Renal Section, Evans Biomedical Research Center, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Cyanide
(CN)-induced chemical anoxia of cultured mouse proximal tubular (MPT)
cells increased the kinase activity of c-Src by approximately
threefold.
4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a specific inhibitor of c-Src, prevented Src activation. CN also
increased the permeability of MPT cell monolayers, an event ameliorated
by PP2. During CN treatment, the proteins of the zonula adherens (ZA;
E-cadherin and the catenins) disappeared from their normal location at
cell-cell borders and appeared within the cytosol. CN also resulted in
the appearance of c-Src at cell-cell borders. PP2 prevented these
CN-induced alterations in the distribution of ZA proteins and c-Src. CN
also increased the association of c-Src with -catenin and p120 and
induced a substantial increase in tyrosine phosphorylation of both
catenins. PP2 prevented the CN-induced phosphorylation of these
catenins. In summary, we show that CN-induced chemical anoxia activates
c-Src and induces its translocation to cell-cell junctions where it
binds to and phosphorylates
-catenin and p120. Our findings suggest
that these events contribute to the loss of the epithelial barrier
function associated with chemical anoxia.
ischemia; tight junction; Src kinase; E-cadherin; catenins; p120ctn; renal tubular cells
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INTRODUCTION |
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CHEMICAL ANOXIA, A TERM THAT refers to the depletion of cell energy stores using mitochondrial inhibitors such as antimycin (10, 51), rotenone (16), or cyanide (CN) (37, 46), is widely used as a model of reversible injury to cultured cells (34). A number of investigators have shown that chemical anoxia can reversibly impair the integrity of the junctional complex, an effect that manifests functionally as loss of transepithelial electrical resistance and an increase in permeability of the epithelial monolayer (7, 10, 30, 44). The junctional complex comprises at least three structures: the zonula occludens (ZO), the zonula adherens (ZA), and desmosomes (11, 18, 21, 22). The ZO (also called the tight junction) is the component of the junctional complex that represents the physical barrier to paracellular flux of molecules and ions across the epithelium (11). However, the integrity of the ZO is dependent on the formation of an intact ZA, which lies immediately basal to the ZO and mediates cadherin-dependent cell-cell adhesion (22).
The ZA consists of a complex of proteins consisting of E-cadherin and
members of the catenin family of proteins. E-cadherin is a
transmembrane cell-cell adhesion molecule that mediates cell-cell adhesion by the homophilic binding of the extracellular domains expressed on adjacent cells (1, 19, 33). The catenin
family consists of a number of isoforms including -,
-,
- and
p120 catenin (p120ctn) (1, 2). The
- and
-catenins compete for binding to a single site on the cytoplasmic
tail of E-cadherin called the "catenin-binding domain"(CBD), so
each E-cadherin molecule binds to either
- or
-catenin in a
mutually exclusive manner (1, 19). Both
- and
-catenin bind to
-catenin, which, in turn, binds to the actin
cytoskleleton either directly (42) or indirectly via the actin binding protein
-actinin (29, 38). By contrast,
p120ctn (p120) binds to the juxtamembrane (JMB) domain of
the cytoplasmic tail of E-cadherin and is present in all E-cadherin
complexes containing either
- or
-catenin (49, 53).
Thus the cytoplasmic tail of E-cadherin has at least two distinct
protein-binding epitopes in its cytoplasmic tail: one, the CBD, that
associates with either a
- or
-catenin, and another, the JMB,
that binds to p120 (2).
We have previously reported that sublethal injury induced in tubular
cells by CN-induced chemical anoxia leads to tyrosine phosphorylation
of -catenin and that phosphorylation of components of the ZA
contributes to the disruption of ZA and the loss of the epithelial
permeability barrier associated with sublethal injury
(44). These findings are consistent with several lines of
evidence that have implicated protein tyrosine kinases and protein
tyrosine phosphatases in the normal modulation of E-cadherin-catenin complex formation and disassociation (5, 6, 9, 11, 13,
32). Furthermore, activation of the Src family of tyrosine kinases has been implicated in tyrosine phosphorylation of ZA proteins
and cell-cell adhesion in keratinocytes and other nonrenal epithelial
cells (9, 28, 48).
In this study, we provide entirely novel evidence that CN-induced
sublethal injury to renal tubular cells activates c-Src and induces its
translocation to the ZA. In addition, activated c-Src tyrosine binds
to, and tyrosine phosphorylates, -catenin and p120. Finally, we
demonstrate that these events contribute to the reversible loss of
cell-cell adhesion and the increase in epithelial permeability
associated with sublethal injury.
