|
Originally published In Press as doi:10.1074/jbc.M003172200 on July 31, 2000
J. Biol. Chem., Vol. 275, Issue 41, 32289-32298, October 13, 2000
Cell Volume-dependent Phosphorylation of Proteins of
the Cortical Cytoskeleton and Cell-Cell Contact Sites
THE ROLE OF Fyn AND FER KINASES*
András
Kapus §,
Caterina
Di Ciano,
Jianguo
Sun,
Xi
Zhan¶,
Leung
Kim ,
Tai Wai
Wong**, and
Ori D.
Rotstein
From the Department of Surgery, The Toronto General
Hospital and University of Toronto, Toronto, Ontario M5G 1L7, Canada,
the ¶ Department of Experimental Pathology, The Holland
Laboratory, American Red Cross, Rockville, Maryland 20855, the
NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and the ** Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New Jersey 08543
Received for publication, April 13, 2000, and in revised form, July 12, 2000
 |
ABSTRACT |
Cell volume affects diverse functions including
cytoskeletal organization, but the underlying signaling pathways
remained undefined. We have shown previously that shrinkage induces
Fyn-dependent tyrosine phosphorylation of the cortical
actin-binding protein, cortactin. Because FER kinase was implicated in
the direct phosphorylation of cortactin, we investigated the
osmotic responsiveness of FER and its relationship to Fyn and
cortactin. Shrinkage increased FER activity and tyrosine
phosphorylation. These effects were abolished by the Src family
inhibitor PP2 and strongly mitigated in Fyn-deficient but not in
Src-deficient cells. FER overexpression caused cortactin
phosphorylation that was further enhanced by hypertonicity. Exchange of
tyrosine residues 421, 466, and 482 for phenylalanine prevented
cortactin phosphorylation by hypertonicity and strongly decreased it
upon FER overexpression, suggesting that FER targets primarily the same
osmo-sensitive tyrosines. Because constituents of the cell-cell
contacts are substrates of Fyn and FER, we investigated the effect of
shrinkage on the adherens junctions. Hypertonicity provoked
Fyn-dependent tyrosine phosphorylation in  -catenin,
 -catenin, and p120Cas and caused the dissociation
of -catenin from the contacts. This process was delayed in
Fyn-deficient or PP2-treated cells. Thus, FER is a volume-sensitive
kinase downstream from Fyn, and the Fyn/FER pathway may contribute to
the cell size-dependent reorganization of the cytoskeleton
and the cell-cell contacts.
| |
INTRODUCTION |
Changes in cell volume or cell shape are known to affect a variety
of functions including the activity of ion transporters, the
organization of the cytoskeleton, and the transcription of certain
genes (1-3). Although these responses are important for the
maintenance of cellular homeostasis and integrity, little is known
about the volume-dependent signaling pathways that convert the initial alteration to the compensatory or regulatory effector functions.
We and others have noted previously that one of the earliest cellular
responses to hyperosmotic stress is a dramatic increase in protein
tyrosine phosphorylation (4, 5). This phosphotyrosine accumulation is
triggered by cell shrinkage and not by hyperosmolarity per
se, and in fibroblastic cell lines it occurs predominantly in
bands of 80-90 and 110-130 kDa. Our recent studies aimed at the
identification of targets of the hypertonic phosphorylation and the
respective tyrosine kinases showed that one of the major substrates for
osmotic phosphorylation is cortactin (6), an 80-85-kDa cortical actin
filament cross-linking protein, and preferred substrate for Src family
kinases (7-10). Cortactin has a unique structure composed of six and a
half tandem repeats responsible for actin binding, followed by a
serine-threonine-rich domain, a tyrosine-rich sequence, and a
C-terminal SH3 domain (8, 11). These features, together with the
fact that upon cell stimulation cortactin localizes to peripheral
membrane structures such as ruffles and lamellipodia (8, 12), suggest
that this protein may be an important organizer of the cortical
cytoskeleton dynamics. In favor of this notion, overexpression (13) or
tyrosine phosphorylation (11) of cortactin has been associated with
increased cell motility and invasiveness.
Within the Src family, Fyn kinase seems to play a specific role in the
osmotic cortactin response. This conclusion is based on our earlier
findings that 1) Fyn but not Src itself is activated by osmotic shock
and 2) the hypertonic cortactin phosphorylation is substantially
reduced in Fyn-deficient but remains intact in Src-deficient cells (6).
Recent observations, however, suggest that the direct phosphorylation
of cortactin might be catalyzed not (only) by Src-type kinases but also
by FER kinase, a member of the c-FES family (14). FER contains a unique
kinase domain, a central SH2 domain, and two regions with homology to
coiled-coil oligomerization domains (15, 16). Little is known about the mechanism of FER activation apart from the fact that it coincides with
the tyrosine phosphorylation of the enzyme. This might be brought about
by the activated platelet-derived growth factor receptor (15),
by autophosphorylation following FER oligomerization (15-17), and by
as yet unidentified tyrosine kinases (16). Recent studies show that the
primary substrates of FER include key constituents of the cell-cell
contact apparatus, such as -catenin, and a related molecule,
p120Cas (15, 18). These proteins link the transmembrane
adhesion receptor cadherins to the actin skeleton (19), and their
tyrosine phosphorylation is thought to be a crucial mechanism in the
regulation of adherens junctions (20). A large body of evidence
suggests that, besides FER, various Src family kinases also play a
direct or indirect role in the phosphorylation of these contact
elements (21-24). In most cases, increased tyrosine phosphorylation of
the junctional proteins has been associated with the disassembly of the
contacts (22, 24-28).
