Direct association of RhoA with specific domains of PKC-{alpha}

Haiyan Pang and Khalil N. Bitar

Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan

Submitted 28 July 2004 ; accepted in final form 23 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)-{alpha} and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC-{alpha} involved in direct interaction with RhoA, His-tagged PKC-{alpha} proteins of individual domains and different combinations of PKC-{alpha} domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC-{alpha} in this study. The data indicate that RhoA directly bound to full-length PKC-{alpha}, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC-{alpha} [PKC-{alpha} (C1)] (70.48 ± 20.78% above control), PKC-{alpha} (C2) (72.26 ± 29.96% above control), and PKC-{alpha} (C4) (90.58 ± 26.79% above control), but not to PKC-{alpha} (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-{alpha}, PKC-{alpha} (C2, C3, and C4), and PKC-{alpha} (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC-{alpha} (C1, C2, and C3) or to PKC-{alpha} (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC-{alpha} (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-{alpha}. The studies using cells transfected with truncated forms of PKC-{alpha} indicate that PKC-{alpha} (C1 and C2), PKC-{alpha} (C1, C2, and C3), and PKC-{alpha} (C2 and C3) did not associate with RhoA. Only full-length PKC-{alpha}, PKC-{alpha} (C2, C3, and C4), and PKC-{alpha} (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC-{alpha} with RhoA may require the C4 domain.

domains; histidine; fusion proteins


RHO IS A SMALL GUANOSINE TRIPHOSPHATASE (GTPase) involved in smooth muscle cell proliferation and migration (2, 19, 21, 39). Rho also regulates smooth muscle contraction (12, 41). Activation of RhoA leads to the formation of contractile bundles of actin and myosin, known as stress fibers, and of focal adhesions (1, 19). Rho regulates myosin light-chain (MLC) phosphorylation through its effectors, Rho-associated kinase (Rho kinase), and the myosin-binding subunit (MBS) (14, 17, 18). Activated Rho kinase phosphorylates MBS, thereby inactivating myosin phosphatase (3, 18). Rho kinase has been shown to phosphorylate the 20-kDa regulatory light chain of myosin (MLC20) directly (16). Activation of Rho/Rho kinase is essential for agonist-induced contraction of gastrointestinal smooth muscle (31, 42).

Protein kinase C (PKC) is a family of multifunctional, phospholipid-dependent protein kinases involved in various signal transduction processes, including cell growth and differentiation events (28). Mammalian PKC-{alpha} consists of 672 amino acids and is distributed in all tissues (25). PKC-{alpha} plays important roles in the control of major cellular functions (26). It also plays an important role in smooth muscle contraction (13, 46).

PKC is characterized by 1) an NH2-terminal regulatory domain, which mediates membrane association and activation; 2) a small central hinge region; and 3) a COOH-terminal catalytic domain containing the active site (26). There are four conserved domains of PKC: C1–C4. The C1 domain contains a Cys-rich motif, which forms the diacylglycerol (DAG)/phorbol ester-binding site. The C2 domain contains the Ca2+-binding site. The C2 domain mediates Ca2+-dependent binding to the membrane lipid phosphatidylserine (PS), which induces a conformational change that activates the enzyme. The C3 domain possesses the binding site for ATP, which is the phosphate donor for phosphotransferase activity. The C4 domain is the catalytic domain that possesses the binding site for substrates (36).

Several studies have demonstrated a convergence between PKC and Rho GTPase-regulated signaling pathways. Evidence has shown that this might involve a close association between PKC and Rho GTPases (5, 6, 11, 30, 39). Previous studies conducted in our laboratory have shown that RhoA modulates agonist-induced signal transduction cascades in smooth muscle contraction and that RhoA colocalizes on membranes with the actin-binding protein heat shock protein 27 (HSP27) as observed using confocal microscopy (45). PKC-{alpha} also translocates to the membrane in two ways: through its association with HSP27 and with RhoA on the membrane during agonist-induced contraction. PKC-{alpha} and RhoA coimmunoprecipitate in the particulate fraction of colon smooth muscle cells in response to different contractile agonists (3). The association of RhoA with PKC-{alpha} is due to a direct interaction between these two molecules (33). Cross-talk between the PKC-{alpha} pathway and the RhoA pathway may be the key signaling event of the contractile activation of smooth muscle. The aim of this study was to determine the domains of PKC-{alpha} that are involved in direct interaction with RhoA. Therefore, His-tagged individual domains and different combinations of domains of PKC-{alpha} were constructed. The proteins produced from these constructs and fusion proteins glutathione-S-transferase (GST)-RhoA were used to perform an in vitro binding assay. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC-{alpha} in this study. The data derived from the in vitro binding assay indicated that RhoA directly bound to full-length PKC-{alpha}. RhoA bound to the C1 domain of PKC-{alpha} [PKC-{alpha} (C1)], PKC-{alpha} (C2), and PKC-{alpha} (C4), but not to PKC-{alpha} (C3). RhoA bound to the different combinations of PKC-{alpha} domains PKC-{alpha} (C2, C3, and C4), PKC-{alpha} (C3 and C4), and PKC-{alpha} (C1 and C2), but not to PKC-{alpha} (C1, C2, and C3) or to PKC-{alpha} (C2 and C3). These results suggest that the C3 domain could exert an inhibitory effect on the binding of RhoA with PKC-{alpha} (C1, C2, and C3) or PKC-{alpha} (C2 and C3). On the other hand, when the C1 domain was removed, PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) bound well to RhoA. The constructs of PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) might possibly mimic the active form of PKC-{alpha} in vitro. The studies using smooth muscle cells transfected with truncated forms of PKC-{alpha} have indicated that PKC-{alpha} (C1 and C2), PKC-{alpha} (C1, C2, and C3), or PKC-{alpha} (C2 and C3) did not associate with RhoA. Only full-length PKC-{alpha}, PKC-{alpha} (C2, C3, and C4), and PKC-{alpha} (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC-{alpha} with RhoA may require the C4 domain.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

GST-RhoA protein, full-length PKC-{alpha} protein, and mouse monoclonal anti-RhoA antibody were purchased from Cytoskeleton (Denver, CO). The plasmids pBlueBac/PKC-{alpha} were purchased from the American Type Culture Collection (Manassas, VA). Plasmid pET28a was purchased from Novagen (San Diego, CA). Plasmid pcDNA3.1, Lipofectamine reagent and Lipofectamine Plus reagent, and competent cells Escherichia coli DH5{alpha} and BL21 were purchased from Invitrogen (Carlsbad, CA). Mouse monoclonal anti-PKC-{alpha} antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GST antibody was obtained from Sigma (St. Louis, MO). Mouse anti-His antibody and enhanced chemiluminescence (ECL) detection reagents were purchased from Amersham Biosciences (Little Chalfont, UK). Ni-NTA agarose and Ni-NTA spin columns were purchased from Qiagen (Valencia, CA). Goat anti-mouse IgG (H+L)-horseradish peroxidase-conjugated antibody were purchased from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma.

