Studies of Tyrosine Phosphorylation and Src Family Tyrosine Kinases in the Lens Epithelium

Abstract

purpose. Tyrosine phosphorylation regulates many aspects of cell function; thus, cells that have different roles often display different patterns of tyrosine phosphorylation. Because there is interest in differential function of the anterior and equatorial regions of the lens epithelium, studies were conducted to compare tyrosine phosphorylation in the two zones.

methods. Anterior and equatorial regions of the porcine lens epithelium were collected. Using Western blot analysis, tissue homogenates were probed for tyrosine kinase proteins, phospho-Src, phosphotyrosine, and proliferating cell nuclear antigen (PCNA).

results. Phosphotyrosine immunoblots revealed a marked difference between the pattern of tyrosine phosphorylation in anterior and equatorial regions of the epithelium. Many more bands were detected in the equatorial region, and band density was greater. The abundance of total and active Src family kinases was higher at the equator than at the anterior epithelium. Src kinase activity, which was measured directly by quantifying phosphorylation of a synthetic target peptide using ³²P-γ-ATP, was detectable only at the equator. In organ-cultured lenses, PP2, a specific inhibitor of the Src kinase family, reduced the density of the phosphotyrosine protein bands. The abundance of PCNA, a protein expressed in proliferating cells, also was reduced in PP2-treated lenses.

conclusions. The results suggest that the higher Src family kinase activity at the equator contributes to the higher degree of protein phosphorylation observed in this region. The ability of PP2 to suppress PCNA expression suggests a possible link between the activity of Src family kinases and cell proliferation.

The lens consists of two cell types: fiber cells, which make up the bulk of the tissue, and a relatively small number of epithelial cells that exist as a monolayer covering the anterior surface. Numerous studies have compared the epithelium and fibers and identified differences between the two cell types. Now there is a growing interest in functional differences between the anterior and equatorial regions of the lens epithelium. Epithelial cells at the anterior pole have a flattened appearance and exhibit no mitotic activity. At the equator, the epithelium is more cuboidal, and a small number of cells in this region undergo cell division and fiber differentiation. The anterior and equatorial zone display differences in sodium pump function, gap junction distribution, and growth factor receptor function. ^{1 2 3}  

In the present study, Western blot experiments were conducted to examine protein tyrosine phosphorylation in the equatorial and anterior region of the porcine lens epithelium. Tyrosine phosphorylation regulates many aspects of cell function; thus, cells that have different roles often display different patterns of tyrosine phosphorylation. Predictably, lens epithelium and fibers display a different pattern of phosphorylation bands. ⁴ In the present study, tyrosine phosphorylation in the lens epithelium was found to be markedly higher at the equator. This suggests that there may be differences in tyrosine kinase expression and activity between the anterior and equatorial epithelium. For this reason, studies also were conducted to examine the Src family of nonreceptor protein tyrosine kinases, which have been shown to regulate several aspects of cell function in avian lens epithelium. ⁵ Src, Yes, and Fyn were examined, since these are the three most ubiquitously expressed members of the Src kinase family. ⁶  

Materials and Methods

Peptides and materials used for the Src kinase activity assay and the anti-Src monoclonal antibody were obtained from Upstate Biotechnology (Lake Placid, NY); γ-[³²P]ATP from Amersham Biosciences (Piscataway, NJ); the anti-phosphotyrosine (PY) monoclonal antibody, anti-Yes and anti-Fyn monoclonal antibodies from BD Biosciences (San Diego, CA); the anti-phospho-Src polyclonal antibody from Cell Signaling Technology (Beverly, MA); the anti-PCNA monoclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA); and the EGF receptor specific inhibitor AG1478, the Src-specific inhibitor PP2, and its inactive analogue PP3 from Calbiochem (La Jolla, CA). All other chemicals, unless specified, were obtained from Sigma-Aldrich (St. Louis, MO). 

