* Cutaneous Biology Research Center, Harvard Medical School and Massachusetts General Hospital, Charlestown,
Massachusetts 02129; Department of Biology, Genetics and Medical Chemistry, University of Torino, Torino, Italy 10126; § Wistar Institute, Philadelphia, Pennsylvania 19104;
Molecular Biology Group, Medical Faculty of the University of Halle,
Halle, Germany 06097; and ¶ Division of Rheumatology and Immunology, Brigham and Woman's Hospital, Boston,
Massachusetts 02115
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Abstract |
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In their progression from the basal to upper
differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important
but so far unexplored question is how these early structural events are related to the biochemical pathways
that trigger differentiation. We show here that -catenin,
-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a
time course similar to that of cell junction formation. Together with these changes, there is an increased association of
-catenin and p120-Cas with E-cadherin,
which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific
phosphatase reveals that the strength of
-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine
kinase inhibition suppresses formation of cell adhesive
structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn
tyrosine kinase colocalizes with E-cadherin at the cell
membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of
- and
-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion
similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn
/
mice appears normal, skin of
mice with a disruption in both the fyn and src genes
shows intrinsically reduced tyrosine phosphorylation of
-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically
stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control
of cell adhesion, and this regulatory function appears to
be important both in vitro and in vivo.
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Introduction |
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AS keratinocytes progress from the basal proliferating layer of the epidermis to the immediately adjacent differentiating layer (spinous layer) they lose contact with the extracellular matrix and undergo significant structural changes, such as establishment of close intercellular contacts, desmosome formation and rearrangements of the actin/cytokeratin network. An important but so far unexplored question is how these early structural events are related to the biochemical pathways which trigger differentiation.
Cell-cell contacts among neighboring keratinocytes are
mediated mainly by adherens junctions and desmosomes.
Adherens junctions contain "classical" cadherins, whereas
desmosomes are composed of specialized cadherins, such
as desmoglein(s) and desmocollin(s). Each type of junction is linked via several cytoplasmic proteins to different
elements of the cytoskeleton. Adherens junctions are associated with the actin cytoskeleton and have been shown to
be important for the establishment of cell adhesion and
polarization, while desmosomes interact with keratin filaments and impart mechanical strength to the epithelium
(Cowin and Burke, 1996).
Adherens junctions in epithelial cells depend on the homophilic, calcium-dependent binding of the extracellular
domain of transmembrane cadherins (Takeichi, 1988, 1991
).
The cytoplasmic domain of E-cadherin forms a complex
with
-,
-, and
-catenins, and this association is essential
for the establishment of proper cell adhesion (Nagafuchi
and Takeichi, 1988
; Ozawa et al., 1989
; Nagafuchi et al.,
1991
).
- and
-catenins share a similar structure (65%
identity) (Fouquet et al., 1992
), including a 42-amino acid motif repeated 12 or 13 times, originally described in the
Drosophila segment polarity gene product Armadillo
(Riggleman et al., 1989
).
- and
-catenins bind directly to
E-cadherin in a mutually exclusive fashion (Mathur et al.,
1994
; Stappert and Kemler, 1994
). They also form a complex with
-catenin, a cytoplasmic protein similar to vinculin, which in turn is connected, either directly or indirectly, to the actin network (Knudsen et al., 1995
; Rimm et al.,
1995
). Besides adherens junctions,
-catenin/plakoglobin
is also found in desmosomes, in association with desmosomal cadherins and is thought to modulate their function
(Cowin et al., 1986
; Troyanovsky et al., 1993
). Another
catenin, p120-Cas, originally described as a putative substrate of the activated Src tyrosine kinase (Kanner et al.,
1990
, 1991
; Reynolds et al., 1992
), has been shown to complex directly with E-cadherin (Reynolds et al., 1994
, 1996
; Shibamoto et al., 1995
). In normal cells only a small proportion of p120-Cas is associated with cadherins (Shibamoto et al., 1995
), but in specific circumstances, such as
upon ras transformation, this catenin displays increased affinity for E-cadherin (Kinch et al., 1995
). Interestingly,
p120-Cas lacks the ability to bind
-catenin (Daniel and
Reynolds, 1995
), suggesting that p120-Cas/cadherin complexes are disconnected from the actin cytoskeleton and
thus may account, at least partially, for the poor adhesive
phenotype of Ras-transformed cells.
Elevation of Ca2+ concentrations in epithelial cell cultures induces the rapid translocation of cadherins to cell-
cell borders and consequent adherens junction and desmosome formation (O'Keefe et al., 1987; Lewis et al., 1994
).
These changes are accompanied by a reorganization of the
cytoskeleton, polarization and, in keratinocyte cultures,
stratification. Establishment of adherens junctions has
been shown to play a primary role in these processes. In
particular, inhibition of adherens junction formation by
anti-E-cadherin antibodies (Hodivala and Watt, 1994
;
Lewis et al., 1994
) or by expression of dominant negative
cadherin mutants (Amagai et al., 1995
), suppresses all later
steps, such as desmosome formation, polarization, and
stratification. Whereas calcium can trigger initial formation of cadherin-mediated cell adhesion, a second, temperature-dependent step is required for the strengthening of these interactions (Angres et al., 1996
). In addition, for
stratification to occur, cell contacts need to remain fluid,
as movement of cells within a tri-dimensional structure requires continual disruption and reorganization of intercellular junctions, without loss of adhesive strength (Lewis
et al., 1994
; Tao et al., 1996
). The mechanisms that control
the strength of cell adhesion and modulate its dynamic
state are still poorly understood.
Increased tyrosine phosphorylation of - and
-catenin
(plakoglobin) and p120-Cas has been previously correlated with the decrease of cell adhesion which occurs upon
neoplastic transformation (Matsuyoshi et al., 1992
; Behrens et al., 1993
; Hamaguchi et al., 1993
; Kinch et al., 1995
;
Papkoff, 1997
) or mitogenic growth factor stimulation
(Kanner et al., 1991
; Hoschuetzky et al., 1994
; Reynolds et al.,
1994
; Shibamoto et al., 1994
; for review see Miller and Moon,
1996
). However, in no cases was catenin tyrosine phosphorylation shown to be causally linked to decreased cell adhesion. In fact, additional evidence indicates that loosening of
cell contacts in src-transformed cells cannot be explained by
tyrosine phosphorylation of
-catenin, but is likely resulting from tyrosine phosphorylation of other junctional proteins such as ZO-1, ezrin/radixin/moesin, or some other unidentified proteins (Takeda et al., 1995
). The small Rho/
Rac GTPases have also been implicated in control of cell
adhesion. Interestingly, these molecules appear to play a negative suppressive function in MDCK cells (Ridley et
al., 1995
), but a positive one in keratinocytes (Braga et al.,
1997
), suggesting that control of cell adhesion may differ
significantly among epithelial cells of different types.
Unlike what may occur in oncogene-transformed or mitogenically stimulated cells, we report here that in differentiating keratinocytes, tyrosine phosphorylation plays a
positive role in the strengthening of cell adhesion. At least
two distinct tyrosine kinase activities are induced in the keratinocyte differentiation process (Calautti et al., 1995). One
of these activities is specifically increased by calcium and a
number of other divalent cations from the outside of the
cell, suggesting that an extracellular cation-sensor mechanism is involved. Induction of this kinase activity occurs
within minutes of calcium exposure (Calautti et al., 1995
),
and correlates with the rapid tyrosine phosphorylation of a
ras-GAP associated p62 protein (Filvaroff et al., 1992
, 1994
),
which may be similar, but probably not identical (Medema
et al., 1995
), to the p62-Dok adaptor protein (Carpino et al.,
1997
; Yamanashi and Baltimore, 1997
). The second tyrosine
kinase activity, identified as Fyn, increases only at relatively late times of exposure of keratinocytes to calcium
(within 1-6 h) (Calautti et al., 1995
). In close parallel with
Fyn kinase activation, calcium treatment triggers tyrosine phosphorylation of cortactin, a protein which colocalizes
with subcortical actin at sites of cell movement. A significant role of Fyn in keratinocyte differentiation is indicated
by the fact that this process is altered in keratinocytes with
a disruption of the fyn gene (Calautti et al., 1995
).
