|Molecular Vision 2002;
Received 2 May 2002 | Accepted 14 October 2002 | Published 22 October 2002
The interaction and phosphorylation of tropomodulin by protein kinase Cα in N/N 1003A lens epithelial cells
Lynn M. Wagner,1 Velia M. Fowler,2
Dolores J. Takemoto3
Departments of 1Anatomy and Physiology and 3Biochemistry, Kansas State University, Manhattan, KS; 2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA
Correspondence to: Dr. Dolores J. Takemoto, Department of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, KS, 66506; Phone: (785) 532-7009; FAX: (785) 532-7278; email: email@example.com
Purpose: Tropomodulin, a tropomyosin and actin-binding protein stabilizes tropomyosin-actin filaments and is important in maintaining the elongated shape of lens fiber cells. In this study the role of PKCα-catalyzed phosphorylation of tropomodulin is determined.
Methods: The interaction of PKCα and tropomodulin was measured by immunoprecipitation after activation with either phorbol ester at 200 nM for 60 min or 10 ng/ml EGF for 15 min. Tropomodulin phosphorylation was determined after co-immunoprecipitation using an in vitro [γ-32P] PKC activity assay and by specific reaction with antiphosphothreonine antisera. Changes in tropomodulin interaction with tropomyosin or with the cytoskeleton were measured in a gel overlay assay and by association with a "Triton-insoluble" fraction.
Results: Both phorbol ester and EGF caused an increased interaction of PKCα with tropomodulin. Following activation of PKCα by phorbol ester or by EGF there was an increased phosphorylation of tropomodulin on threonine residues. The phosphorylation of tropomodulin did not affect interaction with tropomyosin as measured by a gel overlay assay. However, there was an increased association of tropomodulin with the "Triton-insoluble" cytoskeletal fraction.
Conclusions: Activation of PKCα by EGF causes an increased phosphorylation of tropomodulin which results in an increase in tropomodulin association with cytoskeletal components. This establishes a signal pathway by which EGF induced activation of PKCα alters the interaction of lens cytoskeletal proteins.
Cytoskeletal proteins provide the structural support for cell shape. In order to retain their elongated state, lens fiber cells rely upon a stable actin cytoskeleton. Several groups have studied the roles of microtubules and microfilaments in lens fiber cell differentiation. They have found that actin reorganization is required for the differentiation of lens epithelial cells [1-4]. A possible cytoskeletal protein that could act in regulating the length and stability of actin filaments in lens fiber cells is tropomodulin. It is an approximately 40 kD protein which binds to actin filaments at the pointed ends where it inhibits both monomer addition and dissociation .
The membrane skeleton consists of a highly cross-linked network of spectrin and short actin filaments, together with associated membrane attachment and actin regulatory proteins . Since cytoskeletal proteins provide the structural support for cell shape, the membrane-associated actin cytoskeleton is likely to play a crucial role in the stabilization and maintenance of the highly elongated fiber cell shape. In epithelial cells, the membrane skeleton is believed to have additional roles in the generation of membrane domains, including apical or basolateral sorting of ion channels, pumps, and adhesion receptors .
Tropomodulin has been identified in cells and tissues that have several of the same phenotypic characteristics such as terminal differentiation, stable, morphology, and specialized structural organization involving actin filaments [2-4]. Lens fiber cells exhibit all of these traits. Tropomodulin protein is not present in undifferentiated cells of the central epithelium and is first detected in the early annular pad of chick lenses . After 10 days of exposure to fibroblast growth factor (FGF), rat lens explants begin to show pronounced structural features that are indicative of fiber cell differentiation . It is at this stage that tropomodulin is first detected . Tropomodulin is localized in these explants at the plasma membrane . This is consistent with the findings in vivo; sectioned rat lenses show membrane-associated tropomodulin .
The presence of actin, tropomyosin, and tropomodulin on nuclear fiber cell membranes implies that capping of tropomyosin-actin filaments by tropomodulin is required throughout the lifetime of the fiber cells . Not only the stabilization of actin filaments in the membrane skeleton by tropomodulin may be important for the mechanical stabilization of lens fiber cell membranes, but this cytoskeletal protein may also be important in maintaining adhesion receptor domains that are critical for minimizing intercellular space to prevent light scattering . The stable tethering of ion channels and pumps by actin filaments in the membrane skeleton may prevent the loss or incorrect positioning of these membrane proteins that are critical for ion homeostasis and lens transparency throughout the life of the lens [13,14].
Protein kinase C is a key enzyme in signal transduction. This enzyme is activated by phorbol esters, substances that are known to interfere with proliferative and differentiation events by promoting oncogenic transformation of cells. Previous studies have demonstrated that several actin-binding proteins such as tubulin, fodrin, dystrophin, spectrin, ezrin, tau, and lipocortins are phosphorylated upon activation of cells by growth factors and are substrates of PKC in vitro [15-21].