Sample Preparation

From porcine eyes kindly donated by Swift Meat Packing Co. (Louisville, KY), lenses were dissected posteriorly. The use of animal tissues was approved by the University of Louisville Institutional Animal Care and Use Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The lens capsule-epithelium was obtained separately from the central zone (anterior region) and periphery (equatorial region) of the anterior lens surface. The anterior epithelium represented roughly 50% to 60% of the anterior surface. The anterior epithelium was dissected free by a curvilinear tear made with surgical scissors. After removal of the anterior epithelium, the remaining equatorial epithelium was then separated from the fiber mass. Western blot analysis for aquaporin 0, a membrane protein expressed in lens fibers but not epithelium, ⁷ suggests fiber contamination is minimal in the equatorial and anterior epithelium homogenates, since aquaporin 0 bands are absent under normal experimental conditions. However, we cannot rule out some degree of fiber contamination, because multiple nonspecific bands as well as an aquaporin 0 band did become visible in blots incubated with primary antibody for 2 days. Contaminating aquaporin 0 band density in the equatorial and anterior samples were similar. 

In specified experiments, lenses were cultured in 4 mL RPMI1640 medium for 24 hours in the presence of the Src kinase inhibitor PP2 (10 μM) or its inactive analogue PP3 (10 μM), before the isolation of the anterior and equatorial epithelium. For Western blot analysis, anterior and equatorial epithelium samples were homogenized separately in either ice-cold RIPA buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, and the protease inhibitors 2 μM antipain, 2 μM leupeptin, 1 μM pepstatin A, 1 μM phenylmethylsulfonyl [PMSF], and 2 μg/mL aprotinin). ⁸ The epithelium samples were homogenized in Src-kinase assay buffer (100 mM Tris-HCl [pH 7.2], 125 mM MgCl₂, 25 mM MnCl₂, 2 mM EGTA, 0.25 mM sodium orthovanadate, and 2 mM dithiothreitol) for the Src kinase activity assay. Protein concentration was determined with a kit (Bio-Rad, Hercules, CA) based on an assay described by Bradford. ⁹  

Western Blot Analysis

After addition of Laemmli buffer ¹⁰ to the epithelial cell homogenate, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in TTBS (30 mM Tris, 150 mM NaCl, 0.5% [vol/vol] Tween-20 [pH 7.4]) for 1 hour and then incubated with primary antibody for another hour. After three washes in TTBS for 5 minutes each, the nitrocellulose membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. After three 5-minute washes, enhanced chemiluminescence substrate (Pierce, Rockford, IL) was applied for 1 minute to the nitrocellulose membrane, and the proteins were visualized by exposure to x-ray film. In some cases, x-ray film was digitally scanned, and band densities were quantified with image-analysis software (Kodak 1D; Eastman Kodak Co., Rochester, NY). 

Src Kinase Activity Assay

Tyrosine kinase activity of the Src kinase family was measured with a commercially available assay kit (Upstate Biotechnology) by quantifying phosphorylation of a synthetic Src substrate peptide (KVEKIGEGTYGVVKK) corresponding to residues 6-20 of p34^cdc2. ¹¹ A similar synthetic peptide in which the tyrosine is replaced by phenylalanine (KVEKIGEGTFGVVKK) was used as a negative control. A sample containing 20 μg of epithelial cell homogenate protein in 40 μL of Src-kinase assay buffer was mixed with 150 μM substrate peptide. In specified experiments, PP3 (10 μM) or PP2 (10 μM) was added to the mixture. After the addition of 115 μM ATP with a trace amount (10 μCi) of [γ -³²P]ATP, the mixture was incubated at 30°C for 20 minutes. The reaction was terminated by adding 20 μL of 40% trichloroacetic acid. Samples were spotted onto P81 phosphocellulose paper (Whatman, Clifton, NJ). After five 5-minute washes with 0.75% phosphoric acid and one 5-minute wash with acetone, the phosphocellulose paper was examined for radioactivity in the presence of scintillation fluid. 