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MATERIALS AND METHODS |
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Reagents.
Protein G-Sepharose beads were obtained from Pierce (Rockford, IL).
Primary antibodies were a rabbit polyclonal anti-c-Src antibody (Santa
Cruz Biotechnology, Cambridge, MA); a mouse monoclonal anti-p120
antibody; a rabbit polyclonal anti--catenin antibody (Sigma, St.
Louis, MO); a rabbit polyclonal anti-
-catenin antibody (Sigma); a
mouse monoclonal anti-focal adhesion kinase (FAK) antibody (Upstate
Biotechnology, Lake Placid, NY); and a mouse monoclonal anti-phosphotyrosine antibody (PY20; Transduction Laboratories, Lexington, KY). Secondary antibodies for immunoblotting were rabbit or
mouse IgG conjugated with horseradish peroxidase (Sigma). Secondary antibodies used for immunofluoresence were donkey anti-mouse or goat
anti-rabbit IgG conjugated with indocarbocyanine (CY3; Sigma). The Src
kinase assay kit was purchased from Upstate Biotechnology, and the Src
kinase inhibitor
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) was from Calbiochem (La Jolla, CA). Rhodamine phalloidin was purchased from Molecular Probes.
Isolation and characterization of a conditionally immortalized
mouse proximal tubular cell line.
We have established a conditionally immortalized mouse proximal tubular
cell line using the "immortoMouse," a transgenic mouse containing
the H-2Kb-tsA58 transgene (47).
This transgene is a temperature-sensitive mutant of the SV40 large T
antigen oncogene. The presence of -interferon in the medium together
with a low incubation temperature (33°C) provides the permissive
conditions necessary for expression of the
H-2Kb-tsA58 transgene. The expression of the
gene is almost completely inhibited (by >95%) under nonpermissive
conditions in which
-interferon is not added to the medium and cells
are incubated at 37°C (47). This study is the first in
which we use this novel cell line.
Passaging of BUMPT-306 cells. The BUMPT-306 cells were passaged under permissive conditions in P100 culture dishes. For all experimental studies, cells were grown to confluence on culture dishes coated with rat tail collagen under permissive conditions. When close to confluence, monolayers were then incubated under nonpermissive conditions for 2 days before experimental studies were begun.
Experimental model of ATP depletion. Cells were incubated in a HEPES-buffered solution (15 mM, pH 7.4) containing (in mM) 134 NaCl, 3.6 KCl, 1.3 KH2PO4, 15 HEPES, 1 CaCl2, and 1 MgCl2. Dextrose (10 mM) was added to the medium of control cells. ATP depletion was induced with medium containing sodium cyanide (NaCN; 5 mM) to inhibit mitochondrial function and an absence of dextrose (to inhibit glycolysis), as described previously (30, 46). ATP depletion followed by recovery was achieved by incubating the cells in CN/no dextrose for 45 min and then incubating the cells in medium containing 10 mM dextrose without CN for an additional 30 min (the CN washout period) (30, 46). In some studies, Src was inhibited in dextrose- and CN-treated cells using PP2 (10 µM), a specific inhibitor of Src (24).
Measurement of cellular ATP content. Cellular ATP was measured using the luciferase assay as previously described (46). ATP levels were measured in control (dextrose-treated) cell monolayers and after 5, 10, 15, 30, and 45 min of incubation with CN. ATP levels at each of these time points are expressed as the percentage of control values.
Immunoprecipitation of c-Src. Cells were lysed in RIPA buffer comprising (in mM) 20 Tris · HCl, pH 7.5, 140 NaCl, 1 NaF, 1 PMSF, and 10 NaPiPO4, as well as 0.5% Na-deoxycholate, 0.1% SDS, 1% Triton X-100, and 10% glycerol. Sodium vanadate [1 mM, a tyrosine phosphatase inhibitor and the protease inhibitors contained in Complete tablets (Boeringer Mannheim)] were added to the lysis buffer just before use. The lysate was harvested and centrifuged at 4°C for 15 min at 2,000 rpm, and the supernatants were saved and kept on ice. Protein G-Sepharose beads were prepared for use by washing with washing buffer [(in mM) 20 Tris · HCl, pH 7.5, 140 NaCl, 1 EDTA, pH 7.5, 1 vanadate, 1 NaF, 1 PMSF, and 10 NaPiPO4, as well as 2% Nonidet P-40 and protease inhibitors] and then diluted in the same buffer. A 500-µg protein aliquot of this supernatant was "precleared" (3) by incubation with non-immune (normal rabbit serum) and beads. We immunoprecipitated c-Src in the supernatant of this precleared sample using a polyclonal antibody to c-Src (Santa Cruz Biotechnology) conjugated to the protein G beads. Immunoprecipitates were washed with washing buffer and used to measure Src kinase activity or to immunoblot for proteins associated with Src.