In this study, tropomodulin was shown to be phosphorylated by PKCα when N/N 1003A lens epithelial cells were exposed to phorbol ester or EGF, or when PKCα was overexpressed for 7 days. At 7 days overexpression of PKCα, N/N 1003A cells begin to elongate and express αA-crystallin, a crystallin not normally expressed in this cell line, thus used as a measure of differentiation . However, the molecular steps between the activation of PKCα and the biological response remain unknown. Tropomodulin is not normally expressed in lens epithelial cells, however, it is expressed in the N/N 1003A lens epithelial cell line. This cell line can be induced to differentiate by prolonged overexpression of PKCα [22,23]. Thus, the N/N 1003A cell line becomes a useful model in studying lens cell differentiation and the relationship between PKCα and tropomodulin.
The N/N 1003A rabbit lens epithelial cell culturing was as described previously  using Dulbecco's Modified Eagles Media (DMEM) supplemented with 10% heat inactivated fetal calf serum (Atlanta Biologicals) and 50 μg/ml gentamicin (Life Technologies). The cells were grown at 37 °C in an atmosphere of 10% CO2 and 90% air. All experiments were conducted with cells near 80-90% confluency (6 x 106 cells/flask) except where stated differently.
The PKCα and PKCγ plasmids were a kind gift from Dr. W. Anderson (NCI). The holo PKCα plasmid was derived from the bovine sequence and the holo PKCγ plasmid was derived from the rodent sequence . In addition, the εMTH (empty vector) was used as a control in stable transfection experiments. The MTH vector is inducible at low levels by 20 μM zinc acetate for 18 to 24 h.
Transfection of N/N 1003A rabbit lens epithelial cells took place when the cells reached approximately 60% confluency. The cells were transfected using Lipofectamine (Life Technologies). The DNA plasmid (5 μg) and Lipofectamine (0.1 mg) in 200 μl of serum free media were used for each transfection. The cells were incubated with the DNA plasmid and Lipofectamine for 12 h at 37 °C. After incubation, media containing twice the amount of fetal calf serum (20%) was added for 24 h. Following this incubation, the media was replaced with media containing 10% fetal calf serum. The transfected cells were selected with 750 μg/ml G418 (Life Technologies) for 6 weeks and grown in half that concentration of G418 thereafter.
Assay for PKC translocation to the plasma membrane
Control N/N1003A cells, N/N1003A cells treated with 200 nM phorbol ester (TPA, for 60 min), 10 ng/ml EGF-treated cells (for 15 min), or 15 ng/ml bFGF-treated cells (for 15 min) were cultured to 90% confluency in 75 cm2 tissue culture flasks and were harvested in ice cold 50 mM Tris, 20 mM MgCl2, 25 μg/ml aprotinin, and 25 μg/ml leupeptin, pH 7.5 and the cell lysate was sonicated. The cell lysate was centrifuged for 60 min at 35,000 rpm (100,000x g) at 4 °C. A 12.5% SDS-PAGE gel containing 25 μg of total cell protein was transferred to a nitrocellulose membrane (OPTI TRAN, Midwest Scientific). The membrane was incubated for 12 h in either anti-PKCα or anti-PKCγ antibody (1:1000, Transduction Laboratories) in 3% powdered milk in distilled water at 4 °C. The membranes were then washed 3 times (10 min each time) in TDN (0.05 M NaCl, 2.0 mM EDTA, 0.01 M Tris). Anti-mouse IgG (1:2500, Promega) in 3% milk in distilled water was added to the membrane for 3 h at room temperature. Membranes were then again washed 3 times (10 min each time) in TDN. The membranes were then developed using SuperSignal Chemiluminescent Substrate (Pierce) and X-ray film (Molecular Technologies).
Immunoprecipitation of tropomodulin and PKC
Control N/N1003A cells, N/N1003A cells overexpressing PKCα or PKCγ for 7 days, 200 nM phorbol ester-treated cells (TPA, for 60 min), or 10 ng/ml EGF-treated cells (for 15 min) 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 μg/ml aprotinin, and 25 μg/ml leupeptin. The lysed cells were homogenized and centrifuged at 13,000 rpm (20,000x g) for 20 min, then the supernatant was collected and antibodies specific for PKCα, PKCγ (5 μg/ml, Transduction Laboratories), or tropomodulin (5 μg/ml) were added to the mixture and incubated overnight at 4 °C with constant mixing. After overnight incubation with PKCα, PKCγ, or tropomodulin antibodies, 20 μl of protein G sepharose beads (50:50 solution in lysis buffer, Sigma) were added to the mixture and the mixture was further incubated for another two h at 4 °C. The mixture was centrifuged at 2000x g for 30 s and the precipitate was collected and washed four times with PBS buffer. The washed precipitate was then mixed with 20 μl Tris-glycine/SDS sample buffer and boiled for five min. Proteins were separated by 7.5% or 12.5% SDS-PAGE, transferred to nitrocellulose membranes for Western blotting as described above and probed with anti-PKCα (mouse, 1:1000), anti-PKCγ (mouse, 1:1000), or anti-tropomodulin (Fowler; rabbit 1749, 1.0 μg/ml) antibody.
