|Molecular Vision 2001;
Received 24 April 2001 | Accepted 12 October 2001 | Published 24 October 2001
PKC-g phosphorylation of connexin 46 in the lens cortex
Suha M. Saleh,1 Larry J.
Zoukhri,3 Dolores J. Takemoto4
1Department of Anatomy and Physiology, 2Division of Biology, and 4Department of Biochemistry, Kansas State University, Manhattan, KS; 3Department of Ophthalmology, Harvard Medical School Boston, MA
Correspondence to: Dr. Dolores J. Takemoto, Department of Biochemistry, 103 Willard Hall, Kansas State University Manhattan, Kansas, 66506; Phone: (785) 532-7009; FAX: (785) 532-7278; email: email@example.com
Purpose: To identify the role of PKC-g in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex.
Methods: The association between PKC-g and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-g were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors.
Results: Co-localization of PKC-g and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-g was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-a was not found in lens cortex or nuclear regions. PKC-g could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-g specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 mM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-a pseudosubstrate inhibitor was not observed.
Conclusions: PKC-g may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-g in control of lens cortical gap junctions is suggested.
PKC-g is a member of the classical group of the PKC family of serine/threonine kinases [1-6]. It plays a regulatory role in memory in the central nervous system [7-9] and in regulation of gap junction intercellular communication in the lens epithelial cells .
The lens is dependent upon intercellular communication through gap junctions which form between epithelial monolayers of cells in culture, between fiber cells, in the whole lens, and between the epithelium and enucleated fiber cells of the whole lens [11-14]. It has been proposed that gap junctions in the lens epithelial cells are regulated, in part, by PKC's through phosphorylation of Cx43 [10,15]. Lens fiber cells are also extensively coupled by gap junctions [16,17] and these fiber cells are classified into two distinct age-defined regions, the fiber cells in the lens cortex and the older fiber cells in the lens nucleus .
The cortical fiber cells are particularly rich in gap junction structures which contain two connexins, connexin 46 and connexin 50 [19-21], which are both essential for the normal function of the lens [22,23]. This is due to the fact that the lens is an avascular tissue which relies upon intercellular communication via gap junctions to provide a pathway for nutrient uptake and waste product removal . Disruption of the Cx46 gene, for example, results in formation of nuclear cataracts which are associated with the proteolysis of crystallins [22,25]. Gap junction channels are unlikely to be directly responsible for the cleavage of these crystallins. Rather, the proteolysis of crystallins may occur via downstream events that result from the lack of cell-cell movement of molecular information via the gap junction channels that are formed by Cx46. It was suggested that phosphorylation/dephosphorylation events control the formation and/or activity of Cx46 hemichannels in the lens cortex . A role for PKC's in gap junction activity of Cx43 in lens epithelial cells has been reported using phorbol-ester activation  and overexpression of PKC's . In lens epithelial cells PKC-g inhibits gap junction activity . In this study a role for PKC-g, through Cx46 phosphorylation in lens fiber cells, was examined.
Preparation of lens sections
Fresh lenses from five to seven month old rats were used in the confocal microscopy experiments. To obtain the lenses, rat eyes were removed within 15 min of animal death and washed with phosphate buffered saline (PBS, 2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4, pH 7.2). The lenses were removed, washed with PBS and fixed for 24 h at 4 °C in fixative containing 2% paraformaldehyde and 0.2% glutaraldehyde in PBS. The fixed lenses were oriented under the dissection microscope so that the anterior of the lens was up. The lenses were then cross sectioned by vibratome into 100 mM sections and fixed again for another 24 h in the same fixative.