The scenario described above offers intriguing possibilities regarding
volume-related signaling: 1) It is conceivable that FER is a
volume-dependent kinase, and if so, it might be involved in
the shrinkage-induced cortactin phosphorylation. 2) The osmotic activation of FER might be related (downstream or upstream) to the
activation of Fyn. 3) Hyperosmotic stress might induce the phosphorylation and reorganization of certain cell-cell contact proteins. An interesting implication of this latter assumption is that
changes in cell size or shape might represent important inputs for the
regulation and coordination of two interrelated functions: cell
migration and cell-cell adhesion.
The aim of the present work was to gain further insight into the
volume-dependent signaling by testing these possibilities. Our results show that FER is phosphorylated and activated by osmotic stress in a Fyn-dependent manner, and it may contribute to
the osmotic cortactin phosphorylation. Moreover, hypertonicity, through Fyn and presumably FER, provokes the tyrosine phosphorylation and
redistribution of cell-cell contact proteins.
| |
EXPERIMENTAL PROCEDURES |
Materials--
Nigericin, [Val5]-angiotensin II,
and ATP were purchased from Sigma. Protease inhibitor mixture
containing 0.8 mg/ml benzamidine HCl, 0.5 mg/ml aprotinin, 0.5 mg/ml
leupeptin, 0.5 mg/ml pepstatin A, and 50 mM
phenylmethylsulfonyl fluoride in pure ethanol was from PharMingen, and
protein G-Sepharose beads were from Amersham Pharmacia Biotech.
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)1 acetoxy methylester
and PP2 were from Calbiochem. Ultra-pure myelin basic protein,
monoclonal anti-phosphotyrosine (4G10), anti-cortactin, and
anti-p120Cas were obtained from Upstate Biotechnology Inc.
Monoclonal antibody against c-Myc(9E10) and polyclonal antibodies
against ERK-2, (E)-catenin, -catenin, VE-,
P-, and N-cadherin were purchased from Santa Cruz Biotechnology. The
anti-FER2 and -FER4 sera used in these studies were described
previously (15). Peroxidase-conjugated anti-mouse and anti-rabbit IgG,
the Enhanced Chemiluminescence Kit, and [ -32P]ATP
(3000 Ci/mmol) were from Amersham Pharmacia Biotech. The fluorescein
isothiocyanate-labeled anti-Goat IgG was obtained from Jackson.
Media--
Bicarbonate-free RPMI 1640 was buffered with 25 mM Hepes to pH 7.4 (osmolarity 290 ± 5 mosM). The isotonic sodium medium (Iso-Na) consisted of 140 mM NaCl, 3 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 5 mM
glucose, and 20 mM Hepes (pH 7.4). When required, the
Iso-Na was made hypertonic by the addition of various amounts of
sucrose (25-600 mM) or 300 mM urea, as
indicated. If not stated otherwise, the hypertonic sodium medium used
in most experiments refers to the isotonic medium supplemented
with 300 mM sucrose (total osmotic concentration is 600 mosM). The hypertonic potassium medium used for the
calibration of the intracellular pH contained 243 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, and 20 mM Hepes. The pH was set to
6.9, 7.4, or 7.9 using the necessary amount of Tris-OH. Osmolarity was
checked with an Osmette osmometer.
Cell Culture--
CHO cells (29) were grown in -minimal
essential medium, containing 25 mM NaHCO3 and
supplemented with 10% fetal calf serum, and 1% antibiotic suspension
(penicillin and streptomycin, Sigma), under humidified atmosphere of
air/CO2 (19:1) at 37 °C. Wild Type (WT), Fyn /,
Src/ fibroblasts were originally isolated from mouse embryos that
were homozygous for disruption in the Src or Fyn gene and were
immortalized with large T antigen (30). These cell lines are identical
with those used in our previous studies (6) and were kindly provided by
Sheila M. Thomas (Fred Hutchinson Cancer Center, Seattle). These cells
as well as the Swiss 3T3 fibroblasts were maintained in Dulbecco's
modified Eagle's medium. All other conditions and treatments were
similar to those used in CHO cells.
Constructs and Cell Transfection--
The plasmid pMyc-cortactin
encoding for the wild type murine cortactin tagged with the Myc epitope
at its N terminus and pMyc-cortactinF421F466F482 encoding for a mutant
version (Pcort) in which the listed residues were exchanged for
phenylalanine were previously described (11). For the expression of the
Hemagglutinin-tagged WT and kinase inactive FER, the plasmids
pCMV-HA-FER(WT) and pCMV-HA-FER(K591R) were used, as described before
(14). Transient transfection with the corresponding plasmids were
performed on CHO cells using the FuGeneTM Reagent (Roche
Molecular Biochemicals), according to the manufacturer's instructions.
Preparation of Cell Extracts--
Confluent cultures were
incubated for 2-3 h in serum- and HCO3-free RPMI 1640 prior to experiments. Cells were preincubated in Iso-Na medium for 10 min and then subjected to various treatments as indicated. The medium
was then aspirated, and the cells were vigorously scraped into ice-cold
lysis buffer (100 mM NaCl, 30 mM Hepes, 20 mM NaF, 1 mM EGTA, 1% Triton X-100, pH 7.5)
supplemented with 1 mM Na3VO4 and
20 µl/ml protease inhibitor mixture. To obtain cell suspension from
cultured cells, confluent monolayers were treated with Ca2+
and Mg2+-free Iso-Na medium supplemented with 5 mM Na2EDTA (5 ml/10-cm dish). After 10 min, the
detached cells (>90%) were vigorously pipetted until single cell
suspension was attained. The suspension was then divided into two equal
parts, and an equal volume of the same isotonic solution or this
solution supplemented with 600 mM sucrose was added
(i.e. final concentration, 300 mM sucrose). After 10 min with slow rotation at 37 °C, the samples were spun (3000 rpm for 3 min), the supernatant was removed, and the pellet was
dissolved in cold lysis buffer.