Phosphorylation of tropomodulin by PKCα
Two approaches were used to detect phosphorylation of tropomodulin in the immunoprecipitates from the N/N 1003A cells. First, tropomodulin or PKCα was immunoprecipitated from N/N 1003A cells treated as described above to activate or inhibit PKC, and the Western blots were probed with anti-phosphothreonine or anti-phosphoserine antibodies (1:1000, Chemicon). Second, the immunoprecipitated tropomodulin still attached to the antibody-beads was used in an in vitro PKC phosphorylation assay. To initiate the PKC assay, 5X PKC substrate solution (250 μM Ac-MBP (residues 4-14), 100 μM ATP, 5 mM CaCl2, 100 mM MgCl2, 20 mM Tris, pH 7.5) or the 5 X inhibitor solution (100 μM PKC (residues 19-36), 20 mM Tris, pH 7.5) was added. To begin the reaction, PKC reaction mixture consisting of 25 μCi/ml [γ-32P] ATP was added for 5 min at 30 °C. The reaction was stopped by 20 μl Tris-glycine / SDS sample buffer and the samples were boiled for five min. Proteins were separated by 12.5% SDS-PAGE, transferred to nitrocellulose membranes, and the blots were exposed for 2 days to x-ray film at -20 °C. In order to determine the level of tropomodulin phosphorylation, the same membrane was probed with anti-tropomodulin antibody and the resulting autoradiogram and the autoradiogram from the [γ-32P] PKC assay were scanned and quantitated (using Un-Scan-It, Silk Scientific, Inc.). The average pixels for each sample lane from the blot probed with tropomodulin antibody was divided by the average pixels for each sample lane from the [γ-32P] PKC assay in order to determine if there was an increase of phosphorylation over control cells.
Tropomyosin gel overlay assay with lens epithelial cells
Tropomodulin was immunoprecipitated from lysates of N/N 1003A cells treated as above to activate or inhibit PKC, and electrophoresed on 12.5% SDS-PAGE gels followed by transfer to a nitrocellulose membrane. The membranes were incubated with 30 μg/ml of purified chicken skeletal muscle tropomyosin (Fowler) in 3% milk containing 2 mM MgCl2 at 4 °C for 2 h. After the incubation period, the membranes were washed 3 times for 10 min each in TDN. The blot was then probed with anti-tropomyosin antibody (Fowler; CH1, 1:1000) overnight. Immunoreactive bands were detected by chemiluminescence (ECL, Pierce). The Western blot was then stripped (using 0.2 M glycine, pH 2.8) and probed with anti-tropomodulin antibody. In order to determine if there is a change in tropomyosin binding when tropomodulin is phosphorylated, autoradiograms from both the blots probed with tropomyosin and tropomodulin antibody were scanned and quantitated (Un-Scan-It, Silk Scientific, Inc.). The average pixels for each sample lane from the blot probed with tropomyosin antibody was divided by the average pixels for each sample lane from the blot probed with tropomodulin in order to normalize the tropomyosin binding changes for any changes in tropomodulin amount.
Triton X-100 fractionation to determine cytoskeletal association of tropomodulin
Control N/N 1003A cells and N/N 1003A cells treated with 200 nM TPA for 60 min or 10 ng/ml EGF for 15 min were harvested in cold CSK buffer (10 mM PIPES, pH 6.8, 100 mM KCl, 300 mM sucrose, 2.5 mM MgCl2, 0.5% Triton X-100, 25 μg/ml aprotinin, and 25 μg/ml leupeptin). The lysate was centrifuged for 60 min at 13,000x g at 4 °C. The resulting pellets and supernatants were loaded onto a SDS-PAGE gel (25 μg protein/lane) and the separated protein was transferred onto a nitrocellulose membrane. The membrane was probed with anti-tropomodulin antibody (1.0 μg/ml) overnight. Immunoreactive bands were detected by chemiluminescence (ECL, Pierce).
Band intensity measurements in Western blots using Un-Scan-It were statistically analyzed using Student's t-test for paired or unpaired values with a sample of less than 30. Values of p<0.05 were considered to be statistically significant.