Results

Examination of tyrosine-phosphorylated proteins using an antibody directed against phosphotyrosine (PY) residues revealed a marked difference between the pattern of tyrosine phosphorylation in anterior and equatorial regions of the epithelium. Many more bands were detected in the equatorial region, and band density was greater. Whereas the equatorial samples displayed a higher abundance of PY-immunoreactive bands compared with the anterior, the abundance of β-actin, a housekeeping protein used as a loading control, was similar in the two regions (Fig. 1) . 

Studies were conducted to examine the Src family of tyrosine kinases. Western blot analyses were performed to probe for Src, Yes, and Fyn, the three most ubiquitously expressed members of the family. Src, Yes, and Fyn immunoblot band densities were higher at the equator than at the anterior epithelium (Fig. 2) . 

The presence of proteins in the Src kinase family does not necessarily signify activity. Src activity was examined in two ways, by Western blot and by direct measurement of tyrosine phosphorylation. The activity of Src family members is regulated by two tyrosine residues, the inhibitory tyrosine at the C terminus of the kinase (Tyr527 of Src) and an activating tyrosine within the catalytic domain (Tyr416 of Src). Western blot studies were conducted with an antibody directed against the activating phosphotyrosine within the catalytic domain. A more dense band was observed at the equator than at the anterior, indicating that the active form of the Src kinase family is higher at the equator (Fig. 3) . To examine the activity of the Src kinase family in a more direct manner, studies were conducted to quantify phosphorylation of synthetic target peptides using γ-[³²P]ATP. In epithelial cells homogenates obtained from the equatorial region, incorporation of ³²P into the Src-specific peptide ([Lys19] cdc2 (6-20)-NH2) was significantly greater than the control in which peptide was omitted (Fig. 4) . Incorporation of ³²P into the Src-specific peptide was also significantly greater than incorporation into a negative control peptide in which the target tyrosine is replaced by phenylalanine ([Lys19, Phe15] cdc2 (6 -20)-NH2). The tyrosine kinase activity toward the Src-specific peptide (total activity minus control activity) was 0.17 pmol PO₄/μg protein per hour. For the anterior epithelium samples, incorporation of ³²P into the Src-specific peptide was similar to incorporation into both the nonpeptide control or negative control peptide (Fig. 4) . The tyrosine kinase activity was below the detection limit of the assay in the anterior region of the lens epithelium. 

Figures 2 to 4show that the equatorial epithelium, which has a higher abundance of tyrosine phosphorylated proteins, also exhibited higher protein expression of Src kinase family members and higher Src family kinase activity than did the anterior epithelium. To examine the potential contribution of the Src kinase family to the observed pattern of tyrosine phosphorylation, equatorial epithelium was isolated from porcine lenses that had been cultured for 24 hours in the presence of the Src kinase family inhibitor PP2. Control lenses were exposed to PP3, a structurally related compound that lacks Src inhibitory activity. Western blot analysis revealed that PP2 markedly reduced the density of multiple phosphotyrosine bands (Fig. 5) . Analysis was not possible at the anterior epithelium because the bands were too faint, even in the control samples. Inhibition of Src family kinase activity by PP2 was confirmed in separate kinase activity assays. Figure 6shows that the addition of 10 μM PP2 to the equatorial lens homogenate abolished Src-specific peptide phosphorylation, whereas 10 μM PP3 had little effect compared with 0.1% dimethylsulfoxide (DMSO; used as a vehicle). In the same assay, PP2 did not detectably alter nonspecific tyrosine phosphorylation of endogenous lens epithelium proteins. 

The effect of a receptor tyrosine kinase inhibitor on tyrosine phosphorylation of the equatorial epithelium was tested. The EGF receptor inhibitor AG1478 was chosen, because it had been used previously on lens cells, ¹² and the EGF receptor has been shown to activate Src family kinases in other cell types. ^{6 13} Intact lenses were exposed to 1 μM AG1478 for 24 hours. Unlike PP2, AG1478 had no detectable effect on tyrosine phosphorylation compared with PP3 (Fig. 7) . 

