|Molecular Vision 2004;
Received 16 June 2002 | Accepted 27 September 2004 | Published 27 September 2004
Differential phosphorylation of connexin46 and connexin50 by H2O2 activation of protein kinase Cγ
Dingbo Lin, Samuel Lobell, Andrea
Jewell, Dolores J. Takemoto
Department of Biochemistry, Kansas State University, Manhattan, KS
Correspondence to: Dolores J. Takemoto, Ph.D., Department of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, KS, 66506; Phone: (785) 532-7009; FAX: (785) 532-7278; email: firstname.lastname@example.org
Purpose: Fiber cell gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) play distinct roles in the avascular lens. The purpose of this study was to determine how protein kinase Cγ (PKCγ) differentially regulates phosphorylation of Cx46 and Cx50 in oxidatively stressed lenses.
Methods: Sprague Dawley rats (six week old) were used in the experiments. PKCγ enzyme activity was analyzed by use of the PepTag assay kit. Phosphorylation of caveolin-1, Cx46, and Cx50 was determined by immunoblotting. Lipid rafts were isolated by continuous sucrose gradient centrifugation. Lipid raft-localization of PKCγ, Cx46, or Cx50 was demonstrated by immunoblotting. Association of caveolin-1 with PKCγ, Cx46, or Cx50 was revealed by co-immunoprecipitation.
Results: H2O2 (100 μM) stimulated PKCγ activation in rat whole lens. Activated PKCγ was recruited into caveolin-1 (Cav-1) containing lipid rafts and this activation enhanced the coimmunoprecipitation of Cav-1, Cx46, and Cx50 with PKCγ. Both Cx50 and Cx46 were associated with Cav-1 in lipid rafts. H2O2 significantly induced threonine (Thr) phosphorylation of Cx46 and Cx50, and serine (Ser) phosphorylation of Cx50. However, There was only a small stimulation of Cx46 phosphorylation at Ser by H2O2, as Cx46 was already phosphorylated.
Conclusions: Activation of PKCγ by H2O2 stimulated differential Ser phosphorylation of Cx50 versus Cx46, within lipid rafts. This suggests that Cx50 and Cx46 may have different functions in lens.
Gap junctions are nonspecific cell-to-cell channels which maintain homeostasis of fluids, ions, nutrients, and facilitate metabolite movement in the avascular lens as it lacks direct access to the blood supply [1,2]. Gap junctions are not uniformly distributed in the lens and may also function as hetero- or homo-hexamers of connexin proteins, referred to as connexons . Rat lens epithelial cells contain connexin 43 (Cx43), and, to lesser extent connexin50 (Cx50), while cortical fiber cells contain connexin46 (Cx46) and Cx50 [1,3]. Fiber cell Cx46 and Cx50 play different roles in lens growth and formation of cataract in knockout mice [4-7]. Therefore, elucidation of the regulation of gap junction proteins Cx46 and Cx50 could be very critical to prevent the initiation of cataractogenesis in humans .
Gap junction activity requires the formation of large numbers of connexons which assemble into functional plaques . Assembly of new plaques has been found to occur through deposition of new connexons at the outside of an existing plaque . Gap junction plaques assemble and disassemble at a rate of about every 2-5 h [10-13]. Phosphorylation of the C-terminus of connexins occurs at numerous serine (Ser) and threonine (The) residues and is catalyzed by various protein kinases [12,14].
Protein kinase Cγ (PKCγ) is an unique isoform of conventional PKC (cPKC) largely enriched in brain cells. It is also found in peripheral nerves, in retina, and in the lens [15-18]. We previously reported that PKCα and PKCγ are predominant isoforms for PKC in lens epithelial cells, whereas PKCγ is the major PKC in the cortex of whole lens . Activation of lens PKCγ by phorbol ester (TPA), insulin-like growth factor-1 (IGF-1), or lens epithelial derived growth factor (LEDGF) caused activation of PKCγ which resulted in decreases in functional gap junctional communication [19-21]. We have also found that a decrease in gap junction plaques signals redistribution of Cx43 in lipid raft domains in lens epithelial cells in culture .