Western blot analysis of PKCα and PKCγ translocation
N/N 1003A cells were treated with 200 nM TPA (a conventional PKC activator) for 60 min, 10 ng/ml EGF, or 15 ng/ml bFGF. Confluent cells were taken up in ice cold 50 mM Tris, 20 mM MgCl2, 25 μg/ml aprotinin, and 25 μg/ml leupeptin, pH 7.5 and sonicated. The lysate was spun down for 60 min at 35,000 rpm (100,000x g). Western blot analysis was performed on the pellet that contained plasma membrane and the supernatant that contained cytosolic components.
Upon activation by TPA, PKCα and PKCγ should translocate from the cytosol to the plasma membrane . In order to determine if PKCα and PKCγ translocate to the plasma membrane in N/N 1003A cells upon activation, TPA was added to the cell culture media for 60 min. PKCα did translocate to the membrane (pellet fraction) upon activation of the enzyme by TPA (Figure 1A, lane 2 versus lane 4). PKCγ also translocated to the membrane (pellet fraction, Figure 1B, lane 2 versus lane 4); however, PKCγ performs a different function when activated with TPA. This involves the regulation of gap junctions by phosphorylating connexin 43 and connexin 46 . Control cells did not show any significant increase in PKCα or PKCγ translocation to the membrane although some PKC was already membrane localized in these samples (Figure 1A, lane 1 versus lane 3; Figure 1B, lane 1 versus lane 3).
To determine whether PKCα or PKCγ translocated to the plasma membrane upon the addition of two growth factors that are present in the ocular fluids, 10 ng/ml EGF or 15 ng/ml bFGF was added to the cell culture media for 15 min. PKCα translocated to the plasma membrane of EGF-treated cells, as represented by the heavier band in the pellet fraction compared to the supernatant fraction that contains cytosolic components (Figure 1C, lane 3 versus lane 6). There was no translocation of PKCα in bFGF-stimulated cells (Figure 1C, lane 2 versus lane 5). PKCγ did not translocate in EGF-stimulated (Figure 1D, lane 3 versus lane 6) nor bFGF-stimulated cells (Figure 1D, lane 2 versus lane 5). Therefore, since only PKCα translocated to the membrane upon stimulation by EGF and not PKCγ by either growth factor, EGF may be unique in that it may be one of the in vivo activators of PKCα. It could be possible that EGF is the in vivo activator of PKCα that would then begin the series of events eventually leading to the phosphorylation of tropomodulin in the lens.
To determine what effect PKC activation in N/N 1003A cells has on tropomodulin association with the membrane, we activated PKC with TPA and separated the plasma membrane fraction from the cytosolic fraction. The pellet fractions were analyzed by Western blot for tropomodulin (Figure 1E). Tropomodulin was present in all the pellet fractions for each sample set and was not present in any of the supernatant fractions (Figure 1E). This membrane localization of tropomodulin has been previously reported [10,11]. The tropomodulin antibody reacted better with the N/N 1003A cell sample treated with TPA as compared to control cell samples because the antibody may react better with a more phosphorylated form of tropomodulin or it might also reflect the increased stabilization of tropomodulin (Figure 1E, lane 1 versus lane 2). Therefore, in summary, the pellet fraction that contains the plasma membrane contains all of the tropomodulin in all tested samples.
Co-immunoprecipitation of tropomodulin with PKCα and not PKCγ
We used a co-immunoprecipitation approach to determine whether PKCα or γ and tropomodulin could be associated in N/N 1003A cells. PKCα was immunoprecipitated from control N/N1003A cells, N/N1003A cells treated with TPA, EGF, or cells overexpressing PKC α by using protein G sepharose in the presence of anti-PKCα antibodies (Figure 2). To test the correct migration of the tropomodulin band and the IgG band, control samples for the immunoprecipitation experiments were analyzed by Western blotting with tropomodulin antibodies (Figure 2A). There were reactive bands at approximately 43 kD representing tropomodulin and IgG (about 50 kD) in samples immunoprecipitated with anti-tropomodulin antibody (Figure 2A, lane 1). Lane 2 (Figure 2A) contains only cell lysate and has only a tropomodulin band. The higher molecular weight IgG band was also detected in the control experiments in which primary antibody but no cell lysate was added to the experimental mixture (Figure 2A, lane 4). This was not observed in lanes without antibody (Figure 2A, lane 3). Recombinant chicken tropomodulin (Figure 2A, lane 5) and N/N 1003A cells (Figure 2A, lane 6) were used as positive controls for the tropomodulin antibody as demonstrated by the band representing tropomodulin at about 43 kD. Lane 7 was a positive control for the migration of the antibody used for the immunoprecipitation (in this case tropomodulin) that consisted of tropomodulin antibody mixed with SDS-PAGE sample buffer (Figure 2A, lane 7). An IgG band was detected, which represented the antibody.