Methods

Cloning of different combinations of PKC-{alpha} domains in expression vectors. The cDNA encoding the human PKC-{alpha} (full length), PKC-{alpha} (C1), PKC-{alpha} (C2), PKC-{alpha} (C3), PKC-{alpha} (C4), PKC-{alpha} (C1 and C2), PKC-{alpha} (C2 and C3), PKC-{alpha} (C3 and C4), PKC-{alpha} (C1, C2, and C3), and PKC-{alpha} (C2, C3, and C4) were amplified by performing PCR using the pBlueBac/PKC-{alpha} clone containing 2.0-kb human PKC-{alpha} cDNA as a template (Fig. 1A). The sense and antisense primers used were as follows: forward primer, 5'-ATG GCT GTT TTC CCG GGC AAC-3', and reverse primer, 5'-TCA TAC TGC ACT CTG TAA GAT-3' (full length); forward primer, 5'-ATG GCT GTT TTC CCG GGC AAC-3', and reverse primer, 5'-TCA TAC TGT GAC ATG GAG CTT-3' (C1); forward primer, 5'-CCT ATG GAT CCA AAC GGG CTT-3', and reverse primer, 5'-TCA GCC AAG TTT GCC TTT CTC-3' (C2); forward primer, 5'-CAA CCT TCC AAC CTT GAC CGA-3', and reverse primer, 5'-TCA TGG ATA GGA AAC GTT GTG-3' (C3); forward primer, 5'-TCC TTG TCC AAG GAG GCT G-3', and reverse primer, 5'-TCA TAC TGC ACT CTG TAA GAT-3' (C4); forward primer, 5'-ATG GCT GTT TTC CCG GGC AAC-3', and reverse primer, 5'-TCA GAT GAC TTT GTT GCC AGC-3' (C1 and C2); forward primer, 5'-CCT ATG GAT CCA AAC GGG CTT-3', and reverse primer, 5'-TCA TGG ATA GGA AAC GTT GTG-3' (C2 and C3); forward primer, 5'-CAA CCT TCC AAC CTT GAC CGA-3', and reverse primer, 5'-TCA TAC TGC ACT CTG TAA GAT-3' (C3 and C4); forward primer, 5'-ATG GCT GTT TTC CCG GGC AAC-3', and reverse primer, 5'-TCA TGG ATA GGA AAC GTT GTG-3' (C1, C2, and C3); forward primer, 5'-CCT ATG GAT CCA AAC GGG CTT-3', and reverse primer, 5'-TCA TAC TGC ACT CTG TAA GAT-3' (C2, C3, and C4), respectively. The restriction site of all forward primers was EcoRI. The restriction site of all reverse primers was XhoI. The PCR products (~2,000, ~530, ~320, ~700, ~340, ~870, ~1,120, ~1,040, ~1,670 and ~1,460 bp) were digested with the restriction enzymes EcoRI and XhoI. The digested PCR products were fused in frame with the NH2-terminal His tag vector pET28a using the restriction sites EcoRI and XhoI. For the purpose of transfection into rabbit colon smooth muscle cells, the PCR products were fused in frame with the pcDNA3.1 vector. pET28a and pcDNA3.1 were transformed into E. coli DH5-{alpha}. After confirmation of the clones by double-digestion release of the inserts and PCR, pET28a-PKC-{alpha} (C1), pET28a-PKC-{alpha} (C2), pET28a-PKC-{alpha} (C3), pET28a-PKC-{alpha} (C4), pET28a-PKC-{alpha} (C1 and C2), pET28a-PKC-{alpha} (C2 and C3), pET28a-PKC-{alpha} (C3 and C4), pET28a-PKC-{alpha} (C1, C2, and C3), and pET28a-PKC-{alpha} (C2, C3, and C4) constructs were transformed into an expression of host E. coli BL21 cells. pcDNA3.1-PKC-{alpha} (C1, C2, and C3), pcDNA3.1-PKC-{alpha} (C1 and C2), pcDNA3.1-PKC-{alpha} (C2 and C3), pcDNA3.1-PKC-{alpha} (C2, C3, and C4), pcDNA3.1-PKC-{alpha} (C3 and C4), and pcDNA3.1-PKC-{alpha} (full length) were transfected into rabbit colon smooth muscle cells. All plasmid constructs were verified by sequencing.



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Fig. 1. Construction and purification of different His-tagged combinations of protein kinase C (PKC)-{alpha} domains. A: PCR amplification of different combinations of PKC-{alpha} domains C1–C4: PKC-{alpha} (C1, C2, and C3), PKC-{alpha} (C2, C3, and C4), PKC-{alpha} (C3 and C4), PKC-{alpha} (C1 and C2), PKC-{alpha} (C2 and C3), PKC-{alpha} (C3), PKC-{alpha} (C4), PKC-{alpha} (C1), and PKC-{alpha} (C2). Regarding cDNA, ~1,670, ~1,460, ~1,040, ~870, ~1,120, ~700, ~340, ~530, and ~320 bp of PCR fragments of different combinations of PKC-{alpha} domains were amplified from a pBlueBac/PKC-{alpha} clone containing human PKC-{alpha} cDNA. Lane 1: PKC-{alpha} (C1, C2, and C3); lane 2: PKC-{alpha} (C2, C3, and C4); lane 3: PKC-{alpha} (C3 and C4); lane 4: PKC-{alpha} (C1 and C2); lane 5: PKC-{alpha} (C2 and C3); lane 6: PKC-{alpha} (C3); lane 7: PKC-{alpha} (C4); lane 8: PKC-{alpha} (C1); and lane 9: PKC-{alpha} (C2). B: purified His-tagged proteins were subjected to SDS-PAGE, followed by Western blot analysis with anti-His antibody. Lane 1: His-PKC-{alpha} (C1, C2, and C3); lane 2: His-PKC-{alpha} (C2, C3, and C4); lane 3: His-PKC-{alpha} (C3 and C4); lane 4: His-PKC-{alpha} (C4); lane 5: His-PKC-{alpha} (C3); lane 6: His-PKC-{alpha} (C1); lane 7: His-PKC-{alpha} (C2); lane 8: His-PKC-{alpha} (C1 and C2); and lane 9: His-PKC-{alpha} (C2C3). C: Western blot analysis with anti-PKC-{alpha} (C) antibody raised against amino acids 373–672 mapping at the COOH terminus of human PKC-{alpha}. Lane 1: His-PKC-{alpha} (C2, C3, and C4); lane 2: His-PKC-{alpha} (C3 and C4); and lane 3: His-PKC-{alpha} (C4). D: Western blot analysis with anti-PKC-{alpha} (H) antibody raised against amino acids 292–317 mapping at the hinge region of human PKC-{alpha}. Lane 1: His-PKC-{alpha} (C1, C2, and C3); lane 2: His-PKC-{alpha} (C2, C3, and C4); lane 3: His-PKC-{alpha} (C2 and C3); and lane 4: His-PKC-{alpha} (C1 and C2).