Src kinase assay.
The immunoprecipitates were suspended in a reaction buffer containing a
synthetic peptide as the Src kinase substrate peptide (150 µM/assay)
and [-32P]ATP and incubated for 10 min at 30°C. The
reactions were stopped by incubating the immunoprecipitates with 40%
TCA (20 µl/tube) at room temperature for 5 min. The beads were
pelleted, and the supernatant (10 µl/tube) was transferred to p81
paper assay squares. The assay squares were washed six times for 5 min
each with 40 ml of 0.75% phosphoric acid and once with acetone. Assay
squares were then transferred to scintillation vials each containing 5 ml scintillation cocktail, and counts incorporated were read on a
scintillation counter. The kinase activity was expressed as picomoles
of phosphate incorporated per minute per milligram protein.
Western blotting.
Lysates immunoprecipitated with c-Src antibody were boiled in 2× SDS
sample buffer for 10 min. The beads were then spun down, and the
supernatant containing the immunoprecipitated proteins were subjected
to SDS-PAGE and immunoblotted with appropriate antibodies to
E-cadherin, pp120, -,
-, and
-catenin, p120, or PY20.
Immunoblots were probed with horseradish peroxidase-conjugated secondary antibodies using chemiluminiscence. Each immunoblot was
stripped and reprobed with the antibody used for immunoprecipitation to
confirm that equal amounts of immunoprecipitated protein were loaded in
each lane.
Immunofluorescence studies. BUMPT-306 cells were grown to confluence on collagen-coated coverslips and treated with NaCN or dextrose for 45 min at 37°C. The cells were fixed in 3.7% paraformaldehyde for 10 min at room temperature (RT). Then, monolayers were washed three times for 5 min each in Tris-buffered saline (TBS; 50 mM Tris, pH 7.6, 150 mM NaCl) and incubated with 1% BSA, prepared in TBS, for 15 min at RT. For visualization of c-Src, E-cadherin, and catenins, cells were then incubated with the appropriate primary antibody diluted in 1% BSA for 45 min at RT. After a washing with TBS, the cells were incubated with Cy3-conjugated secondary antibody for 45 min at RT. Unbound secondary antibody was removed by washing. The F-actin filaments were stained directly with rhodamine phalloidin (Molecular Probes) as described previously (30). After being stained, the cells were mounted on glass slides and photographed using an immunofluorescence microscope and a digital camera.
Measurement of epithelial permeability.
The permeability of epithelial monolayers was determined with methods
previously described by our group (30, 44) by assessing the paracellular flux of tritiated inulin across monolayers of BUMPT-306 cells. BUMPT-306 cells were grown to confluence on 12-mm permeable nitrocellulose inserts (Millicel-HA, Millipore, Bedford, MA).
At the start of all experiments, the inserts were carefully washed with
warm Krebs-Henseleit buffer [KHB; (in mM) 115 NaCl, 3.6 KCl, 1.3 KH2PO4, 25 NaHCO3, 1 CaCl2, and 1 MgCl2]. Then, KHB containing
[3H]inulin was gently layered on the apical aspect of the
monolayers, and the inserts were placed in 24-well plates containing
KHB without added inulin. We determined that the volume of added KHB
exerted no hydrostatic pressure across the monolayer. All experiments were performed in a humidified 95% air-5% CO2 incubator
at 37°C. The amount of inulin [counts/min (cpm)] in the apical and
basal compartments was determined at the end of each 5-min period using a Packard scintillation counter. Epithelial permeability (P
in cm/s ×105) was calculated by the standard formula
P = F/(S ×
C) where F is the rate
of flux of inulin from apical to basal compartments per second,
C is
the inulin concentration gradient, and S is the surface area
of the Millipore insert.
Statistics. Data are presented as means ± SE. All statistical comparisons were done using ANOVA, followed by the Bonferroni correction.