Immunoblotting of keratinocyte cell extracts with anti-phosphotyrosine antibodies indicates that p62 and cortactin are only two of a wider group of proteins which are tyrosine phosphorylated in calcium-induced keratinocyte
differentiation (Filvaroff et al., 1990; Calautti et al., 1995
;
our unpublished observations). We show here that
-,
-catenin (plakoglobin), and p120-Cas become all strongly
tyrosine phosphorylated at early times of calcium-induced keratinocyte differentiation, and that these modifications
are linked to an increased association of
-catenin and
p120-Cas with E-cadherin. Both biochemical and genetic
evidence indicates that tyrosine phosphorylation plays a
fundamental role in the changes in cell adhesion associated
with keratinocyte differentiation, and that the activity of
Fyn and related kinases is involved, both in vitro and in vivo.
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Materials and Methods |
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Cells and Animals
Primary keratinocytes were isolated from newborn Sencar mice or mice
with a disruption of the fyn (Stein et al., 1992), src (Soriano et al., 1991
),
and yes (Stein et al., 1994
) genes, and genetically matched wild-type controls. The yes mutation results in the production of a small amount of a
catalytically inactive fragment of the Yes kinase (Calautti et al., 1995
).
Cells were cultivated in MEM with 4% Chelex-treated fetal calf serum,
EGF (10 ng/ml; Collaborative Research, Inc., Cambridge, MA), and 0.05 mM CaCl2 (low calcium medium), as described (Hennings et al., 1980
; Calautti et al., 1995
). For all experiments, cells were used one week after
plating. Culture medium was changed for the last time 24 h before the experiment. Keratinocyte differentiation was induced by addition of CaCl2
(to 2 mM). Genistein and Tyrphostin 23 were purchased from BIOMOL
(Plymouth Meeting, PA), and then stored in 100-mM aliquots in DMSO
at
20°C. PP1 was a gift of Dr. S. Fuji (Tokyo University, Tokyo, Japan)
and was stored in DMSO (10 mg/ml) at
20°C.
C57B/129 mice carrying single fyn, src, or yes knockout mutations
(Soriano et al., 1991; Stein et al., 1992
, 1994
) were bred to obtain double
heterozygotes for the fyn/src or fyn/yes mutations. Double heterozygotes
were then bred, and among the offspring, the mice carrying a fyn
/
src+/
or yes
/
fyn+/
genotype were intercrossed to obtain src
/
fyn
/
or fyn
/
yes
/
double knockout mice, respectively.
Recombinant Adenovirus Infection of Mouse Primary Keratinocytes
Recombinant adenoviruses expressing either a constitutively active c-Src mutant (Tyrosine 527 to phenylalanine substitution) (Ad-src), or a green fluorescent protein (Ad-GFP) were used at a multiplicity of infection of 100. Detailed description of the generation of Ad-Src will be presented elsewhere. In all cases, infection of primary mouse keratinocytes was performed for 1 h in serum- and EGF-free low calcium medium. Keratinocytes were further incubated for 24 h in low calcium medium, and then either kept in this medium or switched to high calcium medium (2 mM CaCl2) for the indicated times.
Antibodies
Monoclonal antibodies against E-cadherin, -catenin,
-catenin, and
pp120-Cas were purchased from Transduction Laboratories (Lexington,
KY). Anti-
-catenin/plakoglobin mAbs (ascites fluid) were a gift of Dr. P. Cowin (New York University, New York, NY). Anti-desmoglein 3 antibodies were a gift of Dr. J. Stanley (University of Pennsylvania, Philadelphia, PA). Anti-plakophilin 1 rabbit antisera were raised against either
the head or the Arm repeats domain of plakophilin, expressed in Escherichia coli as His-tag fusion proteins. Anti-Fyn and anti-Src polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
HRP-conjugated anti-phosphotyrosine antibodies (RC20H) were purchased from Transduction Laboratories. Anti-phosphotyrosine mAbs
4G10 were purchased from Upstate Biotechnology Inc. (Lake Placid,
NY). Either affinity-purified anti-E1A mAbs (Oncogene Science Inc.,
Cambridge MA) or affinity-purified rabbit IgG were used as unrelated
negative controls.
Immunoprecipitations
Mouse primary keratinocytes were washed twice in PBS containing 1 mM Na3VO4 and lysed for 20 min on ice, either in NP-40 lysis buffer for high stringency immunoprecipitations (0.5% NP-40, 50 mM TRIS-HCl, pH 8.0, 120 mM NaCl) or in CSK lysis buffer for low stringency immunoprecipitations (0.2% Triton X-100, 10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA), supplemented with 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF, 10 µg/ml aprotinin (Sigma Chemical Co., St. Louis, MO), 10 µg/ml leupeptin (Sigma Chemical Co.). Cell lysates were spun for 5 min at 4°C. Samples were normalized for equal amounts of proteins by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Same amounts (0.5-1 mg) were incubated either for 2 h or overnight at 4°C with antibodies (2-4 µg of affinity-purified mAbs or 5 µl of crude ascites fluid). Protein G-agarose beads (60 µl of a 50% suspension; Boehringer Mannheim Corp., Indianapolis, IN) were added and samples were incubated for 45 min at 4°C. Immune complexes were washed five times for high stringency immunoprecipitations and three times for low stringency immunoprecipitations. After the last wash, immune complexes were eluted in boiling Laemmli sample buffer for 3 min, and separated by 7.5% acrylamide/ 0.173% bis-acrylamide SDS-PAGE.
Immunoprecipitations from skin samples were performed as follows: whole skins from newborn mice were snap-frozen in liquid nitrogen and pulverized with a tissue grinder. The pulverized tissues were solubilized with a Polytron homogenizer in NP-40 lysis buffer supplemented with protease and phosphatase inhibitors. Tissue debris was removed by centrifugation for 5 min at 3,000 g. The supernatants were further spun for 45 min at 25,000 g and incubated sequentially with protein G-agarose and protein A-Sepharose beads for 45 min as a preclearing step. Tissue extracts were normalized for total protein content and for E-cadherin protein by preliminary SDS-PAGE and immunoblotting with anti-E-cadherin antibodies. Normalized extracts (2-5 mg of protein) were immunoprecipitated with relevant antibodies and immune complexes were processed as described above for high stringency immunoprecipitations.
Protein-Tyrosine-Phosphatase Treatment of Cadherin-Catenin Complexes
Keratinocytes were lysed in NP-40 lysis buffer supplemented with inhibitors of proteases and phosphatases as described above. E-cadherin and associated proteins were immunoprecipitated with E-cadherin-specific antibodies. The immune complexes were washed twice in NP-40 lysis buffer without phosphatase inhibitors, and twice in phosphatase buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl). The immune complexes were than resuspended in 50 µl of phosphatase buffer supplemented with 1 mM DTT and 1 mg/ml BSA, and incubated for 30 min at 37°C with 25 mU of Yersinia enterocolitica protein tyrosine phosphatase (Boehringer Mannheim Corp.), either in the presence or the absence of tyrosine phosphatase inhibitors (2 mM Na3VO4, 2 mM ZnCl2). The reaction was stopped by the addition of 1 ml NP-40 lysis buffer supplemented with 2 mM Na3VO4, 2 mM ZnCl2. After one wash in this buffer, the complexes were washed three times with 4 M LiCl, 1 mM Na3VO4, and then twice in PBS plus 1 mM Na3VO4. Samples were analyzed by SDS-PAGE and immunoblotting.
Immunoblotting
Immunoblotting of protein gels onto PVDF membranes was performed as
previously described (Calautti et al., 1995). All blots were blocked for 1 h
at room temperature in 5% milk in PBS 0.2% Tween 20, except for anti-phosphotyrosine immunoblots, which were blocked for 1 h in 1% BSA,
10 mM TRIS, pH 7.5, 100 mM NaCl, 0.1% Tween 20. RC20-horseradish-
conjugated anti-phosphotyrosine antibodies were used for immunoblotting at a 1:2,500 dilution. HRP-conjugated anti-mouse or anti-rabbit immunoglobulins (Amersham Corp., Arlington Heights, IL) were used as
secondary antibodies for all other immunoblots at a 1:2,000-1:5,000 dilution, and blots were developed with the ECL system (Amersham Corp.).