 
Purification of His-tagged different constructs of PKC-{alpha} proteins. His-tagged different constructs of PKC-{alpha} proteins were expressed and purified with Ni-NTA agarose according to the manufacturer's instructions. An overnight culture (5 ml) was inoculated in 500 ml of fresh Luria Bertani medium with 100 µg/ml kanamycin. The culture was grown at 37°C with vigorous shaking until the optical density reached 0.6 at 600 nm (~2 h). Isopropylthio-{beta}-D-galactoside (IPTG) was then added to the culture at 1 mM final concentration, and the culture was grown for an additional 3–4 h at 37°C with vigorous shaking. The cells were then harvested by performing centrifugation at 4,000 rpm for 15 min. The cell pellet was resuspended in buffer B (100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M urea, pH 8) at 5 ml/g wet wt. The cells were lysed using gentle vortexing or stirred for 15–60 min at room temperature. The cell lysate was centrifuged at 10,000 g for 20 min at room temperature. The supernatant was mixed with 50% Ni-NTA agarose (4:1 ratio) and rotary shaken for 15–60 min at room temperature. The lysate-resin mixture was loaded into a Ni-NTA spin column and washed twice with a four-bed volume of buffer C (100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M urea, pH 6.3). The recombinant protein was eluted four times with a one-bed volume of buffer D (100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M urea, pH 5.9), followed by four elutions with a one-bed volume of buffer E (100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M urea, pH 4.5). The eluted His-tagged different constructs of PKC-{alpha} proteins were examined using Western blot analysis with anti-His antibody and anti-PKC-{alpha} antibodies (Fig. 1, BD).

Western blot analysis. Purified His-tagged different domains of PKC-{alpha} proteins (20 µg), obtained as described above, or 30 µg of cell lysate from transfected cells and cultured rabbit colon smooth muscle cells were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Western blot analysis was performed using as primary antibodies anti-PKC-{alpha} (C) antibody raised against amino acids 373–672 mapping at the COOH terminus of PKC-{alpha} (1:200 dilution), anti-PKC-{alpha} (H) antibody raised against amino acids 292–317 mapping at the hinge region of PKC-{alpha} (H) (1:200 dilution), or anti-His antibody (1:3,000 dilution). The membrane was incubated in the appropriate dilution of primary antibody for 1 h with rocking at room temperature. The membrane was washed three times for 15 min with Tris-buffered saline with Tween 20 (TBST). The membrane was then incubated in the appropriate dilution of secondary antibody for 1 h with rocking at room temperature, followed by three more 15-min washes with TBST. The membrane was then incubated with ECL reagent for 1 min. The proteins were detected on the membrane by immediately exposing the membrane to film.

In vitro binding assay of fusion protein GST-RhoA with full-length PKC-{alpha} and different His-tagged constructs of PKC-{alpha} proteins. GST-RhoA (25 µg; 0.48 µmol) or 13 µg (0.48 µmol) of GST were mixed with 200 µl of glutathione-agarose beads and rocked for 2 h at 4°C. Unbound GST-RhoA fusion proteins or GST was removed by being washed five times with mercaptoethanol-Triton X-100-PBS (MT-PBS; 150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, pH 7.3, and 1% Triton X-100). All of the washes were retained for further analysis. Full-length PKC-{alpha} protein (10 µg) or 25 µg of His-tagged different constructs of PKC-{alpha} proteins were added to the GST-RhoA-bound beads or GST-bound beads and rocked for 1 h at 4°C. Unbound PKC-{alpha} protein was removed by being washed five times with MT-PBS. All of the washes were retained for further analysis. The bound proteins were eluted twice with a one-bed volume of elution buffer (50 mM Tris·HCl, pH 8.0, containing 10 mM reduced glutathione). From the first, third, and fifth washes and the elution, 10 µl were spotted on three PVDF membranes. The membranes were blocked with 5% nonfat dry milk and immunoblotted with anti-GST antibody, anti-RhoA antibody, and anti-PKC-{alpha} antibodies, or with anti-His antibody, respectively. The spots were detected using ECL.

Dot blot. Dry blotting paper and the PVDF membrane were cut according to the dot blot apparatus size. The cut PVDF membrane was soaked in 100% methanol for 2–3 min followed by equilibration in 1x PBS for 5 min. The cut dry blotting paper was incubated in 1x PBS for 5 min. The wet blotting paper was placed on the dot blot apparatus, followed by the equilibrated PVDF membrane, onto the blotting paper. The dot blot apparatus was clasped with clamps on the sides and was attached to the vacuum pipe. The vacuum was passed at the rate of 1 ml/min. The sample (10 µl) was added into each slot. The clamps were opened to remove the membrane. The membrane was incubated in 5% nonfat dry milk in TBST and rocked for 1 h at room temperature. The membrane was washed three times for 10 min each time with TBST. The membrane was then incubated in the appropriate dilution of primary antibody for 1 h with rocking at room temperature. Again, the membrane was washed three times for 15 min each time with TBST. The membrane was then incubated in the appropriate dilution of secondary antibody for 1 h with rocking at room temperature, followed by three more washes for 15 min each with TBST. The membrane was then incubated with ECL reagent for 1 min. The proteins were detected on the membrane by immediate exposure of the membrane to the film.

Transfection of smooth muscle cells with full-length PKC-{alpha} and different combinations of PKC-{alpha} domains. Rabbit colon smooth muscle cells were cultured in DMEM with 10% FBS and 3% penicillin/streptomycin. Cells were passaged on the day before transfection and allowed to reach 70% confluence on the day of transfection. pcDNA3.1 with full-length PKC-{alpha}, PKC-{alpha} (C1, C2, and C3), PKC-{alpha} (C1 and C2), PKC-{alpha} (C2 and C3), PKC-{alpha} (C2, C3, and C4), and PKC-{alpha} (C3 and C4) cDNA were transfected into the cells using Invitrogen Lipofectamine reagent as described previously (33). Briefly, the cDNA was diluted with serum-free DMEM and mixed with Lipofectamine Plus reagent, followed by incubation at room temperature for 15 min. The Lipofectamine reagent was diluted with serum-free DMEM. Next, the precomplexed DNA and diluted Lipofectamine reagent were mixed and incubated for 15 min. The transfection complex was then overlaid onto the cells. After 4 h of incubation, the medium was changed to culture medium. After the cells reached confluence, they were used for the experiments. The overexpression of full-length PKC-{alpha} and truncated forms of PKC-{alpha} were confirmed by performing Western blot analysis (Fig. 2).



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Fig. 2. Overexpression of full-length PKC-{alpha} and of truncated forms of PKC-{alpha}. Representative blot showing the overexpression of full-length PKC-{alpha} and truncated forms of PKC-{alpha}. Cell lysate (30 µg) from cultured smooth muscle cells and transfected cells were subjected to SDS-PAGE, followed by Western blot analysis with anti-PKC-{alpha} antibody. Lane 1: control (untransfected cells); lane 2: full-length PKC-{alpha} transfected cells; lane 3: PKC-{alpha} (C1, C2, and C3) transfected cells; lane 4: PKC-{alpha} (C2, C3, and C4) transfected cells; lane 5: PKC-{alpha} (C2 and C3) transfected cells; lane 6: PKC-{alpha} (C3 and C4) transfected cells; lane 7: PKC-{alpha} (C1 and C2) transfected cells.