High or prolonged oxidative stress can cause protein oxidation, aggregation, and proteolysis and consequently trigger cataractogenesis in the lens [23-26]. In this report, we show that 100 μM H2O2, at early time periods, activates PKCγ enzyme activity in whole lens. Active PKCγ was recruited into caveolin-1 containing lipid rafts, and coimmunoprecipitated with Cx46 and/or Cx50, and Cav-1. Moreover, H2O2 activation of PKCγ resulted in a stimulated differential phosphorylation of Cx46 and Cx50 at Ser and Thr. Phosphorylation of lens gap junction proteins may alter gap junctional communication which may, in turn, protect lens cells from oxidative stress at early time periods.
Monoclonal antibodies against PKCγ, phospho-Tyr14 of Cav-1 (pY14-Cav-1) and Cav-1 were purchased from BD Biosciences (Palo Alto, CA). Monoclonal mouse anti-Cx50 against the C-terminus (amino acid 290-440) was purchased from Zymed Laboratories (South San Francisco, CA). Polyclonal rabbit anti-phosphoserine and anti-phosphothreonine were purchased from Chemicon (Temicula, CA). Polyclonal rabbit anti-Cx46 against 11 amino acid residues at the C-terminus was purchased from Alpha Diagnostic Intl. Inc. (San Antonio, TX). Protein A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse or anti-rabbit IgG conjugated with HRP and PepTag non-radioactive protein kinase C assay system were purchased from Promega (Madison, WI). Prepared polyacrylamide gels (Ready Gels; 4-15%) were purchased from Bio-rad Laboratories (Hercules, CA). Dulbecco's Modified Eagle Medium (DMEM; low glucose), gentamicin, and penicillin/streptomycin were purchased from Invitrogen Life Technologies (Carlsbad, CA). Trichloroacetic acid (TCA), H2O2, and sodium fluoride (NaF) were purchased from Fisher Scientific (Pittsburgh, PA). Na3VO4, methyl-β-cyclodextrin (MβCD), phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail were purchased from Sigma (St Louis, MO).
Male/female Sprague Dawley rats (six week) were purchased from Charles River Laboratories (Wilmington, MA). Rats were sacrificed by CO2. Lenses were taken immediately for fresh use in all assays and were incubated in DMEM with H2O2 as noted. All experiments conformed to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and were performed under an institutionally approved animal protocol.
PKCγ activity assay
PKCγ activity was analyzed using the PepTag assay kit as described by Zhou et al. . Briefly, equal protein amounts of whole cell extracts from whole lens were immunoprecipitated with PKCγ antisera (1:100) at 4 °C for 4 h as previously described . Immunoprecipitated PKCγ/agarose bead complexes were incubated with PKC reaction mixture according to the manufacture's instruction. The reaction mixture also contained 20 mM HEPES (pH 7.4), 1.3 mM CaCl2, 1 mM DTT, 10 mM MgCl2, 1 mM ATP, 0.2 mg/ml phosphatidylserine, and substrate PepTag peptides (PLSRTLSVAAK). The reactions were initiated at 30 °C for 25 min, and were stopped by placing the tubes in a boiling water bath for 10 min. Fluorescent PepTag peptides (PKCγ reaction products) were resolved by 0.8% agarose gel electrophoresis and visualized under UV light. The phosphorylated peptide bands were excised and their fluorescence intensities were quantified by spectrophotometry at 570 nm. The enzyme activity was normalized by calibration of the relative level of phosphorylated substrates to the relative amount of PKCγ in the immunoprecipitation, as determined by western blotting, and activity was expressed as percent of untreated specific PKCγ activity.