Immunofluorescent Staining of Primary Keratinocytes
Mouse primary keratinocytes were plated on collagen-coated glass coverslips, and cells in low calcium medium or at different times after calcium treatment were fixed for 10 min at room temperature with 2% paraformaldehyde in PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS, blocked in 5% goat serum in PBS. The coverslips were stained with primary antibodies for 1 h at room temperature, followed by isotype-specific FITC- or Texas red-conjugated secondary antibodies (Southern Biotechnology Associates Inc., Birmingham, AL) For the analysis of the detergent-insoluble subcellular localization, keratinocytes were pre-extracted for 5 min on ice in 0.2% Triton X-100-CSK buffer supplemented with inhibitors of proteases and phosphatases before fixation in 2% paraformaldehyde in PBS. Conventional fluorescent microscopy was performed with a Nikon FXA microscope (Melville, NY). Confocal microscopy was performed using a TCS 4D scanner (Leica, Heerbrugg, Switzerland) connected to an inverted LEITZ DM IRB microscope (Oberkonen, Germany). Images were processed using a TCS-NT software package.
Dispase Treatment of Mouse Primary Keratinocytes
Duplicate 60-mm dishes of confluent keratinocyte cultures in either low or high calcium medium were washed twice in PBS and incubated in 2 ml dispase solution in PBS (from Bacillus polymyxa; >2.4 units/ml; Boehringer Mannheim Corp.) at 34°C. Cells were analyzed with an inverted transmission microscope at 5-min intervals, for >35 min. Quantification of this assay was performed as follows: after 35 min of dispase treatment, cells from each sample, including single cells released into suspension and detached sheets of cells, were collected by scraping, and then washed twice in PBS. After centrifugation, samples were resuspended in 500 µl of PBS and subjected to mechanical disruption by pipetting 15 times with a 1 ml Pipetman. The remaining aggregates were left to sediment at 1 g for 1 min, and the number of single cells into suspension was determined by counting 10 µl of supernatant with an hemocytometer. The whole samples were then centrifuged, and incubated for 7 min at 37°C in 0.25% trypsin, and 2.5 mM EDTA, to release all cells into suspension. Cells were counted as before and used for assay normalization.
Electron Microscopy
Keratinocytes grown on collagen-coated plastic coverslips were fixed in 1% glutaraldehyde; skin samples were fixed in 1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% picric acid in 50 mM cacodylic acid buffer, pH 7.4. All specimens were postfixed in 1% osmium tetroxide/1.5% potassium ferrocyanide, dehydrated, infiltrated, and then embedded in Epon/ Araldite. Thin sections were cut, stained with uranyl acetate and lead citrate, and then examined with a transmission electron microscope (1200 EX; JEOL USA Inc., Peabody, MA).
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Results |
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Exposure of Keratinocytes to High Extracellular
Calcium Induces Tyrosine Phosphorylation of - and
-Catenin, and Increased Association of
-Catenin with
E-Cadherin Complexes, with a Time Course That
Parallels Cell Junction Formation
As mentioned in the introduction, very little is known about
control of cell adhesion in differentiating epithelia. We
tested whether there is a specific interconnection between
induction of tyrosine phosphorylation and the changes in
cell adhesion that occur during calcium-induced keratinocyte
differentiation. In an initial set of studies, we examined the
cellular localization pattern of total tyrosine phosphorylated
proteins in growing versus differentiating keratinocytes. In
parallel with our previous biochemical data, immunofluorescence analysis with anti-phosphotyrosine antibodies revealed a substantial increase of tyrosine phosphorylation
signal in the differentiating cells, as early as 2 h after calcium exposure. More significantly, calcium treatment resulted in a striking localization of the tyrosine phosphorylation signal at sites of cell-cell contact (not shown). To
test whether -catenin, an essential adherens junction
component, shows an increased localization at tyrosine phosphorylation sites, we focused on the detergent-insoluble submembranous cytoskeleton. Keratinocytes under
growing versus differentiating conditions were pre-extracted
with a 0.2% Triton X-100 buffer before fixation in 2%
paraformaldehyde. Cells were then double stained with
anti-phosphotyrosine and anti-
-catenin antibodies, and
analyzed by confocal microscopy. As shown in Fig. 1 (top
panels), calcium treatment (9 h) resulted in a significant increase of tyrosine-phosphorylated proteins as well as
-catenin associated with the detergent-resistant, membrane-cytoskeleton compartment. The pattern of staining
of anti-phosphotyrosine and anti-
-catenin antibodies was
almost totally overlapping in the calcium-treated cells.
|
To determine whether tyrosine phosphorylation levels
of specific adherens junction components are directly increased with differentiation, extracts from keratinocytes
under basal growing conditions and at various times after
calcium exposure were immunoprecipitated with anti-
E-cadherin antibodies, followed by immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 2 A (left
panel), two tyrosine phosphorylated bands of ~85 and 92 kD
were detected in the anti-E-cadherin immunoprecipitates
of keratinocytes under basal low calcium conditions. Tyrosine phosphorylation of these bands was strongly increased by 9 h of calcium treatment and persisted at elevated levels for 24 h. These bands comigrate and are likely to correspond to - and
-catenin, as judged by reprobing
of the same immunoblot with antibodies specific against
these proteins (Fig. 2 A, right panels). Immunoprecipitation
of keratinocyte cell extracts with antibodies against
- and
-catenins followed by anti-phosphotyrosine immunoblotting, confirmed that tyrosine phosphorylation of
- and
-catenins is directly increased in the differentiating keratinocytes (Fig. 2 B).
|
No direct tyrosine phosphorylation of E-cadherin itself
nor of -catenin could be detected in the anti-E-cadherin
immunoprecipitates. However, even if not directly tyrosine
phosphorylated, the amount of
-catenin found in association with E-cadherin was significantly increased by 9 h of
calcium treatment, whereas the amounts of
- and
-catenins
associated with E-cadherin remained constant (Fig. 2 A,
right panels). Immunoblotting of total keratinocyte cell extracts indicated that the total amounts of
-catenin, as well
as
- and
-catenins and E-cadherin, remained constant up to 48 h of calcium treatment (data not shown).
Besides adherens junctions, -catenin/plakoglobin is
also found in desmosomes, and is thought to modulate
their function (Cowin et al., 1986
; Troyanovsky et al.,
1993
).
-Catenin associated with desmosomal cadherins
may also be increasingly tyrosine phosphorylated in response to calcium. To test this possibility, keratinocyte cell
extracts were immunoprecipitated with anti-desmoglein 3 antibodies, followed by immunoblotting with anti-phosphotyrosine antibodies. A single specific band of ~85 kD
was detected, which became strongly tyrosine phosphorylated by 9 h of calcium treatment (Fig. 2 C). This band is
likely to correspond to
-catenin, as judged by reprobing of the same immunoblot with antibodies against this protein. As in E-cadherin complexes, the amount of desmoglein-associated
-catenin did not appear to vary significantly over time (Fig. 2 C). Tyrosine phosphorylation of
plakophilin 1, another key desmosomal protein that shares
structural homology with
- and
-catenins (Heid et al.,
1994
), was analyzed by direct immunoprecipitation with
anti-plakophilin 1 antibodies followed by anti-phosphotyrosine immunoblotting. Plakophilin 1 was found to be weakly
tyrosine phosphorylated in keratinocytes under low calcium
conditions, and tyrosine phosphorylation of this protein (at
least that recoverable from the detergent-soluble fraction)
did not increase with differentiation (data not shown).
Previous reports showed that elevation of Ca2+ concentrations in epithelial cell cultures induces the rapid translocation of cadherins to cell-cell borders and consequent cell
junction formation (O'Keefe et al., 1987; Lewis et al., 1994
).
