|Molecular Vision 2002;
Received 23 October 2001 | Accepted 11 March 2002 | Published 14 March 2002
Effect of protein kinase Cg on gap junction disassembly in lens epithelial cells and retinal cells in culture
Lynn M. Wagner,1 Suha M.
Saleh,1 Daniel J. Boyle,2
Dolores J. Takemoto3
1Department of Anatomy and Physiology, 2Division of Biology, 3Department of Biochemistry, Kansas State University, Manhattan, KS
Correspondence to: Dolores J. Takemoto, Department of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, Kansas, 66506; Phone: (785) 532-7009; FAX: (785) 532-7278; email: firstname.lastname@example.org
Purpose: To determine the effects of protein kinase Cg (PKCg) on phosphorylation of Cx43, the gap junction protein of lens epithelial cells, and on cell surface assembly/disassembly of Cx43-gap junction complexes.
Methods: Association and phosphorylation of Cx43 by PKCg was determined using co-immunoprecipitation and reaction with phosphoserine antisera. Activation of PKCg was with 200 nM phorbol ester for 30 to 60 min. Effects of specific PKC isoforms was determined after overexpression of either PKCa or PKCg for 24 h in N/N 1003A rabbit lens epithelial cells or in two retinal cell lines, WERI and Y79. Gap junction plaques were counted on the cell surface by immunolabeling of Cx43 using confocal microscopy.
Results: Co-immunoprecipitation of Cx43 with PKCg was observed only in cells over expressing PKCg and in cells activated with phorbol ester. Both overexpression and phorbol ester produced a rapid phosphorylation of Cx43 on serine. Cx43 cell surface gap junction plaques decreased in cells over expressing PKCg and in cells treated with phorbol ester. Similar results were observed using the retinal cell lines, WERI and Y79. The effect of PKCg overexpression was persistent for 7 days but total cell Cx43 was not decreased. Overexpression of PKCa resulted in an increase in cell surface gap junction plaques.
Conclusions: PKCg can be co-immunoprecipitated with Cx43 from lens epithelial cells using phorbol ester activation. PKCg phosphorylates Cx43 on serine and this causes disassembly and loss of gap junction Cx43 from the cell surface. Overexpression of PKCg confirmed that only this PKC isoform caused the loss of cell surface Cx43. Overexpression of PKCa, the other major lens PKC isoform, caused an increase in cell surface Cx43. The presence of PKCg and loss of surface Cx43 from two retinal cell lines, WERI and Y79, upon phorbol ester activation further suggests that activation of PKCg may be a common mechanism for control of cell surface Cx43.
Most cells can communicate with adjacent cells by gap junctions . These membrane structures are clusters, called plaques, of intercellular channels that link the cytosols of adjoining cells and thereby act as direct pathways for the cell-to-cell transfer of small (under 1 kD) molecules [2,3]. The importance of gap junction intercellular communication is particularly obvious in the lens since it is an avascular tissue which relies on gap junctional channels for diffusional distribution of small molecules.
The lens is composed of two cell types; a monolayer of epithelial cells that overlay the anterior face and a core of elongated crystallin-rich fiber cells that are responsible for the refractive properties of the lens . In this tissue, the anterior epithelial cell monolayer, which interfaces with the aqueous humor, contains many of the transporters and ion pumps and helps to control homeostasis throughout the lens via an extensive network of gap junction channels .
A gap junction channel is formed by two hemichannels or connexons which are contributed by each of the adjacent cells. A connexon is an oligomeric unit of six subunits termed connexins (Cxs) . In the lens, gap junction proteins include Cx43, Cx46, and Cx50 [6,7]. While Cx46 and Cx50 are found in lens fiber cells, Cx43 is found in lens epithelial cells.
Many effectors of protein kinases modulate gap junctional communication, and it has been demonstrated that certain connexin proteins are phosphorylated [8,9]. Protein kinase Cg (PKCg) has been identified as an inhibitor of Cx43 gap junction activity in lens epithelial cells in culture . The mechanism of inhibition has not been defined, however, Cx46 is also phosphorylated by PKCg .
Cx43 is one of the major connexins in the body, and it is expressed in many tissues. Previous studies on lens gap junctions have identified Cx43 as epithelial cell specific [12-14]. Cx43 is a phosphoprotein  and its phosphorylation may be coupled to the assembly/disassembly of gap junction plaques, generation of functional channels , and regulation of channel pore size and open-state probability [15,16]. Cx43 has been shown to be serine phosphorylated in lens epithelial cells in situ and in primary cultures of lens cells [8-11,13,16-19].