After analyzing the immunoprecipitated PKCα by staining the blot with Ponceau S to assure equal protein load (not shown), the nitrocellulose membranes were probed with anti-tropomodulin antibody (Figure 2B). The immunoprecipitated N/N1003A cells which were treated with TPA or overexpressed PKCα for 7 days reacted with anti-tropomodulin antibody in a separate and distinct band at about 43 kD (Figure 2B, lanes 1 and 2). The immunoprecipitated N/N1003A cells which were treated with EGF reacted with anti-tropomodulin antibody in a distinct band at about 43 kD (Figure 2D, lane 1). Control cells displayed a reduced tropomodulin band (Figure 2D, lane 2).
PKCγ was immunoprecipitated from control N/N1003A cells, N/N1003A cells treated with TPA, or cells overexpressing PKCγ by using protein G sepharose in the presence of anti-PKCγ antibodies (Figure 2C). After analyzing the immunoprecipitated PKCγ by staining the blot with Ponceau S to assure equal protein load (data not shown), the nitrocellulose membranes were probed with anti-tropomodulin antibody (Figure 2C). Western blots from the N/N1003A cells that were treated with TPA or overexpressed PKCγ for 7 days did not react with anti-tropomodulin antibody (Figure 2C, lanes 1 and 2). The control cells did not show a reactive band for tropomodulin (Figure 2C, lane 3). Thus, this data suggests that tropomodulin is associated with PKCα and not PKCγ.
Co-immunoprecipitation of PKCα and not PKCγ with tropomodulin
To confirm the association of tropomodulin with PKCα, we preformed the reverse experiment. Tropomodulin was immunoprecipitated from control N/N1003A cells, N/N1003A cells treated with TPA, EGF, or cells overexpressing PKCα using anti-tropmodulin antibodies adsorbed to protein G sepharose (Figure 3). A band at approximately 80 kD was detected by antibodies to PKCα in the tropomodulin immunoprecipitates from cells treated with TPA and cells overexpressing PKCα (Figure 3A, lanes 1 and 2). A band at approximately 80 kD was detected by antibodies to PKCα in the tropomodulin immunoprecipitates from cells treated with EGF (Figure 3C, lane 1). From these two experiments, we have confirmed that once PKCα is activated by TPA or EGF, there is a greater interaction between PKCα and tropomodulin compared to control N/N 1003A cells. The control cells did not show reactive bands for PKCα in the tropomodulin immunoprecipitates (Figure 3A, lane 3). The co-immunoprecipitation of PKCα with tropomodulin only under conditions of PKCα activation suggests that phosphorylated tropomodulin may interact more specifically with PKCα.
In contrast, PKCγ was not detected in the tropomodulin immunoprecipitates from cells overexpressing PKCγ, from N/N 1003A cells treated with TPA or from control N/N 1003A cells (Figure 3B). In additional experiments, PKCγ was not detected in the tropomodulin immunoprecipitates for any experimental sample sets except for a light band at about 78 kD for TPA-treated cells (Figure 3B, lane 2). Therefore, the major PKC isoform that interacts with tropomodulin is PKCα and may be responsible for the phosphorylation of tropomodulin.
Phosphorylation of Tropomodulin by PKCα
To investigate directly whether tropomodulin could be phosphorylated by PKCα, we preformed an in vitro [γ-32P] PKC phosphorylation assay on the immunoprecipitated tropomodulin with which the PKCα had been co-immunoprecipitated (n=3). Tropomodulin was immunoprecipitated from N/N 1003A cells treated with 200 nM TPA for 60 min, 10 ng/ml EGF for 15 min, or N/N 1003A cells overexpressing PKCα for 7 days (Figure 4). After the addition of [γ-32P]-ATP and the appropriate reaction mixture (see methods), the tropomodulin/PKCα immunoprecipitates were eletrophoresed on an SDS-PAGE gel and the proteins were transferred to a nitrocellulose membrane. The blot was stained with Ponceau S to determine equal protein loading (not shown). After 2 days of exposure to x-ray film, there was a very heavy band at about 43 kD, representing phosphorylated tropomodulin for cells treated with TPA (Figure 4A, lane 3), with significantly less phosphorylated tropomodulin in cells overexpressing PKCα (Figure 4A, lane 2). There was also a heavy band at about 43 kD representing phosphorylated tropomodulin for cells treated with EGF (Figure 4A, lane 4). The lack of phosphorylation of tropomodulin in immunoprecipitates from control cells (Figure 4, lane 1) was not due to the absence of PKCα since immunoprecipitation and Western blotting of the same samples for PKCα demonstrated that all samples contained the same amount of PKCα (Figure 4C).