It is well established that in a variety of tissues, tyrosine phosphorylation patterns are different in growing and quiescent cells. ¹⁴ A recent study by Choi et al. ¹⁵ has shown that PCNA can be used as a proliferation marker in porcine lens epithelial cells. The abundance of PCNA was analyzed by Western blot. As shown in Figure 8 , PCNA expression was detectable only at the equatorial region of the porcine lens epithelium. After 24-hour exposure of the intact lens to PP2, the band density for PCNA at the equatorial region was significantly reduced compared with PP3-treated controls (Fig. 9a 9b) . PP3 added alone did not elicit a consistent reduction of PCNA band density (Fig. 9c) . Because PP2 treatment reduces PCNA band density, we considered the possibility that extensive proteolysis occurs in the epithelium of PP2-treated lenses. However, it was observed that PP2 treatment of the lens did not cause detectable breakdown of β-actin Western blot bands (Fig. 9a) . 

Discussion

The degree of protein tyrosine phosphorylation reflects a balance between the opposing actions of tyrosine kinases and tyrosine phosphatases. In this respect, the anterior and equatorial regions of the lens epithelium are different. The greater number and greater density of phosphotyrosine-immunoreactive bands observed in the equatorial region fit with the finding of an unequal distribution of Src family nonreceptor tyrosine kinases in the epithelium. Src, Yes, and Fyn proteins were each found to be more abundant at the equator than in the anterior region of the epithelium. A higher abundance of Src does not necessarily mean higher Src kinase activity. Mechanistically, autophosphorylation of a tyrosine within the catalytic domain of the enzyme (Y416 in Src) is believed to play a role in the activation of Src family kinases by inducing conformational changes. ⁶ Therefore, phosphorylation of this tyrosine is often used as a marker of active Src. ^{16 17 18} Western blot using an antibody directed against the autophosphorylating tyrosine of the Src family indicated higher abundance of active Src family kinases at the equator compared to the anterior region of the lens epithelium (Fig. 3) . In this study, the Src kinase proteins did not appear as strong bands in the phosphotyrosine Western blot analysis, perhaps because of the relatively low abundance of the kinase family proteins compared with other tyrosine phosphoproteins in the preparation. Even in studies with exogenously added Lyn kinase, the kinase band was not apparent in phosphotyrosine Western blot analysis. ¹⁹ The regional difference between Src family kinase activity in the equatorial and anterior epithelium was confirmed by an in vitro phosphorylation assay using an Src family-specific peptide substrate. Taken together, the results suggest Src family kinases are not only more abundant at the equator, but the activity is also higher in that region than at the anterior epithelium. Of interest, the expression of ERK, a Ser/Thr kinase that regulates various cellular activity, is also reported to be higher at the equator than in the anterior region of the lens epithelium. ¹²  

Inhibition of Src kinase activity by PP2 reduced the overall degree of tyrosine phosphorylation in the equatorial region of the lens epithelium. In the anterior region, the phosphotyrosine bands were not strong enough to permit detection of an altered pattern in PP2-treated lenses. The results from PP2-treated lenses suggest that Src family tyrosine kinase activity contributes to the maintenance of the higher overall degree of tyrosine phosphorylation observed in the equatorial region. Other tyrosine kinases may also contribute to the observed tyrosine phosphorylation pattern. The higher endogenous level of tyrosine phosphorylation at the equator fits with regional differences in the distribution of receptor tyrosine kinases. de Iongh et al. ²⁰ have shown that FGFR1, the receptor for FGF-1 and -2, has significantly stronger expression at the equatorial region than at the anterior epithelium. Collison and Duncan ¹ have shown that the calcium signaling induced by EGF and PDGF is only observed in the equatorial region of the lens. It is noteworthy that receptor tyrosine kinases can be situated upstream or downstream of Src kinases in a signaling pathway. For example, receptor tyrosine kinases such as PDGF, EGF, and IGF are known to activate Src kinases. ^{6 13} It has also been shown that Src is involved in the transactivation of EGFR by G-protein-coupled receptor agonists. ^{21 22} However, the EGF receptor inhibitor AG1478 failed to alter the tyrosine phosphorylation pattern. Of course this does not rule out cross talk with other receptor tyrosine kinases. 