The precise time course of these events is likely to vary as
a function of cells and culture conditions. It was important
to determine whether the timing of adherens junction and
desmosome formation in our primary mouse keratinocyte
cultures parallels that of increased tyrosine phosphorylation. Accordingly, cells were analyzed at various times after calcium treatment by immunofluorescence with antibodies against either E-cadherin or plakophilin. Extraction
with 0.2% Triton X-100 buffer before fixation ensured detection of these proteins only when assembled into insoluble cell membrane structures. As shown in Fig. 3, by 2 h of
calcium treatment E-cadherin and plakophilin showed only
an incomplete recruitment into membrane-cytoskeleton structures, whereas by 9 h a very strong signal for these proteins was detected at sites of close intercellular junctions.
|
Thus, in mouse primary keratinocytes induced to differentiate by calcium, there is an increased tyrosine phosphorylation of proteins at sites of cell-cell adhesion, and this is
associated with a specific increase in tyrosine phosphorylation levels of - and
-catenins. In contrast,
-catenin is
not directly tyrosine phosphorylated, but its association
with E-cadherin increases, with a time course that parallels
that of
- and
-catenin tyrosine phosphorylation, as well
as that of cell junction formation.
p120-cas Becomes Heavily Tyrosine Phosphorylated in Keratinocytes in Response to Calcium, and Displays Increased Association with E-Cadherin
p120-Cas, another catenin originally described as a substrate of activated Src (Kanner et al., 1990, 1991
; Reynolds
et al., 1992
), can also form a direct complex with E-cadherin
at adherens junctions (Reynolds et al., 1994
; Shibamoto et
al., 1995
). However, in normal cells, only a small fraction
of p120-Cas is associated with cadherins, and this fraction
varies with various cell types (Kinch et al., 1995
; Shibamoto et al., 1995
). To determine whether in differentiating
keratinocytes p120-Cas shows an increased localization at
tyrosine phosphorylation sites in the submembranous cytoskeleton, we performed the same type of immunofluorescence analysis described above for
-catenin: cells either
kept in low calcium medium or switched to high calcium for
9 h were pre-extracted with 0.2% Triton X-100 buffer before
fixation, and were double stained with antibodies against
phosphotyrosine and p120-Cas. Cells were then analyzed by
confocal microscopy. Like
-catenin, in calcium-treated keratinocytes p120-Cas was also recruited into the detergent-insoluble cortical cytoskeleton, and it colocalized with
phosphotyrosine at cell-cell borders (Fig. 1, bottom panels).
To assess whether tyrosine phosphorylation of p120-Cas
is intrinsically increased with differentiation, extracts from
keratinocytes under low versus high calcium conditions
were immunoprecipitated with an mAb that recognizes all
known isoforms of the p120-Cas proteins (Mo and Reynolds, 1996), and the immunocomplexes were analyzed by
anti-phosphotyrosine immunoblotting. As shown in Fig. 2 D,
there was a progressive increase of tyrosine phosphorylation of all four p120-Cas isoforms, which was detectable already by 10 min of calcium exposure, and then became
very pronounced by 9 h. Tyrosine phosphorylation of
p120-Cas remained elevated at least until 24 h. Reprobing
of the same blot with anti-p120 antibodies showed that expression of the two larger isoforms of p120-Cas was only
slightly increased by 24 h of calcium treatment, whereas levels of the smaller forms did not vary significantly over time.
As reported for other cells (Kinch et al., 1995), increased tyrosine phosphorylation of p120-Cas may correlate with a higher affinity of these proteins for cadherin-
catenin complexes. No p120-Cas/E-cadherin association
could be detected in E-cadherin immunoprecipitates performed under standard stringency conditions (not shown).
However, p120/E-cadherin complexes may have been disrupted by the detergent concentrations used for those experiments. We reexamined this question by performing
anti-E-cadherin immunoprecipitations under milder stringency conditions (lysis in 0.2% Triton X-100 buffer). Immunoprecipitates from keratinocytes in low calcium medium versus cells treated with calcium for 9 h, were
separated by SDS-PAGE, blotted, and then sequentially
probed with antibodies against phosphotyrosine, p120-Cas,
- and
-catenins, and E-cadherin (Fig. 2 E). Under
these conditions, association of all p120-Cas isoforms with
E-cadherin was easily detected, and this association was
strongly increased in the calcium-treated cells, whereas
that of
- and
-catenins remained mostly unaffected. At
least four major tyrosine-phosphorylated bands were detectable in the E-cadherin immunoprecipitate from keratinocytes under low calcium conditions, and tyrosine phosphorylation of these bands was strongly increased upon
calcium treatment (Fig. 2 E). These bands comigrate and
are likely to correspond to the p120-Cas and
- and
-catenins discussed above. In addition, a fifth tyrosine
phosphorylated protein of heavier molecular weight was
detectable in the anti-phosphotyrosine immunoblot, which
appeared only in the calcium-treated sample and whose
identity remains to be assessed.
Thus, calcium treatment of mouse primary keratinocytes induces strong tyrosine phosphorylation of p120-Cas proteins, and this increased phosphorylation correlates with an increased association of all four p120-Cas isoforms with E-cadherin.
Tyrosine Phosphorylation Is Required for the
Increased Association of -Catenin and p120-Cas with
E-Cadherin in Differentiating Keratinocytes
The results of the previous sections showed that exposure
of keratinocytes to high extracellular calcium concentrations has two important consequences: it induces tyrosine
phosphorylation of - and
-catenins and p120-Cas proteins, and at the same time, triggers an increased association of
-catenin and p120-Cas with E-cadherin. An important question is whether the two events are connected.
The isoflavone Genistein inhibits specific tyrosine phosphorylation events associated with calcium-induced keratinocyte differentiation (Filvaroff et al., 1990). Tyrphostins
represent a class of structurally unrelated tyrosine kinase
inhibitors which block EGF-dependent mitogenicity and
revert the transformed phenotype of v-src-transformed cells (Levitzki, 1992
). We initially tested whether treatment of keratinocytes with either Genistein or Tyrphostin
23 could block induction of tyrosine phosphorylation of
-
and
-catenins and p120-Cas in response to calcium, and
whether, in turn, association of
-catenin and p120-Cas
with E-cadherin was affected. Primary keratinocytes were
pretreated for 2 h with Genistein or Tyrphostin 23, or
DMSO solvent alone, and then cells were incubated for
additional 9 h under either low or high calcium conditions.
Cell lysates in 0.5% NP-40 lysis buffer were immunoprecipitated with anti-E-cadherin antibodies and immunoblotted, sequentially, with antibodies against phosphotyrosine,
-,
-, and
-catenins, and E-cadherin. As shown in
Fig. 4 A, Genistein treatment had only limited effects on
basal tyrosine phosphorylation levels of E-cadherin-associated
- and
-catenins, while it blocked almost completely tyrosine phosphorylation of these molecules in
response to calcium. Tyrphostin treatment had more pronounced inhibitory effects on tyrosine phosphorylation of
- and
-catenins, both under basal conditions and after
calcium exposure. In parallel with these effects, both tyrosine kinase inhibitors blocked the increased association
of
-catenin with E-cadherin, which normally occurs in
calcium-treated cells (see Fig. 4 A, top right panel). This effect was specific for the calcium-induced association, as
neither inhibitor affected the basal level of
-catenin associated with E-cadherin in cells under low calcium conditions.
|
To test whether tyrosine phosphorylation is also required
for the increased association of p120-Cas with E-cadherin,
keratinocytes in low versus high calcium conditions ± tyrosine kinase inhibitors, were lysed under mild detergent
conditions (0.2% Triton X-100). Extracts were immunoprecipitated with anti-E-cadherin antibodies and immunoblotted, sequentially, with antibodies against phosphotyrosine, p120-Cas, - and
-catenins, and E-cadherin. As
shown in Fig. 4 B, both inhibitors reduced the basal tyrosine phosphorylation levels of p120-Cas, and blocked
any further increase in response to calcium. Genistein
treatment had little or no effect on the amount of p120-Cas associated with E-cadherin in cells under basal conditions, while it totally prevented the increased association induced by calcium (Fig. 4 B, top right panel). Tyrphostin
treatment caused a reduction of p120-Cas associated with
E-cadherin already in keratinocytes in low calcium medium, and prevented any further increase in cells exposed
to calcium.