Activation of PKC by phorbol ester has been found to inhibit gap junction activity [10,18-20]. The PKC isoform which alters gap junction activity is PKCg .
This study indicates that PKCg can be co-immunoprecipitated from lens epithelial cells with Cx43 and that PKCg mediates the phosphorylation of Cx43 on serine. Furthermore, only activated PKCg can accomplish this. Overexpression of PKCg and not PKCa caused disassembly of gap junctions in lens epithelial cells. This may be a mechanism by which PKCg causes inhibition of gap junction activity. The phorbol ester-induced disassembly of gap junctions in two retinal cell lines suggests that PKCg may also be involved in the control of retinal gap junction assembly.
N/N 1003A rabbit lens epithelial cells were cultured in Minimal Essential Medium, pH 7.2, supplemented with 10% fetal bovine serum and 50 mg/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. Retinal Y79 and Weri cells were grown as suspension cultures in 15% fetal calf serum in RPMI-1640 media. Cells were a gift of Dr. Stephen Pittler, University of Alabama, Birmingham, AL. In some cases, cells were treated with 200 nM phorbol ester (Sigma, St. Louis, MO) for 60 min or 1 mM Calphostin C (Sigma)  for 24 h prior to harvesting.
Transfection and overexpression of PKCg and PKCa
N/N1003A cells were cultured to 50% confluency, then rinsed with serum-free medium and treated with lipofectamine reagent (Life Technologies, Rockville, MD). The cells were transfected with eMTH vector which contained the empty vector, PKCa, or PKCg. eMTH vector is inducible with 20 mM zinc, and it is selectable with Geneticin (G418), as previously described [10,11]. All cells in these experiments were treated with 20 mM zinc at the start of the experiment. The empty vector transformed cells are referred to as control cells in these experiments.
Co-immunoprecipitation of PKCg with Cx43
Control N/N1003A cells (empty vector transfected cells) and N/N1003A cells overexpressing PKCa or PKCg and phorbol ester-treated cells (untransfected) were cultured to 90% confluency in 75 cm2 tissue culture flasks, then, the cells were harvested and lysed on ice with 0.5 ml ice cold cell lysis buffer. The cell lysis buffer contained 20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin. The lysed cells were homogenized and centrifuged at 20,000x g for 20 min, then the supernatant was collected and antibodies specific for PKCg or PKCa (Transduction Laboratories, San Diego, CA; 1:1000 dilution), were added to the mixture which was a final concentration of 5 mg/ml, then, incubated overnight at 4 °C with constant mixing. After overnight incubation with PKCg or PKCa antibodies, 20 ml of protein A sepharose beads (50:50 solution in lysis buffer; Sigma) were added to the mixture and the mixture was further incubated for another two h on ice. The mixture was centrifuged at 11,900x g for 25 s and the precipitate was collected and washed four times with lysis buffer. The washed precipitate was then mixed with 20 ml Tris-glycine/SDS sample buffer and boiled for five min. Proteins were separated by 12.5% SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Cx43 antibody (Chemicon, Temecula, CA; 1:1000 dilution), or other antisera as indicated. Immunoreactive bands were detected by chemiluminescence (ECL, Pierce, Rockford, IL).
Phosphorylation of connexin 43 by PKCg
Control N/N1003A cells (empty vector transfected) and N/N1003A cells overexpressing PKCa or PKCg were cultured to 90% confluency then harvested, rinsed with PBS, and lysed with lysis buffer, which contained 20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin. The cell lysate was centrifuged for 20 min at 20,000x g and the supernatant was used to immunoprecipitate Cx43 as described above. Immunoprecipitated Cx43 was analyzed by 12.5% SDS-PAGE and the separated proteins were transferred to a nitrocellulose membrane and probed with anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibodies (Chemicon; 1:1000 dilution).
Translocation of PKCa and PKCg to membrane fractions
Control N/N 1003A cells (untransfected) or cells treated with either 200 nM phorbol ester (Sigma) for 1 h or 1 mM Calphostin C (Sigma, a commercially available PKC inhibitor ) for 24 h were harvested in 50 mM Tris, 20 mM MgCl2 (pH 7.5) and sonicated. The cell lysate was centrifuged for 1 h at 100,000x g at 4 °C. The supernatant and the pellet were saved and analyzed on a 7.5% SDS-PAGE gel with 20 mg of protein loaded per lane and probed with anti-PKCa or anti-PKCg antibody (Transduction Laboratories; 1:1000 dilution).