Western Blotting and Immunoprecipitation--
Lysates containing
equal amount of protein were clarified by centrifugation at 12,000 × g for 10 min, precleared for 1 h using 40 µl of
50% suspension of protein G-Sepharose beads, and then incubated with
the corresponding antibodies for at least 1 h. Immunocomplexes
were captured using 40 µl of protein G-Sepharose, and the beads were
washed four times with lysis buffer containing 1 mM
Na3VO4. Immunoprecipitated proteins were
diluted with Laemmli sample buffer, boiled for 5 min, and subjected to
electrophoresis on 7.5 or 10% SDS-polyacrylamide gels, as specified
under the figures. The separated proteins were transferred to
nitrocellulose using a Bio-Rad Mini Protean II apparatus. To check
effectiveness of transfer and similarity of protein amount, lanes were
visualized by staining with Ponceau S. Blots were blocked in
Tris-buffered saline containing 5% bovine serum albumin and incubated
with the corresponding primary antibody. Binding of the antibody was
visualized by the relevant (anti-mouse, rabbit, or goat),
peroxidase-coupled secondary antibodies (1:3000 dilution) using the
enhanced chemiluminescence method.
Densitometry--
Quantification of the bands was performed
using a Bio-Rad GS-690 Imaging Densitometer and the Molecular Analyst
program as in Ref. 6.
Immunocomplex Kinase Assays--
In vitro FER
activity was determined essentially as described previously, using
[Val5]-angiotensin II as substrate (15). Cell lysates
obtained from iso- or hypertonically treated WT and Fyn/ cells and
containing equal amount of protein (500-1000 µg) were subjected to
immunoprecipitation using the anti-FER2 antibody. The precipitates were
washed with kinase buffer (50 mM Tris-HCl and 10 mM MnCl2, pH 7.7) supplemented with 0.25 mM Na3VO4. The beads were then
incubated for 10 min at 30 °C in 25 µl of kinase buffer containing
5 mM [Val5]-angiotensin II, 5 µM ATP (potassium salt), and 10 µCi of
[-32P]ATP/sample. The reaction was terminated by the
addition of 35 µl of ice-cold stop solution (20% trichloroacetic
acid and 5 mM ATP). 35 µl of this mixture was layered
onto P81 phosphocellulose squares, and after extensive washing with
0.85% phosphoric acid, radioactivity bound to the filters was measured
by scintillation counting. The background, i.e. the activity
detected in samples that had been incubated identically but without the
substrate, was determined in each experiment and subtracted. Results
were normalized to the amount of protein content of the cell lysate.
For the determination of ERK-2 activity, lysates containing equal
amounts of protein were immunocomplexed using the anti-ERK-2 antibody.
The beads were washed with kinase buffer and preincubated with 20 µg
of myelin basic protein/sample for 5 min on ice. The samples were then
placed in a 30 °C waterbath, and the reaction was initiated by the
addition of 30 µl of assay buffer containing 10 mM
MgCl2, 50 mM Tris-HCl (pH 7.4), 40 µM ATP, and 1.2 µCi of [-32P]ATP. 15 min later the reaction was terminated by 30 µl of 2× Laemmli buffer,
and the samples were boiled and subjected to SDS-polyacrylamide gel
electrophoresis and autoradiography.
Immunfluorescence Microscopy--
Confluent cultures grown on
coverslips were preincubated for 10 min in Iso-Na medium and treated as
specified under the figures. The cells were then fixed with 4%
paraformaldehyde for 30 min in the same iso- or hypertonic medium as
used for the experiment. The coverslips were extensively washed with
phosphate-buffered saline, incubated with 100 mM glycine in
phosphate-buffered saline, permeabilized with 0.1% Triton for 20 min
at room temperature, and blocked with 1% donkey serum plus 1% bovine
serum albumin for 1 h. Subsequently, the samples were incubated
with anti--catenin (1:100) for 1 h, followed by washing and
incubation with fluorescein isothiocyanate-labeled secondary antibody.
The coverslips were washed and mounted on glass slides using the
Anti-fade kit (Molecular Probes). The staining was visualized using a
Leica Immunofluorescence microscope, and pictures were taken with the
WinView® software.
Measurement of Cytosolic pH--
Changes in pHi were
monitored fluorometrically using the indicator dye BCECF, as described
before (29). Confluent cultures of WT and Fyn/ cells grown on glass
coverslips were loaded with 1 µM BCECF acetoxy
methylester for 10 min in Iso-Na medium. Ratio fluorometry was
performed on small populations of cells (6-12 cells/measurement) using
a DeltaRAM illumination system from Photon Technologies, Inc. in the
dual excitation (490 ± 5 nm/440 ± 5 nm), single emission
(530 ± 30 nm) configuration. Each coverslip was mounted to form
the bottom of a thermostatted, perfusable Leydig chamber into which 0.5 ml of isotonic medium was added and the basal fluorescence was
recorded. The medium was then rapidly exchanged (by addition of 10 times 0.5 ml in less than 20 s) to a hypertonic solution. After
each measurement, fluorescence was calibrated in terms of
pHi by sequential perfusion of the chamber with
nigericin-containing calibration media (see above). Data analysis was carried out using the Felix® software.
