interaction of PKC and Cx43 which results in phosphorylation of Cx43 on Ser-368 (37). PKC directly phosphorylates Cx43 on Ser-368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication (37). We have shown that treatment of cells with C1 synthetic peptides results in the activation of endogenous PKC resulting in phosphorylation of Cx43 on Ser-368 (Fig. 7). This was accompanied by a decrease in gap junction dye transfer and plaques.
We provide additional evidence that PKC binds to the 14-3-3 isoform (Fig. 2) through the C1B domain of PKC. Upon activation of PKC after LEDGF or IGF-1 exposure, PKC is released from 14-3-3 (Fig. 2B). PKC contains two potential binding sites for 14-3-3. The synthetic peptides corresponding to these binding sites were synthesized and shown to be the sites of interaction of PKC and 14-3-3 by both in vivo and in vitro incubation (Figs. 3, 4, 5). Either C1B1 or C1B5 appears to compete and is not additive, suggesting that competition at either site on PKC may be sufficient to remove endogenous PKC from the 14-3-3. Removal of the PKC from the 14-3-3 could result in an increased interaction with membrane proteins through an as yet undefined mechanism. The PKC could undergo a conformational change, or potential phosphorylation sites could be exposed after removal from the 14-3-3. There are several potential phosphorylation sites within the PKC/14-3-3-binding sites that could be masked when the enzyme is docked to the 14-3-3. Future studies will entail mutations at these regions.
Among the 14-3-3-binding partners, some proteins, like Raf-1, bind to almost all 14-3-3 isoforms (35, 38). We tested for specificity of the peptides with Raf-1-14-3-3 interactions (Fig. 3C). Neither the C1B1 nor C1B5 peptide altered the interaction of Raf-1 and 14-3-3, indicating that these peptides were specific for PKC/14-3-3 interactions. We have demonstrated for the first time that C1B1 and/or C1B5 regions of PKC are essential for the interaction with 14-3-3. This model is similar to that proposed for the regulation of Raf-1 activity (38) in which 14-3-3 binds to inactive Raf-1 at the consensus sequence RSXpSXP to maintain it in an inactive state, and disruption of this sequence binding is sufficient to permit activation of Raf-1 (37). In addition, Wang et al. (39), using random peptide phage display technology to identify and characterize the peptide for 14-3-3 proteins, demonstrated that an 18-mer (R18) peptide inhibited the interaction of 14-3-3 with Raf-1. Both Raf-1 and PKC are then covalently modified by phosphorylation, although PKC appears to accomplish some of this via autophosphorylation. Under normal conditions in a cell, this would not occur (competition with a synthetic peptide), thus DAG is the usual activation signal. Our results, however, demonstrate that synthetic peptides may prove useful to activate endogenous PKC and, thus, inhibit gap junctions. This would be useful in preventing ischemic stroke damage which occurs partially via open gap junctions (40).
We have demonstrated that the activity and localization of endogenous PKC is regulated by the binding of 14-3-3 to the C1B domain in the N-terminal region of PKC, specifically at residues 101–112 and 141–151. Competition assays showed that the physical interaction of 14-3-3 and PKC can be competed in vitro with C1B5 peptides (Fig. 5). Once the binding of PKC to 14-3-3 is prevented by competition with synthetic peptides, in vivo, the endogenous PKC is removed from 14-3-3, enzyme activity increases, and translocation to the membrane increases. At present we are not sure of the order of this sequence of events. PKC-14-3-3 complexes may be in equilibrium with a constant on and off PKC conformation, and the changes initiated upon competition with excess peptide (i.e. phosphorylation and translocation) may prevent the necessary interaction of the enzyme with the 14-3-3 required to sequester this enzyme away from a target. This could expose membrane-binding sites long enough for enzyme/substrate interactions to occur. The demonstration that PKC interacts with Cx43 and regulates gap junction activity suggests that synthetic peptides of the PKC C1 domain can provide a novel and specific method of causing gap junction inhibition. In our hands, peptides used at 100 µM only resulted in a level of 4.7 nmol/cell. This is sufficiently high enough to result in some activation of PKC. However, almost total gap junction activity was decreased. This is because activation of an enzyme with high catalytic rates such as PKC would result in high phosphorylation of Cx43 (1, 2), the substrate; thus, an enzyme activation amplified cascade may result. Therefore, a 50% competition of 14-3-3/PKC interactions may be sufficient to close the gap junctions. A 50% effect was observed by either peptide at 100 µM, added exogenously to live cells. This lack of total PKC activation is most likely due to the fact that cells have a rapid and robust PKC turn-off mechanism including, among other things, dephosphorylation of PKC by phosphatase (41).
In brain tissues PKC appears to prevent brain ischemia and is suggested as a target for ischemic preconditioning (40, 42–44). However, the role of PKC in ischemic preconditioning was not clearly understood until recently (1, 2), when we demonstrated that PKC is involved in gap junction control. Ischemic damage occurs in part through open gap junctions that allow passage of small molecules (about 1 kDa or less) from damaged cells to healthy ones, resulting in cell death of neighboring cells. Thus, control of gap junctions is essential in the prevention of ischemic damage (45–47). Our data using synthetic peptides of the C1 domain of PKC suggest that this approach can be used in preventing ischemic damage, because these peptides specifically target PKC and inhibit gap junction activity.
The results of this study have important implications both for the understanding of how PKC regulates gap junction activity and for designing novel approaches to modulate gap junction control. Future studies will be directed toward identification of amino acids within the C1B1 and/or C1B5 peptides, which play a direct role in the interaction with 14-3-3. Site-directed mutagenesis of Ser-107 in the C1B1 peptide and Ser-145 in the C1B5 peptide will be examined for the analysis of 14-3-3-binding sites to the endogenous PKC and the roles of phosphorylation at these residues. Moreover, we will examine the effect of these mutants on the inhibition of gap junction activity. Clearly understanding the role of the C1B binding domain is a key to understanding how PKC is involved in the control of gap junction activity in vivo.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, KS 66506. Tel.: 785-532-7009; Fax: 785-532-7278; E-mail: dtak{at}ksu.edu.
1 The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; IGF, insulin-like growth factor; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PA, phosphatidic acid; SL/DT, scrape loading/dye transfer; Cx43, connexin 43; LEDGF, lens epithelium-derived growth factor.
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