 
Immunoprecipitation and immunoblotting. Confluent transfected rabbit colon smooth muscle cells were treated with acetylcholine for 4 min and then were washed three times with cold PBS. Lysates from the transfected cells were analyzed for protein content using Bio-Rad protein assay reagent. Anti-RhoA antibody (4 µl) was added to 500 µg of protein sample in 500 µl of lysis buffer and rocked overnight at 4°C. A protein G-Sepharose bead slurry (50 µl; 50% concentration) was added to the overnight mixture and rocked at 4°C for 2 h. The beads bound with proteins were then collected by performing centrifugation at 14,000 g for 3 min at 4°C. The supernatant was discarded, and the bead pellet was washed three times at room temperature with TBS bead wash buffer (20 mM Tris·HCl and 150 mM NaCl, pH 7.6) (33). The beads were then resuspended in 25 µl of 2x Laemmli sample buffer and boiled for 5 min. Proteins from the immunoprecipitates were separated onto SDS-PAGE gel and transferred to a PVDF membrane. The membrane was immunoblotted with anti-PKC-{alpha} antibody.

Data Analysis

Dot blot spots were quantified using a densitometer (model GS-700; Bio-Rad Laboratories), and spot volumes (absorbance units x mm2) were calculated and expressed as a percentage of the total volume. Spot data were within the linear range of detection for each antibody used. The control spot intensity was standardized to 100%. The spot intensities of eluted fractions of the PKC-{alpha} were compared with the control and expressed as the percentage change from the control. All of the means were compared and analyzed using Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Construction of Different His-Tagged Combinations of PKC-{alpha} Domains

To determine the domains of PKC-{alpha} that bind to RhoA, different His-tagged constructs containing individual domains and different combinations of the domains of PKC-{alpha} were constructed (Fig. 3). cDNA were amplified from the pBlueBac/PKC-{alpha} clone containing 2.0-kb human PKC-{alpha} cDNA (Fig. 1A). The PCR-amplified fragments of different constructs were inserted into pET28a at the EcoRI and XhoI sites to express as NH2-terminal His-tagged proteins. The clones were confirmed to be in the correct open reading frames by sequence. The His-tagged different constructs of PKC-{alpha} proteins were expressed in response to induction using 1 mM IPTG.



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Fig. 3. Diagram showing the construction of different combinations of PKC-{alpha} domains. Top bar shows the domain organization of PKC-{alpha}, with the C1 domain (amino acids 1–177), the C2 domain (amino acids 178–289), the hinge region (amino acids 289–311), the C3 domain (amino acids 312–558), and the C4 domain (amino acids 559–672). His-tagged different combinations of PKC-{alpha} fusion proteins were constructed as described in MATERIALS AND METHODS. Histidine was tagged to the NH2 termini of all of the proteins.

 
The expressed His-PKC-{alpha} proteins were purified using Ni-NTA agarose as described in MATERIALS AND METHODS and analyzed using Western blot analysis with anti-PKC-{alpha} (C), anti-PKC-{alpha} (H), and anti-His antibodies. The bands of ~53 kDa [His-PKC-{alpha} (C2, C3, and C4)], ~37 kDa [His-PKC-{alpha} (C3 and C4)], and ~12 kDa [His-PKC-{alpha} (C4)] were detected using both anti-PKC-{alpha} (C) and anti-His antibodies, because the synthesized molecules contained the COOH terminus of PKC-{alpha} (Fig. 1, B and C). The bands of ~60 kDa [His-PKC-{alpha} (C1, C2, and C3)], ~53 kDa [His-PKC-{alpha} (C2, C3, and C4)], ~40 kDa [His-PKC-{alpha} (C2 and C3)], and ~31 kDa [His-PKC-{alpha} (C1 and C2)] were detected using both anti-PKC-{alpha} (H) and anti-His antibodies, because the synthesized molecules contained the hinge region of PKC-{alpha} (Fig. 1, B and D). The bands of ~19 kDa [His-PKC-{alpha} (C1)], ~12 kDa [His-PKC-{alpha} (C2)], and ~25 kDa [His-PKC-{alpha} (C3)] were detected only when anti-His antibody was used, because they lacked the hinge region and the COOH terminus of PKC-{alpha} (Fig. 1B).

RhoA Directly Binds Full-Length PKC-{alpha} In Vitro

To study the direct association of RhoA with PKC-{alpha}, an in vitro binding assay was performed using GST-RhoA fusion protein and purified full-length PKC-{alpha}. GST-RhoA protein was conjugated to glutathione-agarose beads and incubated with full-length PKC-{alpha} protein. The incubation with PKC-{alpha} allowed it to bind to the immobilized GST-RhoA. The GST-RhoA fusion proteins bound with PKC-{alpha} were eluted from the glutathione-agarose beads with reduced glutathione buffer (pH 8.0), as described in MATERIALS AND METHODS. The washed and eluted fractions were examined using dot blot analysis with anti-RhoA, anti-PKC-{alpha} (C), and anti-GST antibodies. The intensity of the dots of PKC-{alpha} was observed in the elution from the glutathione-agarose-bound GST-RhoA fraction. Gluta-thione-agarose beads conjugated to GST alone were used as controls to confirm that the binding of PKC-{alpha} was due to its binding to RhoA but not to GST (Fig. 4). The percentage increase of binding of full-length PKC-{alpha} to RhoA was 82.57 ± 15.26% above control (n = 4, P ≤ 0.01). The data suggest that PKC-{alpha} binds to RhoA directly.



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Fig. 4. Direct association of glutathione-S-transferase (GST)-RhoA with PKC-{alpha}. A: representative dot blot showing the association of PKC-{alpha} with GST-RhoA. GST alone conjugated to glutathione-agarose beads was used as control. Fractions 1–3 were washed free of unbound GST-RhoA protein. Fractions 4–6 were washed free of unbound PKC-{alpha} protein. Fractions 7 and 8 were eluted of GST-RhoA protein bound to glutathione-agarose. Coelution of PKC-{alpha} with GST-RhoA was observed in lanes 7 and 8 [probed with anti-PKC-{alpha} (C) as well as with anti-RhoA and anti-GST antibodies], an indication of direct association of full-length PKC-{alpha} with GST-RhoA. B: densitometric graph representing the percentage increase in the binding of full-length PKC-{alpha} (FL) protein to GST-RhoA fusion protein. The percentage increase in the binding of full-length PKC-{alpha} to GST-RhoA was 82.57 ± 15.27% (n = 4, P ≤ 0.01) above control. GST alone was used as the control.