However, Western blotting of the same samples showed that somewhat more tropomodulin was present in the immunoprecipitates from the PKCα overexpressing and TPA-treated cells (Figure 4B). The reason for this is not clear, but may reflect differences in antibody reaction with phosphorylated vs non-phosphorylated tropomodulin. When the level of tropomodulin phosphorylation in Figure 4A was normalized for the amount of tropomodulin in each of the samples (Figure 4B), this revealed that the level of tropomodulin phosphorylation in TPA-treated cells was 2.2-fold more compared to the control cells. For cells treated with EGF, there was 1.8-fold increase in the level of tropomodulin phosphorylation compared to control cells. After normalization, the level of tropomodulin phosphorylation in cells overexpressing PKCα was much less, only 1.2-fold greater than the control cells. In previous studies overexpression of PKCα under our conditions only resulted in a 1.5-fold increase in PKC activity. Since this is not activated PKCα, the amount of tropomodulin phosphorylation would be low.
To determine whether tropomodulin was phosphorylated on serine or threonine residues, tropomodulin was immunoprecipitated from duplicate samples of control N/N 1003A cells, N/N 1003A cells treated with TPA, and N/N 1003A cells treated with EGF, followed by Western blotting and probing with anti-phosphothreonine (Figure 5A) or anti-phosphoserine antibody (Figure 5B). It was found that the cells that were treated with TPA had a stimulated threonine phosphorylation of tropomodulin (Figure 5A, group 2). Also, the cells that were treated with EGF had a stimulated threonine phosphorylation of tropomodulin (Figure 5C, lane 1). Thus, cells stimulated with EGF showed a phosphorylation of tropomodulin on threonine which is caused by the activation of PKCα.
There was no evidence of serine phosphorylation even when equal levels of protein were present (Figure 5B). This experiment also demonstrates that the tropomodulin may be an in vivo substrate for PKCα in N/N 1003A lens epithelial cells.
Effect of PKC phosphorylation of tropomodulin on tropomyosin binding activity
Previous studies have demonstrated that tropomodulin's tight capping ability of tropomyosin-actin filaments is because tropomodulin binds to both actin and tropomyosin . Tropomodulin has a higher affinity for the pointed ends of actin when tropomyosin is present . The tropomyosin-binding activity of tropomodulin can be assayed by blot overlays, in which tropomyosin protein followed by tropomyosin antibody, is used as a probe to detect tropomodulin:tropomyosin interactions . To investigate whether phosphorylation of tropomodulin affected its tropomyosin-binding activity, tropomodulin was immunoprecipitated from control N/N 1003A cells, N/N 1003A cells treated with TPA for 60 min, N/N 1003A cells treated with EGF for 15 min, or N/N 1003A cells overexpressing PKCα for 7 days followed by Western blotting (Figure 6A). Ponceau S was used to stain the blot to assure equal load of protein per lane (not shown). Tropomyosin binding to immunoprecipitated tropomodulin did not change for any of the samples when they were normalized against tropomodulin levels (Figure 6B). Thus, one can conclude that the phosphorylation of tropomodulin by PKCα has no effect upon tropomyosin binding activity.
Phosphorylation of tropomodulin by PKCα leads to increased association of tropomodulin with the cytoskeleton
We used Triton X-100 insolubility as an empirical measure of tropomodulin association with the cytoskeleton . Control N/N 1003A cells or N/N 1003A cells treated with TPA or EGF were harvested in cold CSK buffer containing 0.5% Triton X-100 and centrifuged at 13,000x g for 60 min at 4 °C. The resulting pellets containing insoluble, cytoskeleton-associated proteins and the resulting supernatants containing soluble protein were loaded onto an SDS-PAGE gel (25 μg protein/lane) and the amount of tropomodulin in each fraction was measured by Western blotting. These experiments showed that in control cells (Figure 7A, lanes 1 and 2), about 68.4±4.4% of the tropomodulin was present in the Triton-insoluble, cytoskeleton fraction (Figure 7B). In contrast, a significantly greater proportion of the tropomodulin was associated with the Triton-insoluble cytoskeleton in TPA-treated cells (Figure 7A, lanes 3 and 4), such that 88.1±3.3% of the tropomodulin was associated with the Triton-insoluble cytoskeleton and 11.9% remained in the soluble fraction. Cells treated with EGF also showed a large proportion of tropomodulin in the Triton-insoluble cytoskeleton such that 79.2±2.9% of the tropomodulin was associated with the Triton-insoluble cytoskeleton and 20.8% remained in the soluble fraction (Figure 7A, lanes 5 and 6). These results indicate that phosphorylation of tropomodulin by PKCα leads to increased tropomodulin association with the cytoskeleton.