Confocal microscopy of gap junction plaques from phorbol ester-treated cells
N/N 1003A lens epithelial cells (untransfected, 1.0x106 total cells), and Weri or Y79 (1.0x105/ml) retinal cells were treated with 200 nM phorbol ester for 1 h or 1 mM Calphostin C for 24 h prior to fixation. The cells were fixed for 10 min with 2.5% paraformaldehyde in phosphate buffered saline (PBS). To label lens Cx43 and PKCg, the primary antisera was diluted in blocking buffer (3% BSA in PBS) and then added to the fixed cells and incubated for 18 h at room temperature with; anti-PKCg (green color, mouse host, Transduction Laboratories; 1:1000 dilution) and anti-Cx43 (red color; rabbit host; Zymed; 1:1000 dilution). The fixed cells were then washed 3 times with blocking buffer and incubated with two types of secondary antisera (working concentration was 7 mg/ml in blocking buffer); Alexa fluor 488 (Molecular Probes, Eugene, OR) which is goat anti-mouse and has an excitation/emission wavelength of 495/519 and emits green, and Alexa fluor 568 (Molecular Probes) which is goat anti-rabbit and has an excitation/emission wavelength of 578/603 and emits red. The cells were then washed with blocking buffer and mounted on slides with 1% glycerol in PBS. Slides were examined using a laser scanning confocal microscope (Zeiss; Thornwood, NY). The cells were examined using a 63x/1.4 oil immersion objective with an excitation filter of KP 600 and the emission filters were BP 515-540 for the green signal and LP 590 for the red signal. The dichroic beam splitter was FT 560, the pinhole was 10, and the scanning time was 8 s. The sample size was between 7 and 11 for control, TPA-treated, and Calphostin C-treated cells. The number of plaques per micrograph were determined using Scion Image software (Scion Corporation; Frederick, MD). After plaque numbers per micrograph were generated for each sample set, the average number of plaques per square micrometer of area analyzed was determined and a Student's t-test for paired or unpaired values with a sample of less than 30 was used to determine significant differences in plaque number between sample sets. Values of p<0.05 were considered to be statistically significant.
Confocal microscopy of gap junction plaques from cells overexpressing PKCa and PKCg
Plaque numbers in N/N 1003A cells overexpressing PKCa, PKCg, or empty-vector control cells were analyzed as described above. The sample size for each time period was between 7 and 11 for 24 h, 3 days, 5 days, and 7 days of PKCa and PKCg overexpression. The working dilution of the primary antisera was as follows; anti-PKCa (mouse host; Transduction Laboratories; 1:1000 dilution) and anti-PKCg (mouse host; Transduction Laboratories; 1:1000 dilution).
Co-immunoprecipitation of PKCg with Cx43
PKCg was immunoprecipitated from empty-vector transfected N/N1003A cells (Figure 1A, lane 1), cells overexpressing PKCg (Figure 1A, lane 2), or PKCa (Figure 1A, lane 3) using protein A sepharose in the presence of PKC antibodies. After analyzing for equal protein load, the nitrocellulose membranes were probed with anti Cx43 antibodies. Only western blots from the N/N1003A cells which overexpressed PKCg reacted with anti-Cx43 antibodies in a separate and distinct band at 43 kD (Figure 1B, lane 2). In all samples there was a reactive band at about 56 kDa which represents the immunoprecipitated IgG fraction of the primary antisera (Figure 1B). These higher molecular weight bands were also detected in the control experiments in which no cell lysate was added to the experimental mixture (Figure 1B, lanes 4,5,6). In additional experiments, PKCg was found to be immunoprecipitable using Cx43 antisera (data not shown). As shown in Figure 1B, PKCg in the empty-vector transfected cells (N/N1003A, lane 1) did not immunoprecipitate Cx43, although these cells do have endogenous PKCg and Cx43. A possible explanation is that the endogenous PKCg in N/N1003A cells is inactive and therefore does not interact with Cx43 until activated. To determine if activation of PKCg is required for interaction with Cx43, control N/N1003A cells (untransfected) were incubated with the PKC activator TPA (12-O-tetradecanoylphorbol-13-acetate, 200 nM), for 60 min  to activate endogenous PKCs. The results demonstrated that the active PKCg, after treatment with TPA, did co-immunoprecipitate with Cx43 in control, untransfected N/N1003A cells (Figure 2A).