 
In Vitro Association of RhoA with Truncated Forms of PKC-{alpha}

As described in MATERIALS AND METHODS, truncated and various overlapping fragments of PKC-{alpha} were expressed in E. coli to define the precise domains of PKC-{alpha} that bind to RhoA (Fig. 1). An in vitro binding assay was performed using purified fragments of different combinations of PKC-{alpha} domains. PKC-{alpha} (C2, C3, and C4) bound to GST-RhoA, while PKC-{alpha} (C1, C2, and C3) did not bind to GST-RhoA (Fig. 5A). The percentage increase in the binding of PKC-{alpha} (C2, C3, and C4) to GST-RhoA fusion protein was 94.09 ± 12.13% above control (n = 4, P ≤ 0.01). There was no difference between the binding of full-length PKC-{alpha} and PKC-{alpha} (C2, C3, and C4) to GST-RhoA. The binding of PKC-{alpha} (C1, C2, and C3) to GST-RhoA fusion protein was 0.47 ± 1.26% above control (n = 4, P ≤ 0.95). There was a significant difference between the binding of full-length PKC-{alpha} (Fig. 4) and PKC-{alpha} (C1, C2, and C3) to GST-RhoA (P < 0.01) (Fig. 5B). These data suggest that the C4 domain of PKC-{alpha} may be essential for the binding of PKC-{alpha} to RhoA. They also suggest that RhoA binding sites may exist on the C4 domain of PKC-{alpha}.



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Fig. 5. In vitro binding assay of GST-RhoA with PKC-{alpha} (C1, C2, and C3) and PKC-{alpha} (C2, C3, and C4). A: representative dot blot showing the nonassociation of PKC-{alpha} (C1, C2, and C3) with GST-RhoA and the association of PKC-{alpha} (C2, C3, and C4) with GST-RhoA. GST alone conjugated to glutathione-agarose beads was used as the control. Fractions 1–3 were washed free of unbound GST-RhoA protein. Fractions 4–6 were washed free of unbound PKC-{alpha} (C1, C2, and C3) protein (left blots) and unbound PKC-{alpha} (C2, C3, and C4) protein (right blots). Fractions 7 and 8 were eluted of GST-RhoA bound to the glutathione-agarose. Coelution of PKC-{alpha} (C1, C2, and C3) with GST-RhoA was not observed in lanes 7 and 8 (left blots), an indication of nonassociation of recombinant PKC-{alpha} (C1, C2, and C3) with GST-RhoA. Coelution of PKC-{alpha} (C2, C3, and C4) with GST-RhoA was observed in lanes 7 and 8 (right blots), an indication of direct association of recombinant PKC-{alpha} (C2, C3, and C4) with GST-RhoA. B: densitometric graph representing the percentage increase in the binding of full-length PKC-{alpha} (FL) (as in Fig. 4), PKC-{alpha} (C1, C2, and C3) (0.47 ± 1.26%, n = 4; P ≤ 0.95), and PKC-{alpha} (C2, C3, and C4) (94.09 ± 12.13%, n = 4; P ≤ 0.01) to GST-RhoA fusion protein. There was a significant difference between full-length PKC-{alpha} (FL) and PKC-{alpha} (C1, C2, and C3) binding to GST-RhoA (**P < 0.01). There was no difference between full-length PKC-{alpha} (FL) and PKC-{alpha} (C2, C3, and C4) binding to GST-RhoA.

 
To further determine the RhoA-binding sites, we deleted the C1 and C2 domains as well as the C1, C2, and C3 domains from PKC-{alpha}. Constructs consisting of C3 and C4 domains and the C4 domain alone were synthesized. We used the synthesized PKC-{alpha} (C3 and C4) and PKC-{alpha} (C4) for an in vitro binding assay. The results indicated that both PKC-{alpha} (C3 and C4) and PKC-{alpha} (C4) bound to GST-RhoA (Fig. 6A). The percentage increase of binding of PKC-{alpha} (C3 and C4) and PKC-{alpha} (C4) to GST-RhoA were 85.10 ± 16.16% (n = 4, P ≤ 0.01) and 90.58 ± 26.79% (n = 4, P ≤ 0.01) above the control, respectively (Fig. 6B). There was no difference in the binding of full-length PKC-{alpha} (Fig. 4), PKC-{alpha} (C3 and C4), and PKC-{alpha} (C4) to GST-RhoA. These data confirm the results obtained previously (Fig. 4) regarding the importance of the C4 domain of PKC-{alpha} in the binding of PKC-{alpha} to RhoA.



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Fig. 6. In vitro binding assay of GST-RhoA with PKC-{alpha} (C3 and C4) and PKC-{alpha} (C4). A: representative dot blot showing the association of PKC-{alpha} (C3 and C4) with GST-RhoA and PKC-{alpha} (C4) with GST-RhoA. GST alone conjugated to glutathione-agarose beads was used as the control. Fractions 1–3 were washed free of unbound GST-RhoA protein. Fractions 4–6 were washed free of unbound PKC-{alpha} (C3 and C4) protein (left blots) and unbound His-PKC-{alpha} (C4) protein (right blots). Fractions 7 and 8 were eluted of GST-RhoA bound to the glutathione-agarose. Coelution of PKC-{alpha} (C3 and C4) with GST-RhoA (left blots) and PKC-{alpha} (C4) with GST-RhoA (right blots) was observed in lanes 7 and 8, indicating direct association of recombinant PKC-{alpha} (C3 and C4) with GST-RhoA and PKC-{alpha} (C4) with GST-RhoA. B: densitometric graph representing the percentage increase in the binding of full-length PKC-{alpha} (FL) (as in Fig. 4), PKC-{alpha} (C3 and C4) (85.10 ± 16.76%, n = 4; P ≤ 0.01), and PKC-{alpha} (C4) (90.58 ± 26.79%, n = 4; P ≤ 0.01) to GST-RhoA. There was no difference among full-length PKC-{alpha} (FL), PKC-{alpha} (C3 and C4), and PKC-{alpha} (C4) binding to GST-RhoA.