Sucrose gradient centrifugation and isolation of lipid rafts
Lipid raft enriched membrane fractions were prepared as previously described . Briefly, whole lenses were extracted with cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.1% protease inhibitor cocktail, 5 mM NaF, 2 mM PMSF, 5 μM Na3VO4, and 1% Triton X-100 and incubated on ice for 30 min. Whole cell lysates were mixed with equal volumes of 80% sucrose in Mes-NaCl buffer containing 25 mM Mes (pH 6.5), 150 mM NaCl, 0.1% protease inhibitor cocktail, 5 mM NaF, 2 mM PMSF, and 5 μM Na3VO4, laid at the bottoms of 12 ml ultracentrifuge tubes, and then overlaid with 8 ml of a 5-35% continuous sucrose gradient in Mes-NaCl buffer containing a double concentration of protease inhibitors (NaF, Na3VO4, and PMSF). The samples were centrifuged at 245,000x g for 22 h at 4 °C with a Beckman swinging bucket rotor (SW41 Ti). Fractions (1 ml each) were collected from the top of each gradient. Protein samples were precipitated with 10% trichloroacetic acid (TCA), separated by 10% SDS-PAGE, and immuno-visualized by western blotting.
For immunoprecipitation assays from Cav-1-containing lipid rafts, fractions 3 to 6 of the sucrose gradients were pooled and resonicated after the addition of 0.1% SDS to the solution.
Western blot and immunoprecipitation
Whole lenses were homogenized with cell lysis buffer followed by sonication. The cell lysis buffer contained 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 0.1% protease inhibitor cocktail, 5 mM NaF, 5 μM Na3VO4, and 2 mM PMSF. After centrifugation at 12,000x g for 20 min, the supernatants were collected and used as whole cell extracts. Western blotting was carried out as described previously . Whole cell extracts or pooled lipid raft containing fractions (fractions 3 to 6, see above) were immunoprecipitated with antisera at 4 °C for 4 h. Immunoprecipitation complexes were resolved by SDS-PAGE and protein patterns were visualized by western blot with particular antisera as indicated.
All analyses represent at least triplicate experiments. The Student's t-test was used for all statistical analysis in this paper. The α level was chosen to be 0.05.
Activation of PKCγ enzyme activity by H2O2
To examine the effect of H2O2 on activation of PKCγ, we tested H2O2 stimulation of PKCγ enzyme activity. Endogenous PKCγ was immunoprecipitated and its enzyme activity was measured by use of the PKC peptide substrate. Enzyme activity was normalized by calibration of the relative level of phosphorylated substrates to the relative amount of PKCγ in the immunoprecipitation, as determined by western blotting, and was expressed as percent of untreated cell PKCγ activity (Figure 1A). The results showed that 100 μM H2O2 for 15 min significantly increased (by 78% above control) activity of PKCγ in rat whole lenses. The results indicated that sublethal dosage of H2O2 (100 μM) activated PKCγ in the lens. As a positive control for initiation of a H2O2 stress response for subsequent lipid raft analysis, the profile of H2O2 stimulated Tyr14 phosphorylation of Cav-1 in lipid raft fractions 3 to 6 was also included (Figure 1B) . The data confirmed that a classic Cav-1 stress response was also observed in whole lens in culture exposed to H2O2.
Redistribution of PKCγ into lipid rafts in response to H2O2
Generally, activated PKCγ translocates to plasma membranes and is recruited into lipid rafts [22,29]. Continuous sucrose density gradient fractionation experiments showed that endogenous lens PKCγ is distributed in fractions 6 to 8. H2O2-stimulated PKCγ redistribution into lighter sucrose density fractions (e.g., fractions 4 and 5, Figure 2A) and cofractionation with Cav-1, Cx46, and Cx50 (Figure 2C) in lipid rafts were consistent with the data from lens epithelial cells exposed to TPA or IGF-1 . Disruption of lipid rafts by methyl-β-cyclodextrin (MβCD, an inhibitor of cholesterol synthesis; 10 mM, 2 h) caused PKCγ to distribute extensively into many fractions (Figure 2A, fractions 6 to 12). This pattern is consistent with disruption of lipid rafts . The data suggest that H2O2 activated PKCγ was recruited into lipid rafts which also contained Cav-1, Cx46, and Cx50.