The Src kinase family and receptor tyrosine kinases have a major influence on proliferation, differentiation, and survival of various cell types. ^{6 23 24 25} Choi et al. ¹⁵ demonstrated recently that PCNA can be used as a proliferation marker for porcine lens epithelial cells growing in the capsular bag model. PCNA is present at high levels in proliferating cells in the S-phase of the cell cycle, whereas normal nondividing cells have low PCNA expression. ²⁶ In the present study, we examined PCNA in epithelium isolated from the intact porcine lens. Cell proliferation is restricted to the equatorial region, ²⁵ and, indeed, in our experimental conditions, PCNA expression was detectable only in the equatorial lens epithelium. Treatment of the intact lens with PP2 resulted in a significant reduction of PCNA immunoblot band density compared with the PP3-treated control. The results suggest a possible link between Src activity and lens cell proliferation. This fits with BrdU incorporation studies by Walker et al., ⁵ who demonstrated that suppression of the Src kinase family by PP1, a synthetic Src kinase family inhibitor very similar to PP2, inhibits proliferation of primary cultured embryonic avian lens epithelium. Studies using other cell types have shown that Src family tyrosine kinases participate in the chain of events by which DNA synthesis is induced by growth factors such as EGF, PDGF, and FGF-2. ^{13 27} These growth factors are known to play a role in lens cell growth. ^{12 25 28}  

The methods used in the present study do not allow us to distinguish phosphotyrosine band density or activity of the Src kinase family exclusively in the proliferative cells and the differentiating cells, because these account for only a small fraction of the equatorial epithelium. Studies on avian lens epithelial cells in primary culture suggest that Src suppresses differentiation by phosphorylating N-cadherin and altering the formation of cell–cell junctions that provide necessary cues for the cells to differentiate. ^{5 29} There have also been reports that tyrosine phosphorylation could influence lens transparency. During the development of selenite cataract, Chandrasekher and Sailaja ³⁰ observed that lens opacification was associated with a decrease in the overall extent of protein tyrosine phosphorylation. On the contrary, Zhou and Menko ³¹ have reported that Src activity, and therefore tyrosine phosphorylation, increases as part of a stress-induced signaling pathway in an avian spontaneous cataract model. Further study is needed if we are to understand the significance of tyrosine phosphorylation in cataract. 

In summary, in the present study, the overall degree of endogenous protein tyrosine phosphorylation was higher in the equatorial region of the lens epithelium than in the anterior. Src family tyrosine kinase activity was detectable only in the equatorial region, and results from studies with the inhibitor PP2 suggest that this tyrosine kinase activity contributes to the endogenous pattern of tyrosine phosphorylation. The change in PCNA expression after Src family kinase inhibition suggests a potential link between cell proliferation and tyrosine phosphorylation. 

 Supported by Grant EY09532 from the National Eye Institute, Research to Prevent Blindness Inc., and The Kentucky Lions Eye Foundation.

 Submitted for publication October 8, 2004; revised January 21, 2005; accepted February 11, 2005.

 Disclosure: S. Tamiya, None; N.A. Delamere, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Tyrosine-phosphorylated proteins in the anterior (AE) and equatorial (EE) porcine lens epithelium. Homogenates from each region of the lens epithelium were probed for phosphotyrosine (PY) residues by Western blot. The phosphotyrosine blot (left) was stripped and reprobed for β-actin (right), which was used as a loading control. The blot is representative of results observed in five separate experiments. Middle: molecular masses in kilodaltons.