We have previously shown that tyrosine kinase inhibitors disrupt several aspects of keratinocyte differentiation
(Filvaroff et al., 1990), and it is still possible that their suppressive effects on
-catenin and p120-Cas association
with E-cadherin complexes are not directly resulting from
inhibition of tyrosine phosphorylation. To test whether tyrosine phosphorylation of E-cadherin complexes can directly affect
-catenin association, keratinocyte extracts
were immunoprecipitated with anti-E-cadherin antibodies, and the immunoprecipitates were treated with a tyrosine-specific phosphatase ± tyrosine phosphatase inhibitors. Preliminary experiments, which included untreated
controls, showed that dephosphorylation of
- and
-catenins
by the Yersinia tyrosine phosphatase was totally blocked
by the inhibitors at the chosen concentrations (not shown).
Immunocomplexes were washed under higher stringency conditions than in the previous experiments using LiCl,
which has been shown to affect charge-based interactions
of stable protein-protein complexes (El-Baradi et al., 1984
).
Samples were then analyzed by SDS-PAGE and sequential
immunoblotting with antibodies against phosphotyrosine,
-catenin,
- and
-catenins, and E-cadherin. Tyrosine
phosphorylation of
- and
-catenins was totally abrogated by phosphatase treatment in the absence of the inhibitor, whereas it remained unaffected in its presence
(Fig. 5).
-Catenin was readily detected in the complexes
treated with phosphatase plus inhibitor, and higher levels
of this catenin were found in the complexes derived from
calcium-treated versus untreated keratinocytes. By contrast, under the same washing conditions, no
-catenin was left in the complexes treated with phosphatase without inhibitor (Fig. 5 A). Levels of
- and
-catenins were also
slightly reduced in these samples, suggesting that tyrosine
phosphorylation may also stabilize association of these
proteins with E-cadherin, even if to a lesser extent than
-catenin (Fig. 5 A). To eliminate this variable, a second
independent experiment was performed, where the E-cadherin immunoprecipitated samples were normalized for
amounts of
- and
-catenin levels before immunoblot
analysis. Even under these conditions, the amount of
-catenin associated with the E-cadherin complexes was found
to be markedly reduced after tyrosine phosphatase treatment (Fig. 5 B).
|
Thus, the increased association of -catenin and p120-Cas with E-cadherin, which occurs in calcium-treated keratinocytes, is blocked by tyrosine kinase inhibition and
the strength of
-catenin association is directly dependent
on tyrosine phosphorylation of E-cadherin-associated proteins.
Inhibition of Tyrosine Phosphorylation Interferes with the Normal Cell Adhesive Function of Differentiating Keratinocytes
To assess whether the biochemical alterations induced by
tyrosine kinase inhibition were associated with altered cell
junction formation, keratinocytes were examined by transmission electron microscopy at 9 h after calcium treatment, at a time when tyrosine phosphorylation of - and
-catenins and p120-Cas is highly induced, and close cell-
cell contacts are formed. Efficient cell-cell junction and
formation was observed in control cells, with cell borders
being brought into close juxtaposition (Fig. 6 A, left; and
data not shown). By contrast, in Genistein-treated cells, only very few and incompletely formed cell junctions were
detected. Cell borders remained quite distant from each
other and were connected by protrusions of neighboring
cells in the form of pseudopod-like structures which met
without forming any kind of organized adhesive structure
(Fig. 6 A, center). As previously reported (Filvaroff et al.,
1990
), calcium-induced reorganization of the actin/keratin cytoskeleton was also prevented. Tyrphostin treatment
also resulted in suppression of cell junctions, but in this
case no pseudopod formation of neighboring cells was detected (Fig. 6 A, right). Widespread suppression of membrane detergent-insoluble structures containing E-cadherin
and plakophilin was confirmed by immunofluorescence analysis of control and tyrosine kinase inhibitor-treated
cells with the corresponding antibodies (Fig. 3). Interestingly, incomplete cell adhesion and membrane protrusions
similar to those of control keratinocytes at 2 h of calcium
treatment were observed.
|
The biochemical and structural alterations of cell junction formation that result from tyrosine kinase inhibition
may be associated with some significant functional defects.
To explore this possibility, we developed a new functional
assay for cohesiveness of cell adhesion. Dispase is a protease that degrades preferentially molecules such as type
IV collagen and fibronectin, which function as important attachment sites for keratinocytes to the underlying substratum (Stenn et al., 1989). Dispase treatment is routinely
used for skin grafting assays, to detach confluent cultures
of well-differentiated keratinocytes as intact sheets of
cells. We reasoned that a lack of cohesive strength may be
revealed under these conditions, when keratinocytes lose
attachment to their support and are connected to each
other only through direct intercellular contacts. Treatment of primary keratinocyte cultures in low calcium medium
with dispase caused detachment of cells as single cell suspension, consistent with the fact that keratinocytes under
these conditions are only very loosely connected with each
other. Conversely, keratinocyte cultures switched to high
calcium medium for either 9 or 24 h were much more resistant to dispase treatment and eventually (after 25-30 min)
detached from the dish as confluent sheet of cells (Fig. 7
A). Genistein-treated cultures switched to high calcium conditions behaved differently from the controls. Already
by 5 min of dispase treatment when control cells were still
unaffected, the Genistein-treated keratinocytes started to
detach, not as a sheet of cells, but in a localized fashion,
generating the appearance of "holes" in the confluent
monolayers (Fig. 7 A). Curiously, one or two cells were selectively left at the center of these holes, the nature of
which remains to be assessed. A similar pattern of cell detachment was also observed with Tyrphostin-treated keratinocytes (data not shown).
|
Quantification was achieved by counting the number of
single cells released by mechanical disruption after a 35-min dispase treatment, normalized for the total number of
cells recovered after subsequent treatment of the same
samples with trypsin (Fig. 8). This assay confirmed at the
functional level our immunofluorescence findings (Fig. 3)
that, in primary mouse keratinocytes cultured under our
conditions, cell-cell adhesion is only marginally increased by 2 h of calcium treatment, whereas it is firmly established by 9 h and beyond (Fig. 8 A). Genistein and Typhostin treatment caused a >50% reduction in strength of
cell adhesion (Fig. 8 B). PP1 is a recently developed inhibitor of tyrosine kinases of the Src family (Hanke et al.,
1996). Concomitant treatment of keratinocytes with calcium and PP1 caused a drastic inhibition of
- and
-catenin tyrosine phosphorylation (not shown) and, at the same
time, a reduction in strength of cell adhesion comparable
to that induced by the other tyrosine kinase inhibitors
(Fig. 8 B), together with the same pattern of cell detachment (not shown).
|
Thus, block of tyrosine phosphorylation has important structural and functional consequences on calcium-induced cell adhesion. Formation of normal cell junctions is prevented, and cohesive strength of confluent keratinocyte cultures, as revealed by a novel dispase-based assay, is significantly reduced.
Expression of an Activated Src Oncoprotein
Suppresses Keratinocyte Cell-Cell Adhesion
through Mechanisms Other Than Disruption of
-Catenin/E-Cadherin Complexes
Previous work with mitogenically stimulated or oncogene-transformed cells established a correlation between increased tyrosine phosphorylation of - and
-catenins and
p120-Cas and decreased rather than increased strength of
cell adhesion (Matsuyoshi et al., 1992
; Behrens et al., 1993
;
Hamaguchi et al., 1993
). However, more recent evidence
has suggested that tyrosine phosphorylation of
-catenin
in src-transformed cells cannot be directly responsible for
loosening of cell adhesion, and tyrosine phosphorylation
of some other proteins must be involved (Takeda et al.,
1995
). It was of interest to determine whether this conclusion applies also to keratinocytes. Accordingly, primary
mouse keratinocytes were infected with a recombinant adenovirus expressing an activated Src kinase, or a green fluorescent protein vector control. At 24 h after infection,
part of the cultures were switched to high calcium medium, and incubation was continued for additional 24 h.
The dispase assay described above was used to evaluate
the strength of cell adhesion of control versus src-transformed keratinocytes, at 24 h after calcium treatment. As
reported for other cells, expression of the Src kinase resulted in a marked disruption of cell adhesion (Fig. 8 C).