Coimmunoprecipitation of Cx50, Cx46, PKCγ, and Cav-1 from lipid raft fractions
Previous reports indicate that most connexin proteins, except Cx50 and Cx26, colocalize with Cav-1 in lipid rafts in a cell tissue culture overexpression system . However, in the whole lens cortex Cx50 was found with Cx46 and Cav-1 in lipid raft fractions. Lipid raft fractionation results shown in Figure 2C indicated that Cx46 somewhat localized to fractions 4 to 6 which contained Cav-1. These fractions are classified as cholesterol rich Cav-1 containing lipid rafts . Cx50 is also localized in these same fractions, suggesting that unlike a tissue culture overexpression system , in intact lens Cx50 does localize to Cav-1 containing lipid rafts. Disruption of lipid rafts by MβCD (10 mM, 2 h), used as a control, caused relocalization of Cx50 or Cx46 to other fractions due to lipid raft disruption. In order to further confirm the association of Cx50 and Cx46 with Cav-1 in lipid rafts, lipid raft fractions 3 to 6 were isolated, pooled, and endogenous Cav-1 was immunoprecipitated with anti-Cav-1 antisera. Coimmunoprecipitation data are shown in Figure 3 and suggest that Cav-1 immunoprecipitated with both Cx46 and Cx50 in lipid rafts in either control or H2O2 treated lenses. Multiple bands were observed in Cx50 samples when lenses were exposed to H2O2, suggesting that phosphorylated Cx50 was also associated with Cav-1. Thus, in whole lens, Cx46, Cx50, and Cav-1 are present in lipid rafts in both control and H2O2 treated samples. However, PKCγ association with the lipid rafts is greatly enhanced after H2O2 stimulation (Figure 3A). The intensity of the western blot bands was digitalized (Figure 3B) and the results suggest that PKCγ association with Cav-1, Cx46, or Cx50 was enhanced by 75% above control when lenses were exposed to H2O2 for 15 min. The association levels between Cx46, Cx50, and Cav-1 were not changed after H2O2 treatment.
Phosphorylation of Cx46 and Cx50 after H2O2 activation of PKCγ
Both Cx46 and Cx50 are phosphoproteins. Changes in their phosphorylation levels may regulate gap junctional cell-to-cell communication in the lens [12,14,18,32-35]. Cx46 phosphorylation is increased during galactosemia and this results in the inhibition of gap junction activity . We previously demonstrated that activated PKCγ caused phosphorylation of Cx43 which, in turn, decreased gap junction activity in lens epithelial cells in culture [20,21]. Therefore, it is hypothesized that activation of PKCγ by H2O2 may cause phosphorylation of Cx46 and/or Cx50 on Ser and/or Thr in the whole lens. Cx46 and Cx50 were immunoprecipitated from the lipid raft fractions 3 to 6 after sucrose gradient fractionation of lens whole cell extracts, and the complexes were resolved by 4-15% gradient gels and immunoblotted with anti-phosphoserine, and anti-phosphothreonine antisera. The intensity of phosphorylated bands was digitalized and graphed. The results in Figure 4 revealed that Cx46 was already phosphorylated on Ser in untreated lenses. H2O2 enhanced Cx46 phosphorylation on Ser to a somewhat higher level (55% above control). In contrast, Cx50 phosphorylation on Ser was not detectable in untreated lenses and H2O2 treatment dramatically stimulated phosphorylation of Cx50 on Ser by 268% above control. H2O2 treatment significantly increased Thr phosphorylation of both Cx46 and Cx50.
Oxygen is required for cell survival, but the cells may also suffer from the toxicity of extra reactive oxygen species (ROS). We mimicked the ROS stress condition by low levels of H2O2 exposure to whole lens in culture. Our findings demonstrate that H2O2 activates PKCγ which, in turn, phosphorylates lens gap junction proteins Cx50 (Thr and Ser) and Cx46 (Thr and possibly on Ser). This may occur within lipid rafts. Differential phosphorylation of lens gap junction proteins may alter gap junctional communication which may, in turn, protect lens cells from oxidative stress at early time periods.