Phosphorylation of Cx43 by PKCg
Cx43 is a phosphoprotein, and its phosphorylation is considered to have a regulatory mechanism in gap junction communication [1,19]. It has been documented that Cx43 is phosphorylated at a serine residue by a PKC isoform [19,20]. In our study, overexpression of PKCg resulted in enhanced serine phosphorylation of Cx43 (Figure 3A, lane 2). No phosphotyrosine (Figure 3A, lane 1) or phosphothreonine was detected in samples of Cx43 which were co-immunoprecipitated with PKCg (Figure 3A, lane 3).
In parallel experiments, total Cx43 was immunoprecipitated with Cx43 antibodies. When Cx43 was analyzed using anti Cx43-immunoprecipitation the Cx43 was found to be phosphorylated on both serine and threonine (data not shown). This suggests that there may be a pool of differentially phosphorylated Cx43, co-immunoprecipitable with PKCg, which is only phosphorylated on serine. Likewise, Cx43 which co-immunoprecipitated with TPA-activated PKCg also showed enhanced phosphorylation of serine on Cx43 (Figure 2B, lane 2).
Disassembly of Cx43 plaques as a result of phorbol ester addition
The addition of 200 nM phorbol ester (for 60 min) to untransfected N/N1003A cells caused PKCg and PKCa to translocate to membrane fractions (Figure 4). Since membrane translocation is known to activate PKCs, the effect on Cx43 gap junction plaques was determined. Control untransfected N/N 1003A cells (n=10) or N/N 1003A cells treated with 200 nM TPA for 60 min (n=10) were fixed and stained with anti-PKCg antibody (green) and anti-Cx43 antibody (red). Control Weri cells (n=8) and control Y79 cells (n=7), or Weri and Y79 cells treated with 200 nM TPA for sixty min (Weri, n=7; Y79, n=8) were also fixed and stained as above. The N/N 1003A cells, and Y79 or Weri cells treated with TPA, all showed a 49-56% decrease in gap junction plaques compared to their paired control cells (Figure 5A and Figure 5B). There was not a significant difference between Calphostin C-treated cells (a PKC inhibitor) compared to control cells for the three cell types. This suggests that under control conditions endogenous PKCs are not active. Although we do not know which PKCs are responsible for the decreased gap junction plaques observed in the Y79 and Weri cells, both contain PKCa and PKCg (Figure 5C). Although there was a decrease in gap junction plaques at the surface of TPA-treated lens epithelial cells and retinal cells, there was not any decrease in total cell Cx43 protein (Figure 6A).
Disassembly of Cx43 plaques as a result of long-term PKCg overexpression
Control (empty-vector transfected) N/N 1003A cells (n=10) or N/N 1003A cells overexpressing PKCg for 24 h (n=11), 3 days (n=10), 5 days (n=9), and 7 days (n=9) were fixed and stained with anti-PKCg antibody (green) and anti-Cx43 antibody (red). The fixed and stained cells were viewed under a laser scanning confocal microscope and the number of Cx43 plaques per square micrometer was counted. The cells overexpressing PKCg showed a significant decrease in gap junction plaques when compared to control cells (24 h overexpression, 73% decrease; 3 days overexpression, 87% decrease; 5 days overexpression, 80% decrease; 7 days overexpression, 84% decrease; Figure 7A and Figure 7B). Despite the decrease in gap junction plaques at the surface of lens epithelial cells, there was not a decrease in total cell Cx43 protein observed for up to 7 days (Figure 6B).
Disassembly of Cx43 plaques as a result of PKCg, not PKCa Overexpression
Control (empty-vector transfected) N/N 1003A cells (n=10) or N/N 1003A cells overexpressing PKCa for 24 h (n=8), 3 days (n=9), 5 days (n=7), and 7 days (n=8) were fixed and stained with anti-PKCa antibody (green) and anti-Cx43 antibody (red). The fixed and stained cells were viewed under a laser scanning confocal microscope. The N/N 1003A cells overexpressing PKCa showed a significant increase in gap junction plaques compared to control N/N 1003A cells (24 h overexpression, 55% increase; 3 days overexpression, 60% increase; 5 days overexpression, 56% increase; 7 days overexpression, 60% increase; Figure 8A and Figure 8C). Comparing cells overexpressing PKCg and cells overexpressing PKCa over the four time periods, there was a significant decrease in gap junction plaques in cells overexpressing PKCg (24 h overexpression, 85% decrease; 3 days overexpression, 93% decrease; 5 days overexpression, 89% decrease; 7 days overexpression, 90% decrease, Figure 8B and Figure 8C). Although there was a decrease in gap junction plaques at the surface in lens epithelial cells overexpressing PKCg, there was not observed a decrease in total cell Cx43 protein observed (Figure 6B). Likewise, no changes in Cx43 protein levels were observed in cells overexpressing PKCa (Figure 6B).