Localization studies and confocal microscopy
A laser scanning confocal microscope equipped with an Argon-Krypton mixed ion laser was used for co-localization of Cx46 and PKC-g. For these experiments, fixed lens sections were incubated in 50 mM glycine in PBS for 10 min to quench any free aldehydes created from the fixation. This was followed by washing the lens sections several times in PBS. Blocking of nonspecific sites was performed by incubating the sections in 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. To label lens Cx46 and PKC-g, the primary antisera was diluted in blocking buffer (3% BSA in PBS) and then added to the lens sections and incubated for 18 h at room temperature. The working dilution of the primary antisera was as follows; anti-PKC-g (1:1000, host: mouse, Transduction Laboratories, San Diego, CA) and anti-Cx46 (1:1000, host: rabbit, a gift from Dr. Larry Takemoto, Kansas State University, Manhattan, KS). Lens sections were then washed several times with blocking buffer and incubated with the secondary antisera which was attached to a fluorochrome and has specific excitation and emission wavelengths. Two types of secondary antisera were used (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) is goat anti-rabbit and has an excitation/emission wavelength of 578/603 and emits red. The sections were washed with blocking buffer and mounted on slides with anti-fading reagent that contained 0.1% r-phenylenediamine in 90% glycerol, pH 8.5. Slides were examined using a laser scanning confocal microscope. The lens sections were examined using a 63x 1.4 oil immersion objective. The excitation filter was KP 600, 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 between 18-20 and the scanning time was 8 s.
For co-localization experiments, lens sections were labeled with two fluorochrome-attached secondary antisera at the same time. Each antiserum had a different wavelength of excitation and emission. After adding the blocking buffer to the lens sections, two primary antisera were added at the same time (anti-PKC-g and anti-Cx46). The lens sections were labeled with the Alexa fluor 488 and Alexa fluor 568 at the same time.
Preparation of cortical fiber cells
Whole rat eyes (three for each data point) were obtained frozen from Pel-Freez (Rogers, AR, frozen immediately after the animal's death). After thawing, the lens were dissected and washed in PBS, pH 7.2, then immediately dissected under the dissecting microscope. Fresh lenses were initially used and results of immunoprecipitation and phosphorylation were found to be identical to the data obtained with frozen lenses. However, since approximately 500 lenses were used in these experiments, frozen lenses were used for most studies.
Using both dissection scissors and forceps, the lens capsule, along with the epithelial monolayer, was carefully removed, and the cortical fiber cells were dissected away from the nuclear fibers cells and washed in 500 ml PBS. The isolated cortical fiber cells were then used as controls or incubated with the pseudosubstrate inhibitory peptide in Minimal Essential Medium at 37 °C for 1 h, and then lysed with lysis buffer (see below) on ice and used for experiments. Cortical fiber cell preparations contained cells from the outer cortex and bow region. Pseudosubstrate inhibitors were synthesized as previously described [26-30].
Co-immunoprecipitation and phosphorylation of Cx46
Cortical fiber cells from three lenses per data point were dissected and prepared as described above. Lysis of the fiber cells was performed on ice by adding 0.5 ml lysis buffer per sample (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) to the dissected cortical regions from three lenses. The lysed cells were homogenized and centrifuged at 13,000 rpm (20,000x g) for 20 min, then the supernatant (containing about 10% of total PKC-g and 50% of total Cx46) was collected (approximately 300 mg total protein). To reduce the non-specific adsorption, the supernatant was pre-cleared by incubation with protein G agarose beads and pre-immune sera (1:1000) in 2.5% bovine serum albumin (BSA) at 4 °C for 15 min. The samples were then centrifuged and the pellet was discarded. The cleared supernatants (approximately 200 mg total protein) were then mixed with anti-Cx46 antibodies at 5 mg/ml and incubated overnight at 4 °C with constant mixing. Protein G agarose beads (20 ml, 1:1, v/v, in lysis buffer) were added to the samples and they were incubated for another 2 h at 4 °C. The samples were washed three times with lysis buffer and centrifuged at 10,000 rpm (11,900x g) for 25 s. The immunoprecipitates (approximately 5 mg total protein) were mixed with 20 ml of SDS sample buffer, boiled for 5 min and eluted proteins were analyzed on 12.5% SDS-PAGE and western blotting. The transferred proteins on the nitrocellulose membranes were probed with anti-PKC-g, anti-phosphoserine, anti-phosphotyrosine, anti-phosphothreonine, or anti-Cx46 antibodies at 1:1000 and visualized by enhanced chemiluminescense (ECL, Pierce, Rockford, IL).