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RESULTS |
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Effect of CN on cell ATP content. The ATP content of control BUMPT-306 cells was 27 ± 2 ng/mg cell protein. After treatment of cells with CN in the absence of dextrose for 5, 10, 15, and 45 min, cellular ATP content fell to 71 ± 4, 45 ± 1, 21 ± 1, and 5 ± 1% of control levels, respectively (all P < 0.01 vs. control levels). These values are comparable to those we obtained in response to CN treatment of primary cultures of mouse proximal tubular cells (30, 46) and of Madin-Darby canine kidney cells (46).
ATP depletion inceases c-Src kinase activity.
The activity of c-Src increased within 5 min of ATP depletion and
remained elevated for the duration of the 45-min period of ATP
depletion (n = 5) (Fig.
1). PP2 (10 µM), a specific inhibitor of Src kinase (43), completely inhibited the increase in
c-Src activity observed after 10 min of CN treatment (n = 6) (Fig. 1).
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Inhibition of c-Src kinase activity ameliorates the increase in
epithelial permeability associated with ATP depletion.
The permeability of epithelial monolayers was assessed by measuring the
flux of tritiated inulin across monolayers of cells grown on permeable
supports using methods previously described by our group (30,
40) (see MATERIALS AND METHODS for details). Cells
grown to confluence on permeable supports were incubated with dextrose
or CN in the presence and absence of 10 µM PP2. The basal
permeability of dextrose-treated monolayers was 8.3 ± 1.5 cm/s × 105 and remained constant for the duration
of the experiment (60 min; n = 5) (Fig.
2). The permeability of monolayers
treated with dextrose and PP2 (25 µM) was comparable to those treated
with dextrose alone (n = 5) (Fig. 2). In monolayers
treated with CN, permeability rose from the basal value of 8.3 ± 1.5 to 27.3 ± 3.1 cm/s × 10
5 within 10 min
and remained relatively constant at that value for the duration of the
experiment (P < 0.01 vs. dextrose, ANOVA for repeated
measures; n = 8) (Fig. 2). In the presence of CN and
PP2, permeability rose from 10.5 ± 1.7 to 14.5 ± 2.4 cm/s × 10
5 by 10 min and remained relatively
unchanged for 60 min (P < 0.02 vs. CN alone and
dextrose+PP2). Thus inhibition of c-Src kinase activity with PP2
ameliorated, but did not completely prevent, the CN-induced loss of
epithelial barrier function. We conclude that impaired barrier function
of BUMPT-306 cells, induced by CN-induced chemical anoxia, is partly
mediated by activation of a member of the c-Src family of kinases.
However, because PP2 completely inhibited activation of c-Src activity
(Fig. 1) without completely reducing epithelial permeability to control
levels (Fig. 2), we infer that factors in addition to Src activation must contribute to the loss of permeability associated with CN.
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Immunofluoresence studies of the effects of CN on ZA proteins,
actin, and c-Src.
Immunofluoresence techniques were used to examine the distribution of
E-cadherin, as well as - and
-catenin, in BUMPT-306 cells
subjected to chemical anoxia in the presence and absence of PP2. In
control, dextrose-treated cells, E-cadherin was localized predominantly
at cell-cell borders as expected for a normal epithelial monolayer.
CN-induced chemical anoxia caused a marked decrease in the amount of
E-cadherin at cell-cell junctions and an increase in the protein within
cytosolic aggregates (Fig. 3). PP2
largely prevented the disruption of normal E-cadherin localization
induced by CN (Fig. 3). Comparable results were obtained when the
effect of CN and CN+PP2 on the distribution of other ZA proteins was studied (
- and
-catenin; data not shown).
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Effects of CN on coimmunoprecipitation with ZA- and focal
adhesion-associated proteins with c-Src.
BUMPT-306 cells were treated with dextrose, dextrose+PP2, and CN for 5, 10, 15, and 45 min, and CN (for 10 min)+PP2. Lysates of each monolayer
were subjected to coimmunoprecipitation using a monoclonal antibody to
v-Src, and the immunoprecipitates were subjected to SDS-PAGE and then
probed by Western blotting for p120, -catenin, E-cadherin, or FAK.
ATP depletion increased the association of Src with all three ZA
proteins (p120,
-catenin, and E-cadherin) (Fig.
6). The increased association of Src with all the ZA proteins was prevented by pretreatment of cells with PP2
(Fig. 6). Similar results were obtained when p120,
-catenin, E-cadherin, and FAK were immunoprecipitated from lysates obtained from
dextrose-, CN-, and CN+PP2-treated cells and probed for Src (data not
shown). In contrast to the effects of CN on Src association with the ZA
proteins, CN had no effect on the degree of association of Src with FAK
(Fig. 6).