PKCg has been detected in the central nervous system where it is thought to be involved in the formation of neural plasticity and memory [21-24]. It is also present in the lens . Although PKCg has been found to inhibit gap junction dye transfer activity in lens epithelial cells , no previous work has been done to determine how PKCg alters gap junction activity.
In this study the association between lens PKCg and Cx43 was further investigated. This work was prompted by reports of TPA inhibition of gap junction dye transfer activity [18-20]. Although TPA does activate PKCs, it has other unrelated effects. Therefore, it was necessary to identify a PKC isoform as the effector of gap junction inhibition. This was done using overexpression of PKCg . The current work extends these findings by demonstrating an immunoprecipitable interaction of Cx43 with PKCg. This interaction enhances serine phosphorylation of Cx43. However, total Cx43 is phosphorylated on both serine and threonine. This suggests that there may be a pool of Cx43 which is predominantly serine phosphorylated which may interact with PKCg. Furthermore, this PKCg appears to be under tight regulatory control, since co-immunoprecipitation of Cx43 with endogenous PKCg is not observed unless PKCg is activated by a phorbol ester such as TPA.
It can be proposed that PKCg inhibits the gap junctions in lens epithelial cells through interaction with and phosphorylation of Cx43 on serine. Phosphorylation of Cx43 may be coupled to the assembly of gap junction plaques and/or generation of functional channels , or to regulation of channel pore size and open-state probability [26-28]. The physiological effector of lens epithelial cell PKCg remain unknown.
Previous studies have shown that TPA, a potent activator of conventional PKCs, can affect gap junction assembly by affecting the trafficking of connexins to the plasma membrane or connexin-connexin assembly at the plasma membrane [10,29-31]. These studies have demonstrated that TPA significantly reduced the assembly of gap junctions. Overexpression of PKCg also significantly decreases cell surface gap junction plaques. Confocal microscopy confirmed that gap junction activity  and plaques (this paper) were significantly reduced for TPA-treated cells and cells overexpressing PKCg compared to that of control cells, while there were no changes in the levels of total cellular Cx43 protein. These results support a number of other reports that TPA treatment decreases cell communication with little or no changes in Cx43 expression [30-32]. There is a direct correlation between the effects of TPA-treated cells and the overexpression of PKCg. TPA will activate conventional PKCs, of which PKCg is a member, thus increasing the amount of PKCg at the plasma membrane. Overexpression of PKCg will cause an increase in PKCg activity throughout the cell . Both overexpression and TPA activation reduced cell surface gap junction plaques at early time periods. This suggests that the reported decrease in gap junction dye transfer  after these treatments is due to a loss of cell surface Cx43.
PKCa appears to provide a positive signal to the gap junction assembly/disassembly process. Even at early time periods the increase in gap junctions after PKCa overexpression could result from either enhanced assembly or prevention of disassembly or both. Since overexpression of PKCa does not induce Cx43-PKCa co-immunoprecipitation or phosphorylation of Cx43 the effect of PKCa may be more indirect. Long-term overexpression of PKCa induces lentoid body formation and expression of crystallins in addition to the observed plaque increase . Therefore, long-term overexpression of PKCa may cause other cellular changes besides increased cell surface Cx43.
However, the overriding signal, once PKCa and PKCg are both activated by TPA, is PKCg, resulting in decreases in gap junctions at the surface after only 60 min. PKCg is thought to be a PKC isoform which is specific to neurally-derived cells. The retinal cell lines, Y79 and Weri, also exhibit decreased surface gap junctions after TPA-treatment, and both contain PKCa and PKCg. It will be of interest to determine if PKCa and PKCg exert reciprocal controls on gap junction assembly in other neurally-derived cells as well.
The authors thank Dr. John Reddan (Oakland University) for providing the N/N1003A cells, Dr. Wayne Anderson (NCI) for providing PKCg and PKCa plasmids, Dr. Peggy Zelenka (National Eye Institute), and Dr. Anna Zolkiewska (Kansas State University) for their useful technical advice, and Dr. Steven Pittler (University of Alabama at Birmingham) for providing the Weri and Y79 cells. We gratefully acknowledge the support of the Kansas State University Cancer Center for travel support and NIH Grant EY13421. This is publication 02-130-J from the Kansas Agricultural Experiment Station.
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