Cell Volume Measurements--
Confluent CHO cells were detached
using the Iso-Na medium supplemented with 5 mM
Na2EDTA and suspended in Iso-Na, hypertonic sodium medium,
or Iso-Na containing 300 mM urea for 10 min. The median
cell volume was then determined by electronic sizing using a Coulter
Counter model ZM equipped with a Channelyzer.
Other Methods--
Protein concentration was determined by the
BCA assay (Pierce) using bovine serum albumin as standard. Data are
presented as representative immunoblots of at least three similar
experiments or as the means ± S.E. of the number of experiments
indicated (n).
| |
RESULTS |
The Effect of Hypertonicity on FER Kinase Phosphorylation and
Activity--
To assess whether FER kinase might be involved in the
volume-dependent phosphorylation of cytoskeletal proteins,
we first investigated whether FER itself undergoes tyrosine
phosphorylation upon cell shrinkage. To address this question, various
cell types (CHO cells, embryonic fibroblasts, Swiss 3T3 cells) were
challenged with isotonic or hypertonic solutions, and following cell
lysis, FER kinase was immunoprecipitated from the Triton X-100-soluble fraction and probed with anti-phosphotyrosine. Under isotonic conditions FER was only minimally tyrosine phosphorylated, whereas osmotic shock (300 mM sucrose) induced a rapid and robust
phosphotyrosine accumulation in the kinase in each fibroblastic cell
line tested (Fig. 1, A and
B for CHO cells, and C for embryonic fibroblasts; data not shown for 3T3). Phosphorylation was also evoked when tonicity
was increased by sorbitol, or to a somewhat lesser extent by NaCl,
indicating that the type of osmolyte was not critical as long as
membrane-impermeant molecules were used (not shown). In contrast, the
membrane-permeable urea, which elevates both intra- and extracellular
osmolarity but causes only marginal volume change, failed to induce
significant FER phosphorylation (Fig. 1B). This finding
indicates that the critical stimulus for hypertonicity-induced FER
phosphorylation, similar to that of cortactin, is a reduction in cell
volume and not an increase in the osmolarity per se.
View larger version (49K):
[in this window]
[in a new window]
|
Fig. 1.
Volume-dependent tyrosine
phosphorylation of FER kinase. CHO cells (A and
B) or wild type embryonic fibroblasts (C) were
serum-deprived and then challenged with isotonic (ISO) or
hypertonic (HYP) solutions. If not stated otherwise,
hypertonicity was added in the form of 300 mM sucrose
(final osmolarity, 600 mosM) for 10 min. Where indicated,
CHO cells were preincubated with 10 µM PP2 and then
exposed to the hypertonic solution containing the same drug
concentration. After treatment the cells were lysed, and FER kinase was
immunoprecipitated (IP) as described under "Experimental
Procedures." The precipitates were subjected to SDS-polyacrylamide
gel electrophoresis and Western blotting (WB) using the
indicated antibodies. A, blots were first probed with
antiphosphotyrosine (Anti-PY), and then the filters were
stripped and reprobed with anti-FER. B, comparison of the
effect of impermeable (300 mM sucrose, SUC) and
permeable (300 mM urea) osmolytes on cell volume and FER
phosphorylation. C, the dependence of the shrinkage-induced
FER response on time (left panels) and osmotic concentration
(right panels). The blots were reprobed first with anti-FER
and second with anti-cortactin.
|
|
The more detailed analysis of the dependence of FER phosphorylation on
time and osmolarity was performed in embryonic fibroblast, because
previously we used the wild type and Fyn-deficient versions of this
cell type to show that Fyn kinase is involved in hypertonic cortactin
phosphorylation (6). Fig. 1C demonstrates that in these
cells, FER phosphorylation triggered by 300 mM sucrose
became clearly visible after 2 min and increased further with time, for as long as 30 min. Moreover, FER was sensitive to small changes in
medium osmolarity; a 25-50 mosM increase in the external
osmotic concentration for 10 min was sufficient to elicit detectable
tyrosine phosphorylation, and the response became stronger with
increasing hypertonicity. Changes in osmolarity did not affect the
amount of immunoprecipitated FER.
To test whether the increased phosphorylation of the kinase is
associated with a rise in its activity, FER was immunoprecipitated from
lysates obtained from isotonically or hypertonically treated cells, and
in vitro kinase assays were performed using angiotensin II
as a preferred FER substrate (15). Cell shrinkage resulted in  70%
increase in FER activity (Fig.
2A, panel WT), an
activation that is similar in magnitude to the effect of
platelet-derived growth factor-induced FER stimulation is Swiss 3T3
cells (15). Taken together, these data show that FER is an
osmosensitive kinase that is both phosphorylated and activated by a
decrease in cell volume.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
The contribution of Fyn kinase to the osmotic
activation (A) and phosphorylation
(B) of FER kinase. A, FER kinase was
immunoprecipitated (IP) from equal amount of lysates
obtained from isotonically (I) or hypertonically
(H, 300 mM sucrose, 10 min) treated WT and
Fyn-deficient (Fyn/) fibroblasts, as in Fig. 1. FER activity was
determined by an in vitro kinase assay using angiotensin II
as substrate (see "Experimental Procedures"; n = 4 for each condition; p < 0.01 for isotonic medium
versus hypertonic medium in the WT). B, FER
immunoprecipitates prepared from the same cell types and from
Src-deficient (Src/) fibroblasts were probed with
antiphosphotyrosine (anti-PY) and then with anti-FER.