Lens fiber cells require a stable elongated morphology that has been previously shown to involve specific actin regulatory proteins [8,29]. Tropomodulin is one such regulatory protein that binds to the pointed ends of actin filaments to prevent assembly/disassembly, thus creating a stable actin cytoskeleton. Tropomodulin contrasts with other lens actin-binding proteins that have been studied because it is expressed so late. Actin binding proteins spectrin, tropomyosin, calpactin, and α-actinin are present in both undifferentiated and differentiated lens fiber cells [30,31]. Even more interesting, tropomodulin expression is very similar to other known lens differentiation markers such as crystallins , α3 and α8 connexins , α6 integrin , and the lens intermediate filament proteins filensin and phakinin [34-36]. There is evidence that tropomodulin is restricted to postmitotic, long-lived cells with stable actin cytoskeletons, including striated muscle, erythrocytes, and neurons . Tropomodulin has not been detected in dividing, undifferentiated cells, such as lens cells from the germinative zone  or motile fibroblasts . Thus, the presence of tropomodulin may be a requirement of specialized differentiated cells such as lens fiber cells to maintain stable, long-lived actin filament arrays.
In the oldest lens cells, the nuclear fiber cells, microtubules and vimentin intermediate filaments are not present and the proteins are completely degraded [38-40]; however, tropomodulin and tropomyosin are not proteolyzed in the nuclear fiber cells of the adult lens . Lens-specific intermediate filament proteins such as phakinin and filensin are partially proteolyzed in nuclear fiber cells of adult bovine lenses although they remain associated with the plasma membrane [40,42]. Therefore, the nuclear fiber cells of adult lenses may have just actin filaments on the plasma membrane as the only intact cytoskeletal system.
Not only does tropomodulin assemble onto the lateral plasma membrane during lens fiber cell differentiation, but tropomodulin also is assembled onto a special structure at the apical and basal ends of newly differentiated fiber cells . The reasoning for a tropomodulin-containing cytoskeletal structure at the ends of young fiber cells could lie in the fact that a tropomodulin-containing structure might be important for apical and basal cell contacts with the epithelium or lens capsule. As new fiber cells push older fiber cells inward, their apical and basal ends slide past the epithelium or capsule . Perhaps accommodative focusing of the lens may require extra tensile strength at fiber ends; polygonal arrays of actin filament bundles at the epithelium-fiber cell interface have been proposed to be important for resisting tension during lens focusing . Also, a specialized structure may be important for regulating ion homeostasis, therefore, maintaining lens transparency, by clustering water channels and ion pumps to the apical and basal ends of fiber cells .
Lens epithelial cells normally do not have tropomodulin present, however, the N/N 1003A rabbit lens epithelial cell line does. Although the morphological changes in this study do not reproduce all the lens structural features, the data presented here replicate some of the changes in molecular composition of newly differentiated lens fiber cells. In cells that overexpress PKCα for 7 days or for extended time periods (in which cells begin to elongate), there is a significant increase of tropomodulin expression. The theory is that as the lens epithelial cells begin to elongate because of overexpression of PKCα, tropomodulin becomes expressed in order to stabilize the elongating epithelial cells. This result has been demonstrated in previous studies using whole lenses in which, at the annular pad of the lens, tropomodulin becomes expressed .
The PKC family can phosphorylate a large number of proteins and is involved in various cell functions. PKC is now recognized as a major regulatory enzyme and it has been implicated in the control of a wide variety of physiological processes. There are numerous endogenous substrates, including cytoskeletal proteins that can be phosphorylated by PKC. PKCα has been previously shown to initiate lens epithelial cell differentiation in culture by overexpression of the enzyme [22,23].
Because of PKCα's role in the initiation of fiber cell differentiation and the role that tropomodulin plays in differentiated lens fiber cells, one possible function of PKCα phosphorylation of tropomodulin is that it regulates lens fiber cell elongation. From this study, it was determined that only PKCα, and not PKCγ, phosphorylated tropomodulin. Previous studies from our laboratory suggest that PKCγ is the major PKC isoform in regulating gap junctions in that it phosphorylates serine residue(s) of Cx 43 . From Triton X-100 fractionation experiments done in this study, it was determined that the phosphorylation of tropomodulin by PKCα leads to an increase in the proportion of tropomodulin associated with the cytoskeleton. This could potentially lead to an increase in tropomodulin capping of actin filaments and subsequent actin filament stablization.
How might this work? First, the increase in tropomodulin association with the cytoskeleton following activation of PKCα does not appear to be due to a direct effect on tropomodulin binding to tropomyosin (Figure 6). Instead, it may be due to an increase in the capping affinity of phosphorylated tropomodulin for pure actin filament pointed ends. Interestingly, complexes of tropomodulin and actin without tropomyosin have been isolated from lens fiber cells . This would not have been expected based on the relatively weak binding of tropomodulin to pure actin pointed ends in the absence of tropomyosin . It is tempting to speculate that the tropomodulin in these complexes from the lens fiber cells might have been phosphorylated. This could potentially provide a mechanism to further stabilize the actin filament cytoskeleton in the long-lived lens fiber cells.