PKCγ is an important enzyme in the regulation of cell gap junctional signaling in the lens [19-22,36]. We have observed that PKCγ is present in lens epithelial cells and is the primary sensor of changes in diacylglycerol (DAG) at low/physiological levels, rather than PKCα . Like PKCα, the PKCγ regulatory region contains C1 and C2 domains. NMR structural analysis shows that PKCγ, like PKCα, has two zinc-finger motifs which are enriched with cysteine (Cys) residues in the C1 domain, C1A and C1B . Both C1A and C1B domains of PKCγ have high affinity for DAG, whereas only the C1A domain of PKCα has high affinity for DAG binding [20,38]. Both C1A and C1B domains are involved in DAG stimulated PKCγ activation at a basal intracellular calcium level . Activation of PKCα by either DAG- or phorbol ester-binding with C1 domains is due to the binding-triggered Zn release and consequent conformational change of the C1 domain [37,39]. Reactive oxygen, such as H2O2, passes through lipid bilayers as well as aqueous channels in plasma membranes, and triggers the release of Zn from purified PKCα in vitro [39,40]. Our data demonstrate that transient H2O2 (100 μM) exposure increases PKCγ enzyme activity (Figure 1). Furthermore, activated PKCγ translocates into lipid rafts (Figure 2A and Figure 3) and has enhanced association with Cav-1, Cx46, and/or Cx50 (Figure 3).
We propose that oxidation of the thiol groups of Cys residues in the C1A and/or C1B domains of PKCγ by reactive oxygen species may cause a change in the structure of this region, just as DAG or TPA binding to this region causes a transient activation . This structural change could subsequently induce PKCγ activation by transient H2O2 exposure in the whole lens. Transient activation of PKCγ could provide an initial protective effect on lens through rapid inhibition of gap junctions. This, in turn, would prevent relay of damaging signals, but not H2O2, through open gap junctions, to adjacent cells such as is observed in brain during ischemia. However, prolonged exposure at comparable H2O2 concentrations (for example, 2 h) could cause development of cataracts in rat lens . It will be an interesting subject for future work to investigate regulation of gap junctions at longer periods and their role in cataractogenesis in the lens.
The lens is an avascular organ which relies upon gap junctions to provide a pathway for nutrient uptake and waste product removal [2,14]. Cx50 and Cx46 are the only gap junction proteins in the lens fiber cells of rats . In whole lens fiber cells, we observe that PKCγ phosphorylation of Cx46 is increased after galactosemia in the region of the cortical fiber cells where Cx50 and Cx46 are not yet C-terminally truncated (results in a loss of the PKCγ phosphorylation site) . In older enucleated lens fiber cells of the inner cortex, Cx46 and Cx50 are regulated by C-terminal truncation, a process which occurs by calpain cleavage [42,43]. Calpain cleavage of the C-terminus of Cx50 or Cx46 probably results in the loss of the PKCγ phosphorylation site. In this current report we employed commerically available Cx50 and Cx46 antisera, which were raised against the carboxy-tails located between residues 290 to 440 of Cx50 and 11 amino acid residues of Cx46. Therefore, only non-truncated outer cortical Cx46 and Cx50 are identified in this study. The data shown in Figure 2, Figure 3, and Figure 4 suggests that Cx50, along with Cx46, colocalizes with Cav-1 in lipid rafts in the newer cortical fiber cells of whole lens outer cortex. However, since the antisera would not recognize truncated Cx46 or Cx50, we do not have information regarding Cav-1 association with connexins in mature fiber cells.
Amino acid sequence analysis shows that both Cx46 and Cx50 have perfect consensus sequences for Cav-1 binding (θXθXXXXθ, where θ is an aromatic amino acid [Phe, Tyr, or Trp]) in the very beginning residues of the first transmembrane sequence (rat Cx46 or Cx50 amino acid sequence, W25LTVLFIF32) for binding to a Cav-1 scaffolding domain . The presence of lipid rafts in the lens has been recently studied [22,45]. However, there is only a single publication on the association of Cx50 in Cav-1 containing lipid raft domains . The authors studied localization of connexins to Cav-1 containing lipid rafts by using overexpression in tissue culture cells and found that almost all connexin proteins were localized into lipid rafts with Cav-1 except Cx26 and Cx50. This difference from our data could be due to the use of only single connexin proteins. Cx50 may form some heteromeric channels with Cx46 in the lens . Therefore, colocalization of Cx50 in Cav-1 containing lipid rafts may be at least partially due to Cx46 association in whole lens. These may also be unique lens proteins which allow Cx50 to associate with Cav-1 which are not found in overexpression systems or in cells in culture. It will be of interest in the future to determine how lipid rafts contribute to regulation of Cx46/50 gap junctions in the lens.