WB, Western blotting.
|
|
The Involvement of Fyn Kinase in the Osmotic Activation of
FER--
The upstream signaling pathways leading to FER activation are
not known. Because we have shown that Fyn kinase is involved in the
osmotic phosphorylation of cortactin (6), it was conceivable that Fyn
and FER constitute elements of the same signaling pathway or,
alternatively, their osmotic activation may be due to independent routes. To address this question, first we tested whether PP2, a Src
family inhibitor, interferes with the osmotic FER phosphorylation. The
structural basis of the selectivity of PP2 is that it binds to a
threonine residue shared among Src kinases but absent in most other
nonreceptor tyrosine kinases (31). Fig. 1B shows that the
drug completely abolished the hypertonic FER phosphorylation. To assess
the specific involvement of Fyn, we compared the hypertonicity-induced phosphorylation in wild type, Fyn-deficient, and Src-deficient cells
(Fig. 2B). In Fyn/ cells, the shrinkage-induced FER
phosphorylation was strongly reduced compared with the wild type. In
contrast, in Src/ cells (which have been shown to overexpress Fyn
(6)), FER phosphorylation was increased even under isotonic conditions, and it was further stimulated by hypertonicity. The observed
differences in phosphorylation were not due to differences in FER
expression, because a similar amount of FER was immunoprecipitated from
each cell type (Fig. 2B, lower panel).
Importantly, the differential phosphorylation was in complete agreement
with direct activity measurements; the basal FER activity was the same
in the wild type and Fyn/ cells, but hypertonicity failed to elicit
a significant rise in the latter cell type (Fig. 2A).
Collectively these data suggest that Fyn kinase is one of the major
upstream contributors to the hypertonic FER activation, although its
role is not exclusive in this process. On the other hand, Src kinase is
not necessary for FER stimulation.
The impaired osmotic responsiveness observed in Fyn/ cells may be a
general feature of this cell line (which would suggest a defect in the
putative volume sensor), or it may be specifically restricted to the
FER pathway. To answer this question, we investigated whether two well
known osmotic responses, the activation of the ERKs (4) and the
hypertonic stimulation of the Na+/H+ exchanger
(29), were also altered in the Fyn/ cells. Shrinkage induced strong
and essentially similar activation of ERK-2 in WT and Fyn/ cells.
In accordance with this, similar ERK phosphorylation was observed in
both cells, when whole cell lysates were probed with a phospho-specific
ERK antibody (Fig. 3A). Fig.
3B shows that hyperosmolarity induced a sizeable cytosolic
alkalinization in both cell types, and the response was entirely
blocked by HOE-694, a highly specific inhibitor of the NHE-1 isoform of
the exchanger. Neither the rate nor the extent of the intracellular pH
change was different in the two cell types. Taken together, these
results show that Fyn/ cells are capable of responding to osmotic
stress, and their refractoriness seems to be specific for the osmotic FER activation. Moreover, neither Fyn nor FER appear to be involved in
the shrinkage-elicited activation of ERK-2 or NHE-1.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Fyn-deficient cells remain viable and
osmotically responsive. A, WT and Fyn/ cells were
treated with isotonic (I) or hypertonic (H,
Iso-Na plus 300 mM sucrose) solutions for 10 min and lysed.
Aliquots of the whole cell lysates were subjected to electrophoresis
and subsequent Western blotting (WB) using a
phospho-specific ERK antibody (upper panel). The majority of
the lysate was processed for immunoprecipitation using an anti-ERK-2
antibody. Immunocomplex kinase assays were performed applying myelin
basic protein (MBP) as substrate. The complexes were
subjected to SDS-polyacrylamide gel electrophoresis, followed by
autoradiography (lower panel). B, WT and
Fyn-deficient cells were grown on glass coverslips and loaded with the
fluorescent indicator BCECF. Intracellular pH was monitored on small
cell populations consisting of 6-12 fibroblasts. Where indicated by
the arrow (HYP), the isotonic sodium medium was
replaced by a hypertonic solution (Iso-Na plus 300 mM
sucrose). To verify that the observed intracellular alkalinization was
mediated by the Na+/H+ exchanger, cells were
first pretreated for 1 min with 5 µM HOE 694, a selective
blocker of NHE-1, and then challenged with the hypertonic medium
supplemented with the same concentration of the inhibitor.
|
|
The Role of FER in Cortactin Phosphorylation--
Although the
above observations were suggestive of the involvement of FER in the
osmotic cortactin phosphorylation, they did not explicitly show that
FER is capable of mediating this reaction in situ. To gain
further insight into the relationship of FER and cortactin, we first
tested whether these molecules can form a complex in the lysates
obtained from isotonically and hypertonically treated cells. To this
end, the immunoprecipitates obtained using the anti-FER-2 antibody were
reprobed with anti-cortactin. The lower panel of Fig.
1C shows the anti-FER-2 precipitates contained cortactin.
The amount of co-precipitated cortactin was similar in control and in
hypertonically treated samples, suggesting that these proteins bind
each other constitutively and probably independent of FER activation.