Alternatively, the increased amount of tropomodulin in the cytoskeleton after activation of PKCα in the lens cells could be due to an increase in tropomodulin binding to another (non-actin, non-tropomyosin) cytoskeleltal protein in the lens fiber cells. A possible candidate might be filensin, since tropomodulin has been reported to interact in vitro with the lens cell-specific intermediate filament protein, filensin [28,46].
PKC regulates many aspects of cellular structure and function . There has been considerable evidence suggesting that PKC regulates cytoskeletal interactions. Phorbol esters, which are potent activators of PKC, produce profound changes in morphology and organization of the cytoskeleton in many cell types [48,49]. Phorbol esters can also cause specific PKC isoforms to translocate to the cytoskeleton [50,51]. The ability of PKC to alter cytoskeletal protein function in vitro, its phosphorylation of these proteins in situ, and its translocation to the cytoskeleton upon activation suggest that the enzyme is involved in cytoskeletal regulation in the lens. For example, α3, β1, and α6 integrins become phosphorylated in a PKC-dependent manner [52,53]. Integrin-dependent cell adhesion, through integration of cell signaling pathways and cytoskeletal reorganization, influences cell growth, death, and differentiation . α6 Integrin is known to be a marker of lens fiber cell differentiation .
There are several actin-associated proteins that are phosphorylated by PKC. Profilin, a regulator of actin polymerization, is known to be an in vivo substrate of PKC [55,56]. Adducin, a calmodulin-binding protein, is also a substrate for PKC . This protein is localized at spectrin-actin junctions in erythrocyte cytoskeletons  and co-localizes with spectrin at sites of cell-cell contact in epithelial cells . Adducin exhibits in vitro activities of promoting association of spectrin with actin [59,60] and capping the fast-growing ends of actin filaments . Calponin is a cytoskeletal protein that binds to F-actin, tropomyosin, and calmodulin , and smooth muscle calponin is known to be an excellent substrate for PKC in vitro [63,64]. The phosphorylation of calponin by PKC results in loss of its activity via dissociation of calponin from actin . Among actin-binding or actin-associated proteins, filamin , troponin , talin , and vinculin [19,65] have been identified in vitro as PKC substrates.
In general, epidermal growth factor (EGF) has been shown to be a potent mitogen for normally amitotic central epithelial cells in organ culture or in cultures derived from central epithelial cells [67,68]. Of all the growth factors, EGF is the most effective in promoting not necessarily differentiation, but cell proliferation . EGF has been implicated in promoting the appearance of lentoid bodies cultured from human lens epithelial cells [69,70]. EGF has been detected within the aqueous humor and could be an exogenous source for lens stimulation , although its cellular origin could not be determined. There have been previous studies that name some possible endogenous sources of EGF in human and rat lenses. Lenticular EGF was found in regions that include the most peripheral epithelial cells plus superficial cortical fibers [72,73]. To date, a role for EGF in regulating the cell division responsible for continuous lens growth remains to be determined.
PKC activation by a growth factor promotes changes in actin filament organization. Most of the work has been done using EGF in a number of different cell types and comparing the results with the activation of PKC by TPA. EGF induces changes in actin structure in human epidermoid carcinoma A-431 , KB cells , primary rabbit corneal endothelial cells , and also in human retinal pigment epithelial cells , while similar changes have been stimulated by TPA in epithelial African green monkey kidney (BSC-1) cells , chick embryo fibroblast , various transformed fibroblast cell lines , canine thryroid epithelial cells , and human T lymphocytes . In all these cells types, changes in actin organization were accompanied by increased membrane ruffling, increased lamellipodial and filopodial extension, and increased motility. Therefore, the stimulation of individual cell migration that occurs upon exposure of these cell types to EGF is a partial consequence of PKC-induced cytoskeletal alterations that promote cell motility.
Membrane ruffling involving cytoskeletal reorganization is a cellular response regulated by growth factors such as EGF and IGF-1 [83-85]. Previous studies have shown that EGF, IGF-1, and insulin induce membrane ruffling in human epidermoid carcinoma (KB) cells [83,86,87]. IGF-1 (and insulin) has been shown to induce membrane ruffling through PKC-independent pathway in human epidermoid carcinoma (KB) cells . However, in the same cell type, EGF induced membrane ruffles through PKC-dependent pathway . From our studies and those of others, it is clear that EGF elicits a large number of cellular responses by activating PKC that have to do with the changes in the actin cytoskeleton.
The authors thank Dr. John Reddan for providing the N/N1003A cells (Oakland University) and Dr. Wayne Anderson (NCI) for providing PKCγ and PKCα plasmids. We gratefully acknowledge the support of NIH Grant EY13421. This is publication 02-131-J from the Kansas Agricultural Experiment Station.
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