Figure 2.

 

Western blot of Src kinase family members, Src, Yes, and Fyn in anterior (AE) and equatorial (EE) porcine lens epithelium. The blot is representative of results observed in four separate experiments. Left: molecular masses in kilodaltons.

Figure 3.

 

Active Src family kinases in anterior (AE) and equatorial (EE) porcine lens epithelium. Homogenates from each region of the lens epithelium were probed by Western blot using an antibody directed against the autophosphorylating tyrosine within the catalytic domain of Src family kinases. The blot is representative of results observed in four separate experiments. Left: molecular masses in kilodaltons.

Figure 4.

 

Measurement of Src family kinase activity in anterior (AE) and equatorial (EE) porcine lens epithelium. Homogenates from both region of the lens epithelium were mixed with buffer only (no peptide control), an Src-specific peptide ([Lys19] cdc2 (6-20) NH₂), or a non–tyrosine-phosphorylatable peptide ([Lys19, Phe15] cdc2 (6-20) NH₂) in the presence of γ-[³²P]ATP. Incorporation of ³²P was measured with a scintillation counter and the results expressed as a percentage of the no-peptide control. The data are the mean ± SE of five independent experiments. *Significant difference (P < 0.05) from the no peptide control and from the nontyrosine peptide control.

Figure 5.

 

Tyrosine-phosphorylated proteins in equatorial epithelium of porcine lenses cultured for 24 hours in the presence of PP2 or PP3. Homogenates were probed for phosphotyrosine (PY) residues by Western blot. The phosphotyrosine blot (left) was stripped and reprobed for β-actin (right), which was used as a loading control. The blot is representative of results observed in five separate experiments. Middle: molecular mass in kilodaltons.

Figure 6.

 

The effect of a specific Src kinase family inhibitor PP2 or its inactive analogue PP3 on equatorial lens epithelium Src family kinase activity. Homogenates from the equatorial region of the lens epithelium were mixed with an Src-specific peptide ([Lys19] cdc2 (6-20) NH₂) or a non–tyrosine-phosphorylatable peptide ([Lys19, Phe15] cdc2 (6-20) NH₂) in the presence of 0.1% DMSO (vehicle), 10 μM PP3, or 10 μM PP2. After the addition of γ-[³²P]ATP, incorporation of ³²P was measured with a scintillation counter and the results expressed as a percentage of the non–tyrosine-phosphorylatable peptide. The data are mean ± SE of four independent experiments.

Figure 7.

 

Tyrosine-phosphorylated proteins in equatorial epithelium of porcine lenses cultured for 24 hours in the presence of PP3, PP2, or a specific inhibitor of EGF receptor AG1478. Homogenates were probed for phosphotyrosine (PY) residues by Western blot. Left: molecular mass in kilodaltons.

Figure 8.

 

PCNA expression in the anterior (AE) and equatorial (EE) porcine lens epithelium. Homogenates from each region of the lens epithelium were probed for PCNA by Western blot. The PCNA blot (top) was stripped and reprobed for β-actin (bottom), which was used as a loading control. The blot is representative of results observed in three separate experiments. EGF-stimulated A431 cell lysate was used as a positive control (+ve).

Figure 9.

 

PCNA expression in the equatorial epithelium of PP3- and PP2-treated lenses. (a) Western blot for PCNA (top) representative of results observed in five separate experiments. The PCNA blot was stripped and reprobed for β-actin (bottom), which was used as a loading control. (b) Relative pixel density of digitized PCNA blots normalized for protein loading. The results were obtained from the epithelium of PP3- and PP2-treated lenses. Results are the mean ± SE of five separate experiments. *P < 0.05 between PP3 and PP2 band densities. (c) Western blot showing PCNA band density in PP3-treated lenses compared with lenses exposed to vehicle (0.1% DMSO) alone.