Parallel cultures were analyzed for E-cadherin complex
formation by immunoprecipitation with anti-E-cadherin antibodies and sequential immunoblotting with antibodies
against phosphotyrosine,
-,
-, and
-catenins and E-cadherin.
- and
-catenins were very highly tyrosine phosphorylated in the Src-expressing keratinocytes, whereas
their relative amounts remained unchanged (Fig. 9). Importantly, similar levels of
-catenin were found in the
E-cadherin complexes from Src expressing versus control keratinocytes, and calcium treatment caused a similar increase of
-catenin association in the two types of cells
(Fig. 9). At least two other prominent tyrosine phosphorylated bands were evident in the src-transformed keratinocytes, which were not detected in the control (more
bands were evident after prolonged exposure of the autoradiograph). One of these bands comigrates and is likely to correspond to E-cadherin. The other, with a size of ~60
kD, was found in Src-expressing keratinocytes after calcium treatment (Fig. 9). Reprobing of the same immunoblot with anti-Src antibodies revealed that this latter protein comigrates and is likely to correspond to constitutively
active Src itself, which increasingly associates with E-cadherin complexes in a calcium-dependent manner.
|
Thus, suppression of cell adhesion in src-transformed
keratinocytes is not because of disruption of E-cadherin/
-catenin complexes, but correlates with tyrosine phosphorylation of additional proteins not phosphorylated in
response to calcium, including E-cadherin itself.
The Fyn Tyrosine Kinase Is Involved in Catenin Phosphorylation and Modulation of Adhesive Function in Differentiating Keratinocytes in Culture
An important question raised by the above results is which
kinase(s) are responsible for tyrosine phosphorylation of
specific catenins in calcium-induced keratinocyte differentiation. In our previous work, we demonstrated that at
least two distinct tyrosine kinases are activated in calcium-induced keratinocyte differentiation, one of which corresponds to Fyn, a specific Src family member (Calautti et al.,
1995). An intriguing possibility was that Fyn may be at
least partially responsible for tyrosine phosphorylation of
- and
-catenins and p120-Cas in differentiating keratinocytes. Consistent with this possibility, immunofluorescence analysis of detergent-extracted keratinocytes showed
that the after calcium treatment the Fyn kinase colocalizes
with E-cadherin at sites of cell adhesion (Fig. 10).
|
The tyrosine phosphorylation state of E-cadherin-associated molecules was analyzed in primary keratinocytes
derived from mice with a disruption of the fyn gene versus
genetically matched wild-type controls. Cells were either
kept in low calcium medium or exposed to high calcium
concentrations for various amounts of time. Cell extracts
were immunoprecipitated with antibodies against E-cadherin or p120-Cas, followed by immunoblotting with anti-phosphotyrosine antibodies (Fig. 11 A, top panels). The
same immunoprecipitates were then blotted with antibodies
against - and
-catenins and p120-Cas, to verify amounts
of the respective proteins (Fig. 11 A, bottom panels). Tyrosine phosphorylation of all three catenins was found to
be reduced in fyn-deficient keratinocytes already under
basal conditions. In response to calcium, tyrosine phosphorylation of
- and
-catenin increased in fyn-deficient
cells, but to a much lesser extent than in the wild-type controls. Tyrosine phosphorylation of p120-Cas was also
strongly reduced in the fyn-deficient cells, with phosphorylation of the high molecular weight isoforms remaining close to undetectable even in cells under high calcium conditions. The decreased levels of catenin tyrosine phosphorylation appear to be specific for cells lacking the Fyn kinase, as they were not observed in cells with a disruption
of the related yes or src kinase genes (Fig. 11 A; data not
shown).
|
This reduction of tyrosine phosphorylation in fyn-deficient keratinocytes may have structural and/or functional consequences similar to those caused by tyrosine kinase inhibition. In fact, relative to wild-type controls, electron microscopy of fyn-deficient keratinocytes treated with calcium showed a drastic reduction in cell junction formation (Fig. 6 B). Cell-cell borders remained widely separated and were connected by pseudopod-like protrusions remarkably similar to those observed with Genistein-treated cells. A functional impairment in cell adhesion was also revealed by the dispase assay with wild type and fyn knockout cultures tested at 9 h of calcium treatment (Fig. 7 B). Under these conditions, the fyn-deficient keratinocytes detached in an uneven fashion, similar to that was observed with tyrosine kinase inhibitor-treated cells.
Thus, in cultured keratinocytes, basal and calcium-induced
tyrosine phosphorylation of - and
-catenin and p120-Cas
is partially dependent on the Fyn kinase. Lack of this kinase has important structural and functional consequences
that share substantial similarities with those induced by tyrosine kinase inhibitors.
Fyn and Src Kinases Are Important Determinants of
-Catenin Tyrosine Phosphorylation and Cell Adhesion
in Keratinocytes In Vivo
Unlike the significant alterations found in fyn-deficient
keratinocytes in culture, the epidermis of fyn knockout
mice shows a partial reduction of differentiation marker
expression (Calautti et al., 1995) but looks otherwise normal. One possible explanation for this discrepancy between the in vitro and in vivo situations could be the capability of other Src family members to compensate for Fyn
function in the intact epidermis. We investigated this possibility as it relates to E-cadherin function, by analyzing the skin of mice with a concomitant disruption of the fyn/
yes and fyn/src genes in comparison with that of wild-type
and single knockout littermates. More than 90% of mice
with double fyn/src, and ~70% of mice with fyn/yes homozygous mutations die soon after birth (Stein et al.,
1994
), thus limiting our analysis to newborn animals.
Direct biochemical analysis of keratinocytes in the skin
is complicated by the presence of other cell types. However, we took advantage of the fact that E-cadherin is preferentially expressed in keratinocytes, to directly immunoprecipitate this protein from total skin extracts and evaluate
the in vivo tyrosine phosphorylation pattern of E-cadherin
associated proteins by anti-phosphotyrosine immunoblotting. In an initial set of experiments, we compared E-cadherin complexes derived from wild-type newborn skin versus cultured keratinocytes at 9 h of calcium treatment.
Immunoprecipitates were normalized for the same amounts
of E-cadherin protein and immunoblotted sequentially
with antibodies against phosphotyrosine and - and
-catenin. Interestingly, the skin-derived E-cadherin complexes
contained a single prominent tyrosine phosphorylated band as opposed to the two bands present in the complexes from cultured keratinocytes (Fig. 11 B, left panel).
This different tyrosine phosphorylation pattern reflected
the fact that similar amounts of
-catenin were found to be
associated with E-cadherin both in vitro and in vivo. By
contrast, the association of
-catenin/plakoglobin with E-cadherin is markedly reduced in vivo, possibly because of a
preferential association of this catenin with desmosomal cadherins in the skin (Fig. 11 B, right panel).
When the skin of single and double knockout mutant
mice was analyzed, similar amounts of E-cadherin-associated -catenin were found in all cases (Fig. 11 C, bottom
panel). Tyrosine phosphorylation levels of this catenin
were essentially the same in wild-type mice and mice with
single fyn, yes, or src mutations. By contrast, a very drastic
reduction of
-catenin tyrosine phosphorylation was observed in mice with a concomitant disruption of the fyn and src genes, and, to a lesser extent, in fyn/yes mutants
(Fig. 11 C, top panel).
As discussed above, association of p120-Cas with E-cadherin cannot be detected by coimmunoprecipitation under
standard stringency conditions. For a direct analysis of this
catenin, total skin extracts were immunoprecipitated with
anti-p120-Cas antibodies. Immunoblotting with anti-phosphotyrosine antibodies revealed that levels of tyrosine
phosphorylated p120-Cas were essentially normal in the
skin of single yes knockout mice and were slightly reduced in the skins of mice with a single fyn or src mutations. By
contrast, tyrosine phosphorylation of all forms of p120-Cas
was strikingly reduced in the skin of double fyn/src mutants. A significant but lesser reduction was also observed
in the skin of fyn/yes knockouts (Fig. 11 D, top panel). Surprisingly, however, in contrast to the intrinsically decreased tyrosine phosphorylation of -catenin in the presence of constant protein, p120-Cas protein levels, and not
only their tyrosine phosphorylation state, were drastically reduced in the double knockouts. This was revealed by immunoblotting with anti-p120-Cas antibodies of the p120-Cas immunoprecipitates (Fig. 11 D, bottom panel), as well
as by immunoblotting of total skin extracts (data not shown).