 
To map the precise domain that is responsible for the interaction of the two proteins in vitro, we constructed His-PKC-{alpha} (C1), His-PKC-{alpha} (C2), and His-PKC-{alpha} (C3). The binding results indicated that only PKC-{alpha} (C1) and PKC-{alpha} (C2), but not PKC-{alpha} (C3), bound to GST-RhoA (Fig. 7A). The binding of the fragments, namely, PKC-{alpha} (C1) and PKC-{alpha} (C2), to GST-RhoA was not significantly different from the binding of full-length PKC-{alpha} (Fig. 4). The percentage increases in the binding of PKC-{alpha} (C1) and PKC-{alpha} (C2) to GST-RhoA were 70.48 ± 20.78% (n = 4, P ≤ 0.01) and 72.26 ± 29.96% (n = 4, P ≤ 0.01) above control, respectively (Fig. 7B). The PKC-{alpha} (C3) fragment did not bind to GST-RhoA (0.64 ± 5.18% above control, n = 4, P ≤ 0.99) (Fig. 7B). These results suggest that in vitro binding sites for RhoA may exist on the C1 and C2 domains, but not on the C3 domain, of PKC-{alpha}. To further examine the effect of the C3 domain on the interaction of PKC-{alpha} with RhoA, we constructed His-PKC-{alpha} (C1 and C2) and His-PKC-{alpha} (C2 and C3) and tested their binding to GST-RhoA. The results indicated that PKC-{alpha} (C1 and C2) bound to GST-RhoA (60.78 ± 13.78% above control; n = 4, P ≤ 0.01), while PKC-{alpha} (C2 and C3) did not bind to GST-RhoA (7.45 ± 10.76% above control; n = 4, P ≤ 0.75) (Fig. 8A). There was a significant difference between full-length PKC-{alpha} (Fig. 3) and PKC-{alpha} (C2 and C3) binding to GST-RhoA (P < 0.01) (Fig. 8B). Furthermore, the binding of PKC-{alpha} (C4) to RhoA was better than that of PKC-{alpha} (C1 and C2) (Fig. 6B). Interestingly, the fragments PKC-{alpha} (C3 and C4) and PKC-{alpha} (C2, C3, and C4) also bound to RhoA (Fig. 5B) better than PKC-{alpha} (C1 and C2) alone. Collectively, the data suggest that the C3 domain alone does not represent a binding site for RhoA and that the presence of C3 may interfere with the in vitro interaction of PKC-{alpha} (C1, C2, and C3) with RhoA.



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Fig. 7. In vitro binding assay of GST-RhoA with PKC-{alpha} (C1), PKC-{alpha} (C2), and PKC-{alpha} (C3). A: representative dot blot showing the association of PKC-{alpha} (C1) with GST-RhoA and PKC-{alpha} (C2) with GST-RhoA and the nonassociation of PKC-{alpha} (C3) with GST-RhoA. GST alone conjugated to glutathione-agarose beads was used as the control. Fractions 1–3 were washed free of unbound GST-RhoA protein. Fractions 4–6 were washed free of unbound PKC-{alpha} (C1) protein (left blots), unbound PKC-{alpha} (C2) protein (middle blots), and unbound PKC-{alpha} (C3) protein (right blots). Fractions 7 and 8 were eluted of GST-RhoA bound to the glutathione-agarose. Coelution of PKC-{alpha} (C1) with GST-RhoA (left blots) and PKC-{alpha} (C2) with GST-RhoA (middle blots) was observed in lanes 7 and 8, an indication of direct association of recombinant PKC-{alpha} (C1) with GST-RhoA and PKC-{alpha} (C2) with GST-RhoA. Coelution of PKC-{alpha} (C3) with GST-RhoA was not observed in lanes 7 and 8 (right), an indication of nonassociation of recombinant PKC-{alpha} (C3) with GST-RhoA. B: densitometric graph representing the percentage increase of binding of full-length PKC-{alpha} (as in Fig. 4), PKC-{alpha} (C1) (70.48 ± 20.78%, n = 4; P ≤ 0.01), PKC-{alpha} (C2) (72.26 ± 29.96%, n = 4; P ≤ 0.01), and PKC-{alpha} (C3) (0.64 ± 5.18%, n = 4; P ≤ 0.99) to GST-RhoA. There was a significant difference between full-length PKC-{alpha} (FL) and PKC-{alpha} (C3) binding to GST-RhoA (**P < 0.01). There was no difference among full-length PKC-{alpha} (FL), PKC-{alpha} (C1), and PKC-{alpha} (C2) binding to GST-RhoA.

 


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Fig. 8. In vitro binding assay of GST-RhoA with PKC-{alpha} (C1 and C2) and PKC-{alpha} (C2 and C3). A: representative dot blot showing the association of PKC-{alpha} (C1 and C2) with GST-RhoA and the nonassociation of PKC-{alpha} (C2 and C3) with GST-RhoA. GST alone conjugated to glutathione-agarose beads was used as the control. Fractions 1–3 were washed free of unbound GST-RhoA protein. Fractions 4–6 were washed free of unbound PKC-{alpha} (C1 and C2) protein (left blots) and unbound PKC-{alpha} (C2 and C3) protein (right blots). Fractions 7 and 8 were eluted of GST-RhoA bound to the glutathione-agarose. Coelution of PKC-{alpha} (C1 and C2) with GST-RhoA was observed in lanes 7 and 8 (left blots), an indication of direct association of recombinant PKC-{alpha} (C1 and C2) with GST-RhoA. Coelution of PKC-{alpha} (C2 and C3) with GST-RhoA proteins was not observed in lanes 7 and 8 (right blots), an indication of nonassociation of recombinant PKC-{alpha} (C2 and C3) with GST-RhoA. B: densitometric graph representing the percentage increase of binding of full-length PKC-{alpha} (FL) (as in Fig. 4), PKC-{alpha} (C1 and C2) (60.78 ± 13.78%, n = 4; P ≤ 0.01), and PKC-{alpha} (C2 and C3) (7.45 ± 10.76%, n = 4; P ≤ 0.756) to GST-RhoA. There was a significant difference between full-length PKC-{alpha} and PKC-{alpha} (C2 and C3) binding to GST-RhoA (**P < 0.01), but there was no significant difference between full-length PKC-{alpha} (FL) and PKC-{alpha} (C1 and C2) binding to GST-RhoA.

 
Association of RhoA with Truncated Forms of PKC-{alpha} in Cells Transfected with Different Combinations of PKC-{alpha} Domains

To confirm the binding of truncated forms of PKC-{alpha} with RhoA in cells, full-length PKC-{alpha} and the fragments C1, C2, and C3; C2 and C3; C1 and C2; C2, C3, and C4; and C3 and C4 were subcloned into pcDNA3.1 and transiently transfected into cultured rabbit colon smooth muscle cells. The transfection efficiency was >80%. The overexpression of full-length PKC-{alpha} or the truncated forms of PKC-{alpha} was confirmed using Western blot analysis (Fig. 2).

Confluent transfected cells were stimulated with 0.1 µM acetylcholine for 4 min. The stimulated transfected cells, unstimulated transfected cells, and untransfected rabbit colon smooth muscle cells were immunoprecipitated with anti-RhoA antibody. The immunoprecipitates were then subjected to SDS-PAGE, and the separated proteins were transferred to PVDF membrane. The membrane was immunoblotted with either anti-PKC-{alpha} antibody or anti-RhoA antibody. The band intensity was observed in cells transfected with full-length PKC-{alpha} and in fragments C2, C3, and C4; fragments C3 and C4; and untransfected cells (Figs. 9A and 10A). The association of full-length PKC-{alpha} and fragments C2, C3, and C4 as well as C3 and C4 to RhoA increased upon stimulation with acetylcholine (Figs. 9A and 10A). Acetylcholine induced significant increases in the association of PKC-{alpha} with RhoA in cells transfected with full-length PKC-{alpha}, PKC-{alpha} (C2, C3, and C4), and PKC-{alpha} (C3 and C4) were 139.4 ± 14.5%, 145.8 ± 12.2% and 138.7 ± 18.1%, respectively; (n = 3, P < 0.001) compared with untransfected smooth muscle cells (control) (Figs. 9C and 10C). The amounts of RhoA immunoprecipitated remained constant in all of the experiments (Figs. 9B and 10B). The association of the PKC-{alpha} (C1 and C2), PKC-{alpha} (C1, C2, and C3), and PKC-{alpha} (C2 and C3) with RhoA was not observed (Fig. 10A).