PKC pseudosubstrate peptides: Effect on phosphorylation of Cx46
Lens cortical fiber cells from three lenses for each data point were isolated as described above, washed in PBS, re-suspended in Minimum Essential Medium and incubated with different concentrations of PKC-g or PKC-a N-myristoylated pseudosubstrate peptide. The N-myristoylated pseudosubstrate peptides are cell permeable and have been tested in numerous cell types [27-30]. They inhibit specific PKC isoforms by binding to the pseudosubstrate/substrate site. The sequence for the pseudosubstrate peptide for PKC-g was (FARKGALRQ), and for PKC-a was (DVANRFARKGALRQ). The concentrations of the pseudosubstrate peptides that were added to the cells were 10 nM, 100 nM, and 1 mM. As a control, cortical fiber cells were incubated with media without pseudosubstrate peptide. The fiber cells were incubated with the pseudosubstrate peptide for 1 h at 37 °C. Following incubations, samples were immunoprecipitated as described above.
Effect of PKC-a and PKC-g pseudosubstrate peptides on PKC activity
Cortical fiber cells were isolated and treated with PKC-a/PKC-g pseudosubstrate peptides for 1 h on ice, then the cells were homogenized on ice with modified lysis buffer containing 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 25 mg/ml aprotinin, 25 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). To determine the PKC activity in the cortical fiber cells, a non-radioactive PKC assay kit (Promega, Madison, WI) was used. In this assay, a fluorescent peptide substrate that is highly specific for PKC was used. The assay was initiated by the addition of substrate. The phosphorylation of that peptide by PKC alters its charge from +1 to -1 and that allows the rapid separation of the phosphorylated and nonphosphorylated species of the substrate on an agarose gel at neutral pH. The results are presented as units of PKC activity per ml of sample. One unit of kinase activity is defined as the number of nanomoles of phosphate transferred to a mole of substrate per min per mg protein. The units of PKC activity in each sample are presented as the mean of the results of three different experiments, in triplicate. Substrate concentration was 100 mM ATP, 10 mM MgCl2, and 1 mM peptide. The assay time was 10 min at 37 °C.
Western blot analysis of PKC-g and PKC-a
Whole cortical fiber cells were dissected and washed in PBS, then homogenized and mixed with SDS sample buffer and boiled for 5 min. The samples were analyzed on 10% SDS-PAGE and by western blotting. The transferred proteins on the nitrocellulose membranes were probed with anti-PKC-g and anti-PKC-a antibodies as described previously . After exposure, the X-ray films were scanned and quantitated (Un-Scan-It, Silk Scientific Inc., Orem, UT).
Co-localization of PKC-g and Cx46
Co-localization of PKC-g and Cx46 was examined in different regions of the lens, including the lens epithelium, the bow region, the cortical fibers and the nuclear fibers. In the lens, the nucleated epithelial cells differentiate into fiber cells in the bow region. The mature cells in the inner cortex and nucleus of the lens are enucleated. Epithelial cells contain Cx43, while fiber cells and differentiating lens epithelial cells contain Cx46 and Cx50 . Our results, using confocal immunolocalization, demonstrate the presence of Cx46 (red) in the bow region, cortex and nucleus (Figure 1). PKC-g is localized in the epithelial cells, bow region and cortex (green, Figure 1).
PKC-g co-localized with Cx46 in the bow region and cortex (Figure 2B,C, yellow). Quantitative analysis for the co-localization of PKC-g and Cx46 was determined by calculating the number of co-localized pixels as shown in Figure 2E. PKC-a, a PKC isoform found in lens epithelial cells , is not present in cortical regions of whole lens (Figure 3).
Cx46 is phosphorylated on serine and threonine
Cx46 was immunoprecipitated from the cortical fiber cells and analyzed by SDS-PAGE and western blotting. The transferred proteins on the nitrocellulose membranes were probed with anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibodies. The results indicated that Cx46 is phosphorylated on serine (Figure 4A, lane 1) and threonine (Figure 4A, lane 3) but not on tyrosine (Figure 4A, lane 2). To confirm the immunoprecipitation of equal amounts of Cx46, the western blots were probed with anti Cx46, and the results indicated the presence of equal Cx46 (Figure 4B). This precipitation accounted for approximately 50% of the Cx46. Antisera is polyclonal and not directed to a phosphorylation site.