Lipid rafts are unique plasma membrane microdomains containing many signal transduction proteins, such as G-proteins and a variety of receptors [47,48]. H2O2 stress triggers phosphorylation of Cx50, and, to a lesser extent Cx46, in whole lens lipid rafts (Figure 4). Protein sequence analysis also shows that there are multiple consensus PKC phosphorylation sites (R/KXS/T)  in the rat cytoplasmic C-terminus of Cx43, Cx46, or Cx50. PKCs are typical serine/threonine kinases and PKC primarily phosphorylates Cx43 in Ser 368 with flanking sequences SR/KAS368SRA . The same motif is also found in the C-terminus of rat Cx50 (SKAS430SRA), but not in Cx46. This difference may account for the different phosphorylation profiles for Cx50 versus Cx46 on Ser in response to H2O2 activation of PKCγ (Figure 4). Cx46 was already phosphorylated on Ser in untreated lenses, and H2O2 enhanced this only to a somewhat higher level (55% above control). In contrast, the Cx50 phosphorylation patterns indicate strong enhancement after PKCγ activation (268% above control). This suggests that other protein kinases may also be involved in Cx46 regulation, resulting in higher endogenous Ser phosphorylation of Cx46 in the intact lens. PKCγ may thus regulate these proteins differently in differentiating fiber cells. Recent work using Cx46 and Cx50 knockout mouse models indicates that these proteins have different functions in lens [6,46]. This may be reflected by their different phosphorylation patterns.
We previously reported that after IGF-1 treatment most of the Cx43 proteins in lens epithelial cells were extensively redistributed within lipid rafts and cell surface Cx43 gap junction plaques were decreased in lens epithelial cells in culture . The data presented on the whole lens as shown in Figure 2B do not indicate that Cx50 redistributes as significantly as Cx43 in response to H2O2 stress. Some Cx46 redistribution into fraction 8 and 9 was observed (Figure 2B). This could be due to structural differences in lens fiber cells when compared to lens epithelial cells. Lens fiber cells may not contain all of the necessary membrane structural proteins essential for similar lipid raft redistribution, or Cx50 and Cx46 may associate in lipid rafts with proteins which are unique to fiber cells. In addition, H2O2-treated fiber cells in the peripheral area of the lens may show a different gap junction disassembly and lipid raft redistribution than that observed in fiber cells in the interior part of the differentiating and more mature fiber cells of the lens. Thus, in these whole lens samples we are observing an average lipid raft distribution of many types of fiber cells. We hypothesize that transient stimulation by H2O2 in newer differentiating fiber cells induces cell surface functional large gap junction plaques to disassemble into smaller and less functional plaques due to phosphorylation of Cx50 and/or Cx46 by PKCγ activation. This probably occurs more strongly in the newer, outer cortical fiber cells. To date no information is available to show how gap junctions assemble and disassemble in new or old fiber cells in vivo. Newly developed quantitative freeze fracture-immunolabeling techniques may elucidate these structural changes .
In summary, H2O2 activates PKCγ in the whole lens, which, in turn catalyzes the phosphorylation of Cx50 and/or Cx46 resulting in redistribution of Cx50 and/or Cx46 within lipid rafts. This may result in the disassembly of gap junction plaques and consequent inhibition of gap junctional communication in the lens cortex. PKCγ, thus, could be considered a stress sensor/regulator in the lens.
We thank Dr. Guido Zampighi of University of California at Los Angles, Dr. Peggy S. Zelenka at the National Eye Institute, and Dr. Larry J. Takemoto of Kansas State University for helpful discussions. Funding for this work was provided by NIH-EY13421 to DJT, and by a Fight for Sight Summer Fellowship to AJ. This is publication 04-018-J from the Kansas Agricultural Experiment Station.
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