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Fig. 9. Coimmunoprecipitation of PKC-{alpha} with RhoA in rabbit colon smooth muscle cells transfected with full-length PKC-{alpha} (fl), PKC-{alpha} (C2, C3, and C4), and PKC-{alpha} (C3 and C4) in response to acetylcholine. Cells transfected with full-length PKC-{alpha} or different truncated forms of PKC-{alpha} were treated with 0.1 µM acetylcholine for 4 min. Untreated cultured cells served as controls. Cell lysates were immunoprecipitated with anti-RhoA antibody and were further subjected to SDS-PAGE, followed by Western blot analysis with anti-PKC-{alpha} and anti-RhoA antibodies. A: Western blot analysis showing an increase in the association of PKC-{alpha} with RhoA in cells transfected with full-length PKC-{alpha} (fl) (78 kDa), PKC-{alpha} (C2, C3, and C4) (53 kDa), and PKC-{alpha} (C3 and C4) (37 kDa) in the presence of acetylcholine. Lane 1: control (untransfected cells); lane 2: PKC-{alpha} (fl) transfected cells; lane 3: PKC-{alpha} (fl) transfected cells stimulated with acetylcholine; lane 4: PKC-{alpha} (C2, C3, and C4) transfected cells; lane 5: PKC-{alpha} (C2, C3, and C4) transfected cells stimulated with acetylcholine; lane 6: PKC-{alpha} (C3 and C4) transfected cells; and lane 7: PKC-{alpha} (C3 and C4) transfected cells stimulated with acetylcholine. B: Western blot analysis reprobed with anti-RhoA antibody showing an equal amount of RhoA immunoprecipitated. C: graph showing that stimulation of cells transfected with either full-length PKC-{alpha} (fl), PKC-{alpha} (C2, C3, and C4), or PKC-{alpha} (C3 and C4) with acetylcholine (0.1 µM) resulted in a significant increase in association with RhoA [139.2 ± 14.5%, 145.8 ± 12.2%, and 138.7 ± 18.1%, respectively, for full-length (fl); C2, C3, and C4; and C3 and C4; n = 3; *P < 0.05].

 


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Fig. 10. Coimmunoprecipitation of PKC-{alpha} with RhoA in rabbit colon smooth muscle cells transfected with full-length PKC-{alpha} (fl), PKC-{alpha} (C1 and C2), PKC-{alpha} (C2 and C3), and PKC-{alpha} (C1, C2, and C3) in response to acetylcholine. Cells transfected with full-length PKC-{alpha} (fl) or different truncated forms of PKC-{alpha} were treated with 0.1 µM acetylcholine for 4 min. Untreated cultured cells served as controls. Cell lysates were immunoprecipitated (IP) with anti-RhoA antibody and were further subjected to SDS-PAGE, followed by Western blot analysis with anti-PKC-{alpha} and anti-RhoA antibodies. A: Western blot analysis showing the increased association of PKC-{alpha} with RhoA in cells transfected with full-length PKC-{alpha} (fl) (78 kDa) in the presence of acetylcholine. Association of RhoA with truncated forms of PKC-{alpha} (C1 and C2), PKC-{alpha} (C1, C2, and C3), or PKC-{alpha} (C2 and C3) was not observed. Lane 1: control (untransfected cells); lane 2: PKC-{alpha} (fl) transfected cells; lane 3: PKC-{alpha} (fl) transfected cells stimulated with acetylcholine; lane 4: PKC-{alpha} (C1 and C2) transfected cells; lane 5: PKC-{alpha} (C1 and C2) transfected cells stimulated with acetylcholine; lane 6: PKC-{alpha} (C2 and C3) transfected cells; lane 7: PKC-{alpha} (C2 and C3) transfected cells stimulated with acetylcholine; lane 8: PKC-{alpha} (C1, C2, and C3) transfected cells; lane 9: PKC-{alpha} (C1, C2, and C3) transfected cells stimulated with acetylcholine. B: Western blot analysis reprobed with anti-RhoA antibody showing an equal amount of RhoA was immunoprecipitated. C: RhoA failed to associate with truncated forms of PKC-{alpha} in cells transfected with either PKC-{alpha} (C1 and C2), PKC-{alpha} (C2 and C3), or PKC-{alpha} (C1, C2, and C3) upon stimulation with acetylcholine (99.1 ± 1.2%, 98.4 ± 0.7%, and 96.7 ± 1.9%, respectively, for C1 and C2; C2 and C3; and C1, C2, and C3) (P < 0.05).

 
The data suggest that in smooth muscle cells, full-length PKC-{alpha} and the fragments PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) associated with RhoA in cells and that the association increased upon stimulation with acetylcholine, while the fragments PKC-{alpha} (C1 and C2), PKC-{alpha} (C1, C2, and C3), and PKC-{alpha} (C2 and C3), which lack the C4 domain, did not bind either before or after stimulation with acetylcholine. These data suggest that the functional association of PKC-{alpha} with RhoA may require the C4 domain.


    DISCUSSION
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Agonist-induced contraction of gastrointestinal smooth muscle results in activation of G protein-coupled receptors and subsequent downstream signaling events (4, 22). Acetylcholine stimulates phospholipase D (PLD), which hydrolyzes a major membrane phospholipid, phosphatidylcholine (PC), to generate choline and phosphatidic acid (PA). PA is dephosphorylated to DAG, resulting in the activation of various PKC isoenzymes (23, 24). Because of electrostatic interaction, activation of PKC by anionic lipids (e.g., PLD, PA, DAG) produces only a small change in PKC conformation. PKC bound to the membrane at both the C1 and C2 domains exposes the regions in the catalytic domain, thus resulting in an open conformation (active form) capable of binding to the substrates (27). In smooth muscle cells, PKC can interact with several proteins. Our recent data indicate that acetylcholine induces a significant and sustained increase in the association of tropomyosin with PKC-{alpha} in the particulate fraction of colonic smooth muscle cells (40). The interactions of PKC with other proteins play an important role in the functions of PKC itself and those of the other proteins with which it interacts. Understanding the complexity of these protein-protein interactions will allow the elucidation of isozyme-specific signaling in different cellular systems. Evidence suggests that RhoA is an important signaling protein that mediates various actin-dependent cytoskeletal functions, including smooth muscle contraction (43). There is strong evidence to implicate Rho-kinase as a downstream target in the RhoA-linked pathway (44). Rho kinase phosphorylates myosin phosphatase target protein (MYPT)-1 and inhibits its activity, resulting in an increase in MLC phosphorylation (9, 10, 18). Both PKC and RhoA pathways target the inhibition of MYPT activity and thus maintain the phosphorylated state of MLC20. Previous studies performed in our laboratory showed that PKC-{alpha} and RhoA coimmunoprecipitate in the particulate fraction of colon smooth muscle cells in response to different contractile agonists (3). The association of RhoA with PKC-{alpha} is due to a direct interaction between these two molecules (33). In the present study, we have attempted to map the possible interactive regions of PKC-{alpha} with RhoA.