We tested whether the striking biochemical alterations found in the skins of double fyn/src-deficient mice were associated with defects in keratinocyte cell adhesion detectable at the histological and/or ultra structural level. Newborn mouse epidermis is composed of multiple stratified layers. In contrast to normal skin, a separation of overlying layers was observed occasionally in histological sections of double fyn/src mutant skins (data not shown). Much more widespread and significant alterations were revealed by electron microscopy (Fig. 12), with cells at the border between spinosum and granular layers appearing as the most affected. Similar numbers of well-formed desmosomes were present in the epidermis of wild-type and fyn/src mutant epidermis. However, the tracts of cell membranes between desmosomal junctions, corresponding to sites of adherens junction formation, were usually closely juxtaposed in wild-type skin (Fig. 12 A). By contrast, the interdesmosomal spaces were often widely separated in the double knockouts (Fig. 12 B), generating the appearance of a series of arches or fenestrations between neighboring cells (Fig. 12 C).
|
In parallel with their lesser biochemical alterations, skins of double fyn/yes mutant mice showed none of the ultra structural abnormalities in keratinocyte cell adhesion found in the fyn/src knockouts. However, subtler alterations may also exist in the epidermis of these mice as indicated by their reduced number of layers (data not shown).
Thus, unlike the significant alterations found in culture,
keratinocyte cell adhesion in the epidermis of fyn deficient
mice appears essentially normal. This is likely due to a
functional compensation of Fyn with related kinases. In
fact, in the skin of mice with a double fyn/src knockout
mutation, tyrosine phosphorylation of -catenin is intrinsically reduced, p120-Cas is strongly downmodulated, and
interdesmosomal spaces are largely increased.
![]() |
Discussion |
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We have previously demonstrated that induction of tyrosine phosphorylation is required for keratinocyte differentiation to occur, and that at least two distinct tyrosine kinases are involved, one of which was identified as Fyn. However, only two proteins of ill-defined function were found to be tyrosine phosphorylated in this process, ras-GAP-associated p62 and cortactin, and the specific physiological function(s) of tyrosine kinase activation in keratinocyte differentiation remained to be established. Also, the significance of tyrosine phosphorylation for keratinocyte differentiation control in vivo was not assessed. We show here that induction of tyrosine phosphorylation is involved in at least one key aspect of keratinocyte differentiation, i.e., control of cell adhesion, both in cultured cells and in the intact skin.
E-cadherin-mediated adherens junctions provide a primary determinant of increased cell-cell adhesion in keratinocytes (Hodivala and Watt, 1994; Lewis et al., 1994
;
Amagai et al., 1995
). We have found here that three E-cadherin-associated proteins,
- and
-catenins and p120-Cas,
are directly tyrosine phosphorylated at the onset of calcium-induced keratinocyte differentiation, in parallel with establishment of close intercellular contacts and the beginning of stratification. Consistent with the coordinate control
of adherens junction and desmosome formation (Hodivala
and Watt, 1994
; Lewis et al., 1994
), Desmoglein-associated
-catenin also appears to be increasingly tyrosine phosphorylated in response to calcium. We have found that tyrosine phosphorylation of
- and
-catenins does not affect
their association with E-cadherin. In contrast,
-catenin,
which is not itself tyrosine phosphorylated, becomes increasingly associated with E-cadherin/catenin complexes,
with a time course that parallels that of
- and
-catenin
tyrosine phosphorylation. The increased association of
-catenin with E-cadherin complexes is blocked by tyrosine kinase inhibition, and the strength of
-catenin association depends directly on tyrosine phosphorylation of
E-cadherin complexes.
-Catenin provides a direct bridge
between cadherin-catenin complexes and the actin cytoskeleton (Rimm et al., 1995
). Thus, our findings provide
the first evidence that tyrosine phosphorylation can determine the extent of
-catenin association with E-cadherin
complexes, and provide an attractive explanation for the fact
that in differentiating keratinocytes increased
- and
-catenin tyrosine phosphorylation is associated with an increased
strength of cell adhesion. Control of adherens junction and
desmosome formation is intimately connected (Hodivala and Watt, 1994
; Lewis et al., 1994
; Amagai et al., 1995
).
Our data indicate that desmosomes may also be controlled
by tyrosine phosphorylation, either directly (for instance,
by
-catenin tyrosine phosphorylation) or indirectly (through
modulation of adherens junction formation).
In mitogenically stimulated or oncogene-transformed
cells, increased tyrosine phosphorylation of - and
-catenins has been correlated with a decrease of cell adhesion
(Kanner et al., 1991
; Matsuyoshi et al., 1992
; Behrens et al.,
1993
; Hamaguchi et al., 1993
; Hoschuetzky et al., 1994
;
Shibamoto et al., 1994
; Kinch et al., 1995
). However, additional evidence indicates that loosening of cell contacts in
Src-transformed cells cannot be explained by tyrosine
phosphorylation of
-catenin, but is likely resulting from
tyrosine phosphorylation of some other proteins (Taketa
et al., 1995). A similar conclusion is likely to apply to our
cells. In fact, in keratinocytes expressing a constitutively
active Src kinase, the highly increased tyrosine phosphorylation of
- and
-catenins does not alter their stoichiometry
in E-cadherin complexes, nor the basal or calcium-induced
-catenin association. Given the relatively low tyrosine
phosphorylation levels of
-catenin in normal calcium-treated
keratinocytes, only a tentative tyrosine phosphopeptide
map could be obtained. These results are consistent with
the possibility raised by our other findings that (a) the residues required for basal
-catenin association are already tyrosine phosphorylated in keratinocytes under low calcium conditions and (b) the new residues required for calcium-induced association are not tyrosine phosphorylated
by the constitutively active Src kinase. However, the additional possibility should be considered that any positive
effects of tyrosine phosphorylation of E-cadherin-associated proteins by Src may be counteracted by tyrosine
phosphorylation of other proteins. In fact, we have found
that src-transformed keratinocytes still respond to calcium
but phosphorylate additional proteins, including E-cadherin itself, in an aberrant manner. In addition, constitutively active Src itself was also found to associate with
E-cadherin complexes in a calcium-dependent manner. Thus, by analogy with the results of Takeda et al. (1995)
,
tyrosine phosphorylation of proteins other than
- and
-catenins would seem likely to contribute to suppression
of keratinocyte cell adhesion.
Besides its role in cadherin-mediated cell adhesion, -catenin can play a second independent function in signaling
pathways connected with development and cell fate determination (for review see Gumbiner, 1995
; Miller and Moon,
1996
). A fraction of
-catenin has been reported to localize to the nucleus and to influence, through association
with transcription factors such as LEF-1 or Tcf4, gene expression (Behrens et al., 1996
; Korinek et al., 1997
; Morin
et al., 1997
). Mice with a disruption (van Genderen et al., 1994
) or inappropriate expression (Zhou et al., 1995
) of
the LEF gene exhibit a significantly altered skin phenotype. Future studies will have to address whether even in
keratinocytes
-catenin is involved in complex functions
other than cell adhesion, and whether the extent of this involvement can be controlled by tyrosine phosphorylation.