This finding is in agreement with recent observations made on
platelet-derived growth factor-stimulated 3T3 cells (14). These
observations, however, do not exclude the possibility that the
association occurred only after cell lysis. To test the potential in situ functional relationship between these proteins, we
performed co-transfection experiments. For these studies, we used CHO
cells because the embryonic fibroblasts proved to be poorly
transfectable. To establish whether the overexpression of FER affects
cortactin phosphorylation, the cells were transfected with a cDNA
encoding for the wild type Myc-tagged cortactin alone or along with
cDNAs for the hemagglutinin-tagged FER kinase. Two days after
transfection, the cells were treated with isotonic or hypertonic
solutions, and lysates obtained from these samples were
immunoprecipitated with an anti-Myc antibody. The precipitates were
then probed with anti-phosphotyrosine, anti-Myc, and anti-cortactin.
Fig. 4A shows that, when
expressed alone (or with the empty pCMV vector), Myc-tagged cortactin
behaved exactly as its endogenous counterpart; it was not
phosphorylated under iso-osmotic conditions and became phosphorylated upon hypertonic treatment (first two lanes). Moreover,
immunofluorescence analysis confirmed that the intracellular
distribution of the endogenous and the heterologously expressed
cortactin was indistinguishable (not shown). When FER and cortactin
were co-expressed, strong phosphotyrosine accumulation was detected in
cortactin even under isotonic conditions, and the reaction was further
enhanced in hyperosmotically challenged cells (Fig. 4A,
last two lanes). It is important to note that the
hyperosmotic component of the total cortactin phosphorylation in these
FER-transfected cells was consistently many-fold higher than the level
observed in cells transfected with cortactin alone. This finding
suggests that the increase cannot be accounted for by the osmotic
activation of endogenously expressed tyrosine kinases, implying that
hypertonicity was able to stimulate the transfected FER kinase, and
this effect contributed to the hypertonic cortactin phosphorylation.
Probing the immunonoprecipitates with anti-Myc verified that the
differences in phosphorylation were not due to differential
Myc-cortactin expression (Fig. 4A, lower panel,
and see below). In fact, hyperphosphorylation of cortactin was, in some
experiments, accompanied with a somewhat decreased cortactin level (see
e.g. last lane), a finding that is probably related to the
phosphorylationdependent degradation of this molecule (32). We
also considered the possibility that the robust tyrosine
phosphorylation in FER transfectants could be partially due to the
autophosphorylation of FER that might co-precipitate with cortactin.
However, this is unlikely, because no anti-HA labeling was detected in
the anti-Myc immunoprecipitates (not shown), and the phosphorylated
band perfectly colocalized with cortactin. To assess whether the
response was specific to FER or whether it could be a consequence of a
general increase in tyrosine kinase expression, we co-transfected the
cells with Myc-cortactin and wild type focal adhesion kinase. Although
this maneuver resulted in a huge overexpression of this kinase, it failed to induce cortactin phosphorylation under isotonic conditions (not shown). Because transfection with FER triggered cortactin phosphorylation in otherwise nonstimulated cells, we hypothesized that
some of the upstream signaling pathways to FER may be constitutively active, and this is sufficient to cause cortactin phosphorylation when
FER is overexpressed. In support of this notion, we found that
rendering the cells quiescent by an 18-h serum deprivation largely
reduced the FER-induced basal cortactin phosphorylation, without
affecting cortactin expression (data not shown).
View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
The potential involvement of FER kinase in
the osmotic phosphorylation of cortactin. A, CHO cells
at approximately 40% confluence were transfected with a construct
encoding for Myc-cortactin (1 µg of DNA/10-cm dish) along with an
empty vector or the wild type FER or the kinase-dead FER mutants K591R
(1 µg of DNA/10-cm dish). 48 h later, the cells were treated
with isotonic (I) or hypertonic (H) solutions for
10 min and lysed. Cell lysates were subjected to immunoprecipitation
(IP) using a monoclonal anti-Myc antibody. The precipitates
were then probed with anti-phosphotyrosine (anti-PY;
upper panel) and reprobed with anti-Myc (lower
panel) and anti-cortactin (not shown). B, the
hyperosmotic cortactin phosphorylation observed in control and K591R
FER-transfected cells was quantified by densitometry and normalized to
the level of Myc expression. 100% corresponds to the
phosphotyrosine:Myc density ratio found in hypertonically treated cells
transfected with Myc-cortactin + empty vector (n = 5, p < 0.01). C, cells were transfected with a
construct encoding either for wild type Myc-cortactin
(Myc-cort) or a mutant Myc-cortactin (Myc-Pcort)
in which three tyrosine residues (421, 466, and 482) were replaced by
phenylalanine. After 2 days, the cells were treated with isotonic or
hypertonic solutions, and the tagged cortactin proteins were
immunoprecipitated with the anti-Myc antibody. The precipitates were
tested with antiphosphotyrosine (upper panel) and
subsequently with anti-Myc antibodies (lower panels). Note
that P-cortactin migrates slightly further than the WT. D,
the cells were transfected with Myc-cortactin or Myc-Pcortactin and,
where indicated, with the wild type FER. After isotonic and hypertonic
treatment, anti-Myc immunocomplexes were obtained as above, and the
precipitates were probed with antiphosphotyrosine and later by
anti-Myc. WB, Western blotting.
|
|
Next we asked whether a kinase inactive mutant of FER (K591R) might
interfere with the hypertonic effect. There is a controversy in the
literature as to whether this kinase-dead mutant can exert a potent
dominant negative effect (15, 17), although it has been reported to
inhibit the colony-stimulating factor-induced phosphorylation of
cortactin in the cytosolic fraction (15). In our system, FER K591R
failed to abolish hypertonic phosphorylation. However, we consistently
observed that this construct significantly increased the expression of
the co-transfected Myc-cortactin, perhaps by increasing the stability
of cortactin (Fig. 4A, middle two lanes). When
the level of the hypertonicity-induced phosphorylation was normalized
to Myc-cortactin expression (Fig. 4B), cells transfected with the kinase-dead FER showed a partial but significant reduction (54.8 ± 5.9%, n = 5) in hypertonic cortactin
phosphorylation. After similar normalization, the expression of
kinase-active FER resulted in a 22.8 ± 5.5-fold
(n = 8) increase in the hypertonic cortactin phosphorylation.