Cx46 and PKC-g can be co-immnunoprecipitated
Cx46 was immunoprecipitated from dissected cortical regions of whole lens using anti-Cx46 antisera, analyzed by SDS-PAGE and western blotting, then probed with anti-PKC-g antibodies. PKC-g (about 10% of total PKC-g) in the cortical fiber cells co-immunoprecipitated with Cx46 (Figure 5C,D). This may represent that fraction of PKC-g which is active. In our experience, phorbol ester will activate about 40% of endogenous PKC-g. Thus without exogenous and unnatural activator, only a small fraction of PKC-g may be active and in association with Cx46.
Phosphorylation of Cx46 is inhibited by PKC-g pseudosubstrate inhibitory peptide
To determine the role of PKC-g in phosphorylation of serine and threonine residues on Cx46, an inhibitory pseudosubstrate peptide specific for PKC-g was used to inhibit PKC-g activity in cortical fiber cells. PKC-g activity was determined by both inhibition of total enzyme activity and inhibition of Cx46 phosphorylation. As a control, an inhibitory pseudosubstrate peptide specific for PKC-a was used. PKC-g pseudosubstrate peptide inhibited serine phosphorylation on Cx46 by 73% at 1 mM of PKC-g pseudosubstrate peptide (Figure 5A,B). The pseudosubstrate peptide did not inhibit the co-immunoprecipitation of Cx46 and PKC-g as shown in Figure 5C,D. This suggests that the binding site on PKC-g is not at the pseudosubstrate region.
Threonine phosphorylation on Cx46 was also inhibited by PKC-g pseudosubstrate peptide (36% inhibition at 1 mM; Figure 6A,B) but the effect was not as apparent as it was for serine phosphorylation. The PKC-a pseudosubstrate peptide did not inhibit serine or threonine phosphorylation on Cx46 [data not shown].
PKC-g pseudosubstrate inhibitory peptide inhibits PKC activity
PKC activity was determined in cortical fiber cells with and without treatment with PKC-g or PKC-a pseudosubstrate inhibitory peptides. Cortical fiber cells were incubated with different concentrations of PKC-g or PKC-a pseudosubstrate peptides. PKC-g pseudosubstrate peptide inhibited total PKC activity by 53% at 1 mM. PKC-a pseudosubstrate peptide caused a slight decrease in PKC activity (20% at 1 mM, Figure 7). Residual PKC-g activity could reflect an inability of the pseudosubstrate peptide to permeate into intact cells (note modified lysis buffer). N-myristoylation aids permeability somewhat.
PKC-g has been implicated in inhibition of gap junction channels in the lens epithelial cells, where it is involved in phosphorylation of Cx43 on serine residues . PKC-g can be detected in the lens epithelial cells and in the fiber cells of the bow region and cortex of whole lens. It co-localizes with Cx46 in the outer cortical layers of the bow region, the area where epithelial cells begin to differentiate into fiber cells. Expression of the lens specific Cx46 and Cx50 are also apparent in these regions. PKC-g may be involved in regulation of cortical fiber gap junctions through phosphorylation of Cx46 and/or Cx50. Early studies, using phorbol esters to activate PKCs demonstrated that PKCs may inhibit assembly of gap junction plaques on membranes . Perhaps PKC-g phosphorylation of serine residues on Cx46 changes the assembly/disassembly process of Cx46 complexes. Cx46 from cortical regions was found to be phosphorylated on both serine and threonine. The involvement of PKC-g in serine and threonine phosphorylation on Cx46 was evaluated by inhibiting PKC-g activity in the cortical fiber cells with a PKC-g specific pseudosubstrate inhibitory peptide. The PKC-g pseudosubstrate peptide decreased PKC activity in cortical fiber cells, and inhibited serine phosphorylation on Cx46. It did not inhibit the co-immunoprecipitation of Cx46 and PKC-g, however. Therefore, PKC-g may interact with Cx46 at another site. The PKC-g pseudosubstrate peptide also inhibited threonine phosphorylation on Cx46, but the inhibition was not as great when compared to the inhibition of serine phosphorylation (73% for serine versus 36% for threonine). That may indicate that Cx46 and PKC-g are associated and that PKC-g may modulate the gap junctions in the cortical fibers by phosphorylating Cx46 more specifically on serine. Other kinases in the cortical fiber cells may work along with PKC-g to phosphorylate threonine on Cx46.