The other catenin that is found to be directly tyrosine
phosphorylated in differentiating keratinocytes is p120-Cas. Four isoforms of p120-Cas have been described,
which differ for the presence or the absence of two alternatively spliced sequences (Mo and Reynolds, 1996). All
four isoforms are expressed in cultured keratinocytes and
become increasingly tyrosine phosphorylated in response
to calcium, with kinetics similar to those of
- and
-catenins. However, whereas the stoichiometry of
- or
-catenin in E-cadherin complexes is unaffected by calcium treatment,
p120-Cas displays an increased association with E-cadherin. The association of p120-Cas is likely to be more unstable than that of
- or
-catenin, as it could only be detected by immunoprecipitation under mild stringency. As
with
-catenin association, inhibition of tyrosine phosphorylation suppressed the increased association of p120-Cas with E-cadherin. This situation in differentiating keratinocytes may be similar to that reported for ras-transformed
breast epithelial cells, where tyrosine phosphorylation of p120
was correlated with increased association with E-cadherin
(Kinch et al., 1995
).
p120-Cas binds to E-cadherin in close proximity to the
binding site for - and
-catenins (Daniel and Reynolds,
1995
; Shibamoto et al., 1995
). However, unlike these latter
molecules, p120-Cas does not bind to
-catenin and thus
may function by blocking the interactions of E-cadherin
with this latter protein and, indirectly, with the cytoskeleton (Daniel and Reynolds, 1995
). An attractive working
hypothesis is that tyrosine phosphorylation could play two
complementary functions in the changes in cell adhesion
connected with keratinocyte differentiation. Tyrosine phosphorylation of
- and
-catenins, by promoting
-catenin
association, would enable cadherin-catenin complexes to
become more firmly connected to the actin cytoskeleton,
thus enhancing the strength of cell adhesion. By concomitantly increasing E-cadherin/p120-Cas complex formation, tyrosine phosphorylation of these latter molecules would
ensure fluidity of cell adhesion, thus allowing stratification. Besides the biochemical data, this hypothesis would
be consistent with the functional and structural analysis
discussed below.
Previously used assays of cell adhesion depend on dissociation of cells followed by measurement of their reassociation in suspension and/or after centrifugation (Takeda et al.,
1995; Angres et al., 1996
). The dispase assay that we have
developed provides a useful alternative, in that it allows to
evaluate epithelial cells as confluent layers, without any
previous disruption of already formed cell contacts, and
taking into account the cohesive strength of both adherens
junction and desmosome formation. Using this approach,
we have found that the cohesiveness of keratinocyte cell
adhesion induced by calcium is significantly lessened by tyrosine kinase inhibition, and this is associated with a
strongly reduced number of well-formed cell junctions.
Similar alterations were observed with confluent cultures
of fyn-deficient keratinocytes, in which calcium-induced
tyrosine phosphorylation was strongly decreased, even if
not totally suppressed (as discussed further below). In
both Genistein-treated and fyn-deficient keratinocytes, a
distinguishing feature that accompanies the scarcity of intercellular junctions and separation of cell-cell borders, is
the formation of pseudopod-like extensions, which may
correspond to abortive attempts of cells to establish stable
junctions with each other. Interestingly, adherens junction
formation has been recently reported to depend on a second signaling pathway involving the rho and rac GTPases,
which control actin/cytoskeleton organization and cell motility (Braga et al., 1997
). Thus, it is tempting to suggest that efficient adherens junctions formation involves at least two complementary, and possibly sequential, steps. Active
movement at the cell membrane, leading to bridging protrusions from neighboring cells, would occur in a rho- and
rac-dependent fashion, whereas establishment of fully assembled adherens junctions would be under tyrosine phosphorylation control.
Besides increased strength, fluidity of cell contacts is an
important element in keratinocyte differentiation, allowing these cells to stratify (Lewis et al., 1994; Tao et al., 1996
).
Evidence for tyrosine phosphorylation being involved in
dynamic control of cell adhesion is provided by the strongly
reduced stratification of keratinocytes treated with tyrosine
kinase inhibitors (Filvaroff et al., 1990
) or deficient for the
Fyn kinase after calcium exposure (Calautti et al., 1995
).
Whether or not rho- and rac-dependent pathways are also
involved in the stratification process is obviously a relevant question that remains to be assessed.
A final issue raised by our results is which kinase(s) are
responsible for tyrosine phosphorylation of - and
-catenins
and p120-Cas in differentiating keratinocytes. A specific
involvement of the Fyn kinase is indicated by the fact that
tyrosine phosphorylation of these catenins is significantly
reduced in cultured keratinocytes with a disruption of the
fyn but not yes or src kinase genes. This fact is consistent
with our previous findings that the Fyn kinase is specifically activated in differentiating keratinocytes in culture,
and that a number of differentiation parameters are selectively altered in the fyn-deficient cells (Calautti et al., 1995
).
However, Fyn is not the only kinase to be involved, as a
lower level of basal and calcium-induced tyrosine phosphorylation of these catenins is still observed in the fyn-
deficient keratinocytes. We previously showed that a
second independent tyrosine kinase is activated in calcium-
induced keratinocyte differentiation (Calautti et al., 1995
),
and it is possible that Fyn and the other calcium-induced
kinase cooperate in control of catenin function by phosphorylation. In addition, other Src family members are likely to contribute to catenin tyrosine phosphorylation in
keratinocytes. In fact, we have found that functional compensation of Src family members clearly occurs in vivo, as
tyrosine phosphorylation levels of
-catenin are essentially normal in the skin of newborn mice with a single fyn
knockout mutation, whereas they are significantly decreased in the skin of mice with a concomitant disruption
of fyn and src genes. Interestingly, in these same mice, protein levels of p120-Cas, and not simply its tyrosine phosphorylation, are drastically reduced. This points to a so far
unexplored control of catenin function by src-related kinases, which may be consistent with the already reported
upregulation of p120-Cas expression in src-transformed
cells (Mo and Reynolds, 1996
).
In parallel with the biochemical findings, the skin of the
double fyn/src mutants showed striking ultra structural abnormalities, with widely disrupted interdesmosomal spaces
at sites where adherens junctions are usually found. By
contrast, density and structure of desmosomes were essentially normal. In vitro, our data are consistent with the previous demonstration that impaired adherens junction formation affects desmosome assembly as well (Hodivala and
Watt, 1994; Lewis et al., 1994
; Amagai et al., 1995
). In vivo, the results with the fyn/src mutant skin suggest that control of adherens junctions and desmosome formation are
at least partially independent. This possibility would also
be consistent with the strikingly different composition of
E-cadherin complexes in the in vitro versus in vivo situation, where the ratio of
- to
-catenin appears to be markedly increased.
The skin of mice with a concomitant disruption of the
fyn and yes genes showed none of the alterations in cell
adhesion found in the skin of fyn/src mutants, indicating
that the Src and Yes kinases can compensate for lack of
Fyn to a different extent. In fact, in the fyn/yes knockout
skin tyrosine phosphorylation of -catenin and expression
of p120-Cas were found to be decreased, but to a significantly lesser extent than in the fyn/src mutants. In addition, other more selective biochemical defects may occur in the fyn/src knockout epidermis, which could contribute
to its striking reduction in cell adhesion.
Taken together, our results indicate that tyrosine phosphorylation, and Fyn-related kinases in particular, play an essential function in one critical aspect of keratinocyte differentiation, i.e., control of cell adhesion. This regulatory function is important not only in vitro but also in vivo. More generally, our findings support the notion that in partially redundant regulatory systems, such as that of Src family kinases, the biochemical response of cells in tissues is much more plastic than in culture, so that a combined in vitro/in vivo approach is necessary to dissect the function of any individual component.
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Footnotes |
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Received for publication 29 October 1997 and in revised form 21 April 1998.
Address all correspondence to G.P. Dotto, Cutaneous Biology Research Center, Building 149, 13th Street, Charlestown, MA 02129. Tel.: (617) 724-9538. Fax: (617) 724-9572. E-mail: Paolo_Dotto{at}CBRC.MGH.Harvard.eduWe thank Drs. P. Cowin and J. Stanley for their generous gift of antibodies, and Drs. J. Gonzales, Y. Ge, and M. Ericsson for their help with the mouse colony, confocal microscopy, and electron microscopy, respectively. We also thank Drs. T.W. Wang, David Rimm, and F. Di Cunto for critical reading of the manuscript.
This work was supported by National Institutes of Health Grants AR39190, CA64043, and CA73796 to G.P. Dotto. This work was also supported in part by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement; and by a research fellowship sponsored by Elizabeth Arden Company from the Dermatology Foundation to E. Calautti; and an Arthritis Foundation Investigator Award and funds from the Council for Tobacco Research, U.S.A., to P.L. Stein.
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