To further substantiate the potential role of FER in the osmotic
cortactin response, we investigated whether cortactin phosphorylation induced by hypertonicity or by FER overexpression may occur on the same
tyrosine residues. Previous studies indicated that more than 90% of
the Src-catalyzed, in vitro phosphorylation of cortactin takes place at three critical tyrosine residues, namely
Tyr421, Tyr466, and Tyr482.
Substitution of these amino acids with phenylalanine resulted in a
dramatic decrease in phosphorylation and prevented the concomitant change in the actin cross-linking capability (11). To decide whether
these same residues are involved in the osmotic response, we
transfected the cells with Myc-tagged wild type cortactin, or with a
mutant version, P-cortactin, in which the above-mentioned tyrosine
residues were exchanged for phenylalanine. Hyperosmolarity induced a
strong phosphorylation in WT cortactin, whereas the response was almost
entirely missing in P-cortactin (Fig. 4B). Importantly, the
expression levels of the WT and the mutated cortactin were similar.
These findings suggest that the tyrosine residues 421, 466, and 482 can
be the major targets of the osmotic effects, as well. Next, we compared
the phosphorylation of WT and P-cortactin in control and
FER-transfected cells, under isotonic and hypertonic conditions. As
shown on Fig. 4C, the FER overexpression-induced phosphorylation of WT cortactin was much stronger than the
phosphorylation of P-cortactin. Similarly, the additional
phosphorylation in response to hyperosmolarity was much more pronounced
in the WT than in P-cortactin. These findings imply that FER
overexpression induces phosphorylation of cortactin predominantly (but
not exclusively) on the same tyrosine residues that are the major
targets for the hyperosmotic phosphorylation.
Considered together, these studies have demonstrated that active FER
induces cortactin phosphorylation in situ, and this reaction is further stimulated by hypertonicity. Moreover, kinase-inactive FER
appears to reduce cortactin phosphorylation, and the same tyrosine
residues are critical for the phosphorylation evoked by shrinkage or
FER expression. Thus, FER, presumably downstream from Fyn, seems to be
involved in the osmotic phosphorylation of cortactin.
Cell Volume Affects the Phosphorylation of Constituents of the
Cell-Cell Contact Sites--
FER kinase has recently been implicated
as an important contributor to the phosphorylation of key components of
the cell-cell attachment apparatus, including -catenin and
p120Cas (15, 18). In addition, activation of Fyn kinase was
reported to enhance the phosphorylation of -catenin (23), another
member of the cell contact machinery that associates with the
cadherin--catenin complex and anchors it to the actin
skeleton. It was therefore conceivable that hypertonicity, through the
Fyn/Fer pathway, might promote the tyrosine phosphorylation of these
proteins. To test this hypothesis, we immunoprecipitated the
corresponding molecules from CHO cells and probed the precipitates with
antiphosphotyrosine antibodies. Fig.
5A shows that upon hypertonic
treatment two protein bands (90 and 105 kDa) became tyrosine
phosphorylated in the anti--catenin immunoprecipitates. Reprobing
the blot with anti--catenin indicated that the lower molecular
mass band comigrated with -catenin. Because -catenin and
-catenin are known to form a complex (19, 20), we reprobed the same
blot with anti--catenin as well. Indeed, -catenin was present in
these immunoprecipitates, and it comigrated with the higher molecular
mass band. No major change was detected in the amount of -catenin
that co-precipitated with -catenin in isotonic and hypertonic
samples. To verify that hyperosmolarity induces -catenin
phosphorylation, this protein was directly immunoprecipitated with the
corresponding antibody. As shown on Fig. 5B, osmotic shock
triggered phosphotyrosine accumulation in -catenin. Similar results
were obtained when p120Cas was tested. This molecule also
became tyrosine phosphorylated upon cell shrinkage (Fig.
5C). Thus, we have identified three novel targets for
volume-dependent tyrosine phosphorylation, each of which is
an essential component of the cell contact apparatus.
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Hypertonicity induces phosphorylation of
cell-cell contact proteins -catenin,
-catenin, and p120Cas. CHO cells
were preincubated under isotonic conditions and then further exposed to
isotonic (I) or hypertonic (H) medium (Iso-Na + 300 mM sucrose) for 10 min. In certain experiments (shown
in A, suspended lanes), cells were first detached
using EDTA, and the cell suspension was challenged with isotonic or
hypertonic treatment, as described under "Experimental Procedures."