PKC-g is an isoform of the classical PKC category . This PKC is normally not active and is located in cystolic fractions. Upon activation with natural lipid and calcium, PKC-g will translocate to membrane fractions transiently and will phosphorylate substrates. Prolonged activation by the unnatural lipid class of phorbol esters will cause persistent translocation, activation, cleavage, and down-regulation. Although activation and PKC substrate interactions are transient, PKC-g has a region within the C1 domain which stably binds neural proteins . This may allow co-immunoprecipitation to occur.
Cx46 is a phosphoprotein, however, neither the phosphorylation sites on Cx46 nor the kinases involved in Cx46 phosphorylation are completely identified. Phorbol esters alter the activation and inactivation behavior of the Cx46 hemichannels . It is suggested that phosphorylation/dephosphorylation processes may be involved in controlling the Cx46 hemichannels . This is supported by the results of this paper which indicate that inhibition of PKC-g is associated with a 73% decrease in serine phosphorylation and 36% decrease in threonine phosphorylation on Cx46. It can be suggested that PKC-g phosphorylates Cx46 more specifically on serine residues, while other protein kinases may also phosphorylate Cx46 on threonine.
Phosphorylation of Cx46 has been studied using both recombinant Cx46 in Xenopus laevis oocytes [26,35] and in lens fiber cells . In Xenopus, Cx46 is phosphorylated on serine in response to phorbol ester and this results in a reduction in pH gating [26,35]. The effect is rapid, a large fraction is phosphorylated, and the effect is transient. However, in intact lens the Cx46 has only a low level of phosphorylation and phosphorylation is slow. This reflects the relatively slow metabolic rate in fiber cells . In our experiments, we find that the percent of phosphorylated Cx46 is high (approximately 80%) and PKC-g activity and phosphorylation rates are similar to other cells. It remains to be seen if there is a rapid phosphorylation/dephosphorylation of Cx46 in lens fiber cells. In our work, we measured the phosphorylation of Cx46 at single times, without phosphatase inhibitors. Thus, the data represent rates of phosphorylation and dephosphorylation. The PKC-g being measured is in its naturally active/inactive state since no exogenous activator is added. Nevertheless, the lens has a relatively high endogenous PKC activity, as determined by both the PKC assay and the ability to reduce phosphorylation of Cx46 by the pseudosubstrate peptide. We are not certain why PKC is active in lens fiber cells. However, western blots indicate that this is not due to clipping. Perhaps some other modification of PKC-g control has arisen in fiber cells. The sites of PKC-g phosphorylation have not be identified.
The amino acid sequences of Cx46 and Cx56 are highly homologous (62% identity), therefore it was proposed that Cx46 might contain phosphorylation sites equivalent to that of Cx56 . PKC- and protein kinase-A-dependent phosphorylation sites were found in the C-terminus and in the intracellular loop of the lens Cx56 from chicken . As shown by this paper, serine was identified as a phosphorylated reside for PKC-g. The findings that Cx46 is also phosphorylated on threonine and that the phosphorylation is not abolished by PKC-g inhibition indicates the involvement of other kinases. Protein kinase A may be involved in threonine phosphorylation on Cx46 when considering the high degree of homology between the Cx46 and Cx56 amino acid sequences.
Modulation of cortical fiber gap junctions by PKC-g through phosphorylation of serine on Cx46 may have importance in understanding and developing new methods to reveal the role of PKC-g in loss of normal functioning of lens fiber gap junctions in diabetic cataract. It has been determined that PKC-g levels are considerably lower in diabetic lenses compared to normal lenses . Thus, changes in PKC-g during diabetes could alter gap junction protein phosphorylation and, thus, gap junction activity.
The authors thank Daniel Boyle for his help and advice in the use of the confocal microscope, and Peggy Zelenka for useful advice. We gratefully acknowledge the support of the Kansas State University Cancer Center for travel support and a grant from NEI: EY-13421. This is publication No. 02-134-J from the Kansas Agriculture Experiment Station.
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