His-tagged individual domains and different combinations of domains of PKC-{alpha} were constructed, and their interaction with GST-RhoA was examined. Our in vitro binding data indicate that RhoA binding sites exist on the regulatory domains, C1 and C2, and the catalytic domain, C4, of PKC-{alpha}. Most of the protein binding sequences are localized to the regulatory domain of PKC (20). Short sequences within the C1 region of PKC-{epsilon} were identified as F-actin binding motifs (34). The C1 region in aPKCs was identified as the protease-activated receptor-4 binding site (8). The C1 domains of cPKC, nPKC, and aPKC isozymes were all found to bind the pleckstrin homology domain of Btk of the Tec family in the inactive form (47). Both RACK and RICK binding sites reside partly within the C1 domain. There are at least two protein-protein interaction sites within this phorbol ester-binding domain (36). The C2 domain has dual roles in the regulation of PKC-{alpha} activity. In addition to its proposed lipid or Ca2+/lipid-binding sites, this domain regulates protein-protein interactions. The C2 region contains the RACK binding site (35, 37) and the GAP-43 binding site in PKC-{delta} (7). The C2 region in PKC-{beta} also possesses the pseudo-RACK binding site (38). The RACKs bind PKC in its active conformation and increase the PKC phosphorylation of substrates (36). Rho1p was found to bind to a fragment of PKC1p containing the NH2-terminal pseudosubstrate and the C1 domain. The studies in yeast provide evidence for a GTP-dependent direct interaction between Rho1p and PKC1p (15, 29). In mammalian cells, it was found that one of the sites of interaction of Cdc42 with PKC-{lambda} and PKC-{zeta} was contained within the regulatory domain of PKC-{lambda}. This finding is consistent with a direct interaction between PKC and Rho GTPase (6). In the present study, we found that the RhoA binding sites exist within the regulatory domain, which provides further evidence of the direct interaction between PKC and Rho GTPase.

The COOH terminus is largely conserved among the different PKC isoforms, suggesting the possibility of common binding sites, which bestows a possible role of the catalytic domain in targeting PKC isozymes to specific cellular sites and possible interactions with other proteins. Our data indicate the presence of possible RhoA binding sites in the C1, C2, and C4 domains of PKC-{alpha}. However, PKC-{alpha} (C1, C2, and C3) did not bind to GST-RhoA. Furthermore, the fragments PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) bound to RhoA better than to PKC-{alpha} (C1 and C2). Because the C1 and C2 domains individually and in combination (C1 and C2) could bind to RhoA in vitro (Fig. 8), it is possible that the C3 domain interferes with the binding of C1, C2, and C3 domains with RhoA. However, PKC-{alpha} (C1 and C2) could not be immunoprecipitated with RhoA in cells transfected with constructs of PKC-{alpha} (C1 and C2). It is possible that the secondary structural modifications such as glycosylation of the protein molecules that occur in the eukaryotic translation may affect the structure of the peptides produced. This phenomenon might possibly alter the binding properties of PKC-{alpha} with RhoA in vivo (32). The presence of the C4 domain at the COOH-terminal end of the protein may help to stabilize the structure that may otherwise be altered by C3, which might explain the discrepancies in the observed binding properties of the C1, C2, and C3 and the C2, C3, and C4 fragments. The results of both in vitro binding assays and coimmunoprecipitation studies in transfected cells confirm that the C4 domain may play a role in structural stabilization in the binding of PKC-{alpha} to RhoA. Leinweber et al. (20) found that the C2 and C1B domains of PKC-{alpha} interacted in the intact regulatory domain, because both C2 and C1B bound to the cytoskeletal protein calponin better than to the whole regulatory domain. In a study of regulation of PKC-{alpha} activity, Slater et al. (39) found that the existence of C1-C2 domain interactions retained the PKC-{alpha} molecule in an inactive conformation. This could explain the differences observed in the binding of C1 and C2 in vitro and in cells (i.e., C1 and C2 domains that were expressed in bacterial cells without the glycosylation bound to RhoA in vitro, while a possible inactive conformation present in the cells inhibited its binding with RhoA). This activating conformational change may be required for the C1 and C2 domains to enable their interaction with RhoA in vivo. Our results indicate that in transfected smooth muscle cells, PKC-{alpha} (C1, C2, and C3) and PKC-{alpha} (C2 and C3) did not bind to RhoA, which is consistent with our in vitro binding assay results. In cells, the membrane association of PKC occurs through the C1 or C2 domain (27). The binding of C1 and C2 domains to the membrane may block the RhoA binding sites on PKC-{alpha}, which affects the binding of PKC-{alpha} (C1 and C2) to RhoA in cells (Figs. 8 and 10). Our data suggest that the C3 domain renders inactive conformation to C1 and C2 and that in vitro it prevents RhoA from binding to PKC-{alpha} that lacks the C4 domain. In in vivo situations, recruitment of PKC to membranes by both the C1 and C2 domains results in a remarkably high-affinity interaction that depends on the presence of both DAG (or phorbol esters) and PS. This tight binding results in a release of the pseudosubstrate from the active site, thus allowing substrate binding and catalysis (27).

All PKCs, with the possible exception of PKC-µ, contain an autoinhibitory pseudosubstrate domain that maintains PKC in an inactive conformation by sterically blocking the active site. Activation of PKC is always coupled to removal of this autoinhibitory domain from the active site (27). Newton (26) proposed the model for PKC's regulation. PKC adopts a conformation such that pseudosubstrate occupies the active site of the catalytic domain of PKC. The high-affinity binding of the pseudosubstrate sequence to the catalytic cleft blocks substrate access and hence catalytic activity, thus maintaining it in the inactive form (27). Our results indicate that PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) bound to RhoA in vitro. Furthermore, in cells transfected with these fragments, their association with RhoA increased upon stimulation with acetylcholine. The constructs of PKC-{alpha} (C2, C3, and C4) and PKC-{alpha} (C3 and C4) may mimic the active form of PKC-{alpha}. Our results are consistent with the model in which activation of PKC-{alpha} by phorbol esters or Ca2+ exposes regions in the catalytic domains that interact with PKC-{alpha}-binding proteins (27) and may explain the ability of wild-type PKC-{alpha} to be translocated to the membranes under certain conditions.


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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-042876.


    ACKNOWLEDGMENTS
 
We thank Dr. Sita Somara for technical assistance. We thank Dr. S. B. Patil for useful discussions. The meticulous secretarial assistance of Kelly Smid and Shilow Blea are gratefully acknowledged.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Bitar, Dept. of Pediatrics, Univ. of Michigan Medical School, 1150 W. Medical Center Drive, MSRB I, Room A520, Ann Arbor, MI 48109-0658 (e-mail: bitar{at}umich.edu)

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.


    REFERENCES
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