Where indicated, PP2 was present in the isotonic preincubation for 10 min and during the hyperosmotic exposure. Following cell lysis,
-catenin (A, -cat), -catenin
(B, -cat), and p120Cas
(C) were immunoprecipitated (IP), and
immunocomplexes were subjected to electroctrophoresis on 7.5%
(A and B) or 10% (C) gels. The
separated proteins were probed first with antiphosphotyrosine and then
with the immunoprecipitating antibody. The -catenin immunocomplexes
were also examined with anti--catenin antibodies (lower
panel in A). WB, Western blotting.
|
|
These observations raised the question of whether the phosphorylation
of these proteins is dependent on existing cell-cell contacts or
whether the phenomenon occurs in individual cells as well. Adherens
junctions have been reported to transmit tensile stress between
contracting fibroblasts (33). Thus, it was conceivable that the local
tension generated when neighboring cells start shrinking and move away
from each other may be necessary for the tyrosine phosphorylation of
junctional components. To address this possibility, we compared
tyrosine phosphorylation of - and -catenin in confluent
monolayers and in cell suspensions. To obtain a suspension of
individual cells, the contacts were destroyed by treating the
monolayers with the Ca2+ chelator, EDTA (5 mM
for 10 min), followed by vigorous pipetting, until most cells were
separated from their neighbors as assessed by light microscopy.
Hyperosmolarity induced tyrosine phosphorylation of - and
-catenin both in attached and in suspended cells (Fig. 5A, attached and suspended lanes).
Although the level of phosphorylation appeared slightly less in the
suspended culture than in the attached monolayer, the individual cells
remained clearly responsive to shrinkage. Moreover, at least during
these short term experiments, neither the EDTA-induced disruption of
the cell contacts nor the subsequent shrinkage affected significantly
the binding of -catenin to -catenin.
To test whether the shrinkage-induced tyrosine phosphorylation of the
catenins were mediated by Src family kinases, we used two approaches.
First, we applied the Src family inhibitor, PP2; the drug completely
prevented the hypertonic phosphorylation of each protein (see Figs.
5C and 7A, and not shown for -catenin). Second, we compared the responses in WT and Fyn-deficient fibroblasts. Probing the anti--catenin (Fig.
6A) and anti--catenin
immunoprecipitates (Fig. 6B) with antiphosphotyrosine
antibodies revealed that the phosphorylation of both proteins was
strongly mitigated in the Fyn/ cells.
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
Osmotic phosphorylation of
-catenin and -catenin is
Fyn-dependent. WT and Fyn-deficient fibroblasts were
exposed to isotonic (I) or hypertonic (H) medium
for 10 min. After cell lysis, -catenin (A,
-cat) and -catenin (B,
-cat) were immunoprecipitated (IP)
using the corresponding antibodies. The immunocomplexes were
electrophorosed on 7.5% SDS gels, blotted onto nitrocellulose, and
probed with antiphosphotyrosine (anti-PY) and with the
immunoprecipitating antibodies. The arrows on the
phosphotyrosine blot show where -catenin and -catenin migrate.
WB, Western blotting.
|
|
Volume-dependent Redistribution of
-Catenin--
Tyrosine phosphorylation of the components of the
adherens junctions is known to be associated with significant
reorganization of these structures. In various epithelial cells,
phosphorylation of -catenin was reported to accompany the disruption
of the contacts and the dissociation of its constituents (20, 25-28).
However, in keratinocytes, tyrosine phosphorylation of -catenin was
correlated with the assembly of the junctions (23). Little information is available about the localization and regulation of -catenin in
fibroblastic cells. Therefore, we used immunofluorescence staining to
examine the distribution of -catenin in our cell lines under resting
conditions and after hypertonic challenge.
Under isotonic conditions, confluent CHO cells exhibited a pronounced
cytosolic staining, together with marked -catenin enrichment along
the attaching cell surfaces. After hypertonic exposure for 10 min, the
peripheral -catenin staining almost completely disappeared (Fig.
7B). When the hypertonic
treatment was applied in the presence of the Src family inhibitor PP2
(which abrogates the tyrosine phosphorylation of -catenin (Fig.
7A)), the reduction in the peripheral staining was
substantially mitigated; after 10 min, many of the cell-cell contact
areas were still visualized by the antibody (Fig. 7B).
Dissociation of the components of the adherens junction might be the
result of uncoupling between cadherins and -catenin and/or between
-catenin and -catenin (20, 26-28). Because hypertonicity reduced
the peripheral -catenin staining, we examined whether the
cadherin-catenin binding might be affected by osmotic stress. In
agreement with earlier observations (34), we found that the only major
cadherin expressed in CHO cells is N-cadherin. This protein was
precipitated from lysates of isotonically and hypertonically treated
cells, and the precipitates were probed with anti-N-cadherin and
anti--catenin antibodies (Fig. 7C). Although the amount
of precipitated N-cadherin was similar in the isotonic and hypertonic
samples, significantly less -catenin associated with N-cadherin in
lysates from osmotically challenged cells. This observation supports
the notion that hypertonicity weakens the binding between N-cadherin
and -catenin in CHO cells. Nevertheless, the co-immunoprecipitation
method appeared to be less sensitive in detecting the alteration in
cadherin/catenin association than the immunfluorescence studies
performed on intact cells. Sizable and reproducible dissociation was
detected only after longer (20-30 min) hyperosmotic stress, and at
this time the uncoupling was not effectively inhibited by PP2. This
observation suggests that tyrosine phosphorylation is not the exclusive
mechanisms whereby hypertonicity causes redistribution of junctional
proteins.
View larger version (59K):
[in this window]
[in a new window]
|
Fig. 7.
Hypertonicity-triggered reorganization
of -catenin in CHO cells and the role of
tyrosine phosphorylation in the process. A, CHO cells
were exposed for 10 min to isotonic (ISO) or hypertonic
(HYP) medium in the presence or absence of PP2 (10 µM). -Catenin (-cat) was
immunoprecipitated from the lysates obtained from these differentially
treated cells, and the immunocomplexes were probed with