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Effect of CN on tyrosine phosphorylation of -catenin, p120,
E-cadherin, and FAK.
Finally, we examined the effect of ATP depletion on the degree of
tyrosine phosphorylation of p120,
-catenin, E-cadherin, and FAK. We
immunoprecipitated each of these proteins from lysates of monolayers
treated with dextrose, CN, dextrose+PP2, and CN+PP2 and immunoblotted
the precipitates with a tyrosine phospho-specific antibody (PY20). CN
treatment increased the degree of tyrosine phosphorylation of both p120
and
-catenin, an effect that was completely prevented by
pretreatment of cells with PP2 (Fig. 7). However, we found no tyrosine phosphorylation of immunoprecipitated E-cadherin under control conditions or after exposure of cells to CN
(Fig. 7). In contrast to the effects of CN on phosphorylation of
-catenin and p120, CN induced a fall in tyrosine phosphorylation of
FAK, an effect prevented by PP2 (Fig. 7).
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DISCUSSION |
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An important functional consequence of sublethal ischemic injury to renal tubular cells is loss of the normal barrier function of the tubular epithelium (4, 7, 10, 30, 35, 36). To elucidate the mechanisms responsible for this phenomenon, many investigators have focused their attention on the effects of ATP depletion on the ZO, which represents the physical barrier to paracellular ion flux (15, 20, 50). We have focused instead on the potential contribution of changes in the ZA to the loss of epithelial barrier function associated with ATP depletion (40). The presence of an intact ZA is a prerequisite to the functional integrity of the ZO. The importance of the ZA in tight junctional function has been demonstrated in two ways. Removal of extracellular calcium impairs ZO function (12, 23, 45). Because formation of the ZO itself does not require calcium, these data demonstrate that a structurally intact ZO cannot maintain the epithelial permeability barrier in the absence of a ZA. Also, direct inhibition of ZO assembly using neutralizing antibodies to E-cadherin has also been shown to impair tight junctional integrity and function (11, 21, 22).
We have previously demonstrated in a series of articles that CN-induced
injury to tubular cells increases the permeability of the tubular cell
monolayer and results in the disassociation of the ZA from its normal
location at cell-cell junctions, as evidenced by the withdrawal of
E-cadherin as well as -,
-, and
-catenin to the basolateral
membrane of tubular cells (30, 40, 44). Similar
alterations in the distribution of ZA proteins have been shown by other
investigators after ischemic injury to tubular cells (8,
31, 35). Our group has also reported that chemical anoxia is
associated with increased tyrosine phosphorylation of
-catenin and
that the loss of ZA integrity and of tight junctional function is due,
at least in part, to the changes in regulation of tyrosine
phosphorylation induced by ATP depletion (44). Our findings are consistent with the role of tyrosine phosphorylation of
-catenin as a regulator of cell-cell adhesion under physiological conditions (5, 6, 9, 13).
Some evidence suggests that alterations in ZA phosphorylation and cell-cell adhesion in nonrenal epithelial cells may be due to increased activity of a nonreceptor member of the Src kinase family (9, 48). The purpose of this study was to determine whether Src activation and phosphorylation of ZA proteins contribute to the loss of integrity of the ZA that we (44) and others (8, 31, 35) have previously observed in response to chemical anoxia.
We provide entirely novel evidence that chemical anoxia activates c-Src in tubular cells (Fig. 1). Our data showing that PP2 partly inhibits the loss of epithelial permeability and withdrawal of ZA proteins from cell-cell borders associated with chemical anoxia suggest a participatory role for Src activation in these events (24). Interestingly, PP2 has no effect on the disruption of actin stress fibers (Fig. 4), a well-documented effect of sublethal injury (25, 27, 30). The lack of efficacy of Src inhibition in ameliorating the disruption of actin stress fibers suggests that this alteration in the actin cytoskeleton is not an important event in the loss of integrity of the ZA associated with chemical anoxia. These data are consistent with the notion that the actin ring, a component of the actin cytoskleleton that is relatively preserved during chemical anoxia (Fig. 4), is more relevant to the stability of the ZA than actin stress fibers.
We also provide entirely novel evidence that chemical anoxia induces
translocation of c-Src to the ZA. Immunofluoresence studies show that
CN induces the appearance of cSrc at cell-cell borders, an effect
prevented by Src inhibition with PP2 (Fig. 5). In addition, using
immunopreciptation and immunoblotting we demontrate that CN induces an
increased association of c-Src with the ZA proteins -catenin, p120,
and E-cadherin (Fig. 6). However, CN does not change the degree of
association of c-Src with FAK, a protein localized in adhesion plaques
(Fig. 6).
We also show that CN treatment increases tyrosine phosphorylation of
p120 and -catenin (Fig. 7), both of which are known substrates of
Src (2, 13, 41). The tyrosine phosphorylation of p120 and
-catenin during ATP depletion is inhibited by PP2 (Fig. 7). These
findings are consistent with a role for c-Src in the tyrosine
phosphorylation of
-catenin and p120 during ATP depletion. However,
E-cadherin, unlike p120 and
-catenin, is not tyrosine phosphorylated
under control or CN treatment conditions (Fig. 7). Taken together, our
data suggest that Src binds to and phosphorylates both p120 and
-catenin during chemical anoxia. The apparent increase in the
association of E-cadherin with Src during CN treatment (shown by
immunoprecipitation and immunoblotting studies in Fig. 6) is probably
not due to the direct binding of Src to E-cadherin but rather to the
presence of E-cadherin bound to catenins in complexes of ZA proteins.
In previous studies, -catenin and p120 have both been shown to be
readily phosphorylated by protein tyrosine kinases (2, 13,
41). It is also well established that alterations in the degree
of tyrosine phosphorylation of
-catenin, which binds to the CBD of
the cytoplasmic tail of cadherins (5, 13), is an important
event in modulating cell-cell adhesion. The process of ZA
formation and cell-cell adhesion requires tyrosine dephosphorylation of
-catenin (5), whereas loss of cell-cell adhesion is
associated with an increase in the tyrosine phosphorylation of
-catenin (13). Thus it is likely that c-Src-induced
phosphorylation of
-catenin observed in this study contributes to
the loss of tubular cell-cell adhesion associated with sublethal injury
induced by chemical anoxia.
While there is also evidence to support a role for p120 in modulating
cell-cell adhesion, the mechanisms involved are less well defined than
for -catenin (2). There is evidence that serine/threonine phosphorylation as well as tyrosine phosphorylation of
p120 plays a role in regulating p120 function and cell-cell adhesion
(3, 39). It is well established that Src is able to
tyrosine phosphorylate p120 in vitro and in vivo (41).
Also, growth factors, such as EGF and hepatocyte growth factor (scatter factor), whose receptors have intrinsic tyrosine-specific protein kinase activity, increase the level of tyrosine phosphorylation of p120
as well as of
-catenin (17). However, it has not as yet
been possible to distinguish the functional consequences of tyrosine
phosphorylation of p120 from the effects of phosphorylation of the many
other targets of activated Src, including
-catenin (2).
Given the present uncertainty of the effects of tyrosine phosphorylation of p120 on cell adhesion, we cannot as yet speculate on
the significance of our observation that p120 is tyrosine
phosphorylated by c-Src during chemical anoxia (Fig. 7).
Interestingly, we show that CN decreases tyrosine phosphorylation of adhesion plaque protein FAK, an effect that is the opposite of what happens to phosphorylation of the catenins (Fig. 7). Other investigators have also demonstrated that FAK becomes tyrosine dephosphorylated during ATP depletion (52). While hyperphosphorylation of the catenins is known to be associated with loss of cell-cell adhesion (5, 6, 9, 11, 13, 32), dephosphorylation of FAK causes loss of cell-matrix adhesion (14, 26). It is intriguing that while ATP depletion has opposite effects on the degree of tyrosine phosphorylation of ZA and adhesion plaque proteins, these effects are consistent with the loss of both cell-cell and cell-matrix adhesion, well-known functional effects of sublethal injury (10, 30).
In summary, we have shown for the first time that CN-induced chemical
anoxia activates c-Src and leads to the translocation of the activated
form of c-Src to the ZA. There, c-Src binds to and phosphorylates
-catenin and p120. We also show that these effects of c-Src
contribute to the structural and functional loss of the ZA and to the
increase in epithelial permeability associated with sublethal injury.
Further studies are necessary to define in more detail the effects of
Src-induced phosphorylation that occur in response to chemical anoxia.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-385101, DK-59793, and DK-58306.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. Lieberthal, Renal Section, Evans Biomedical Research Ctr., Rm. 537, 650 Albany St., Boston, MA 02118 (E-mail: wliebert{at}bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 5, 2002;10.1152/ajprenal.00172.2002
Received 1 May 2002; accepted in final form 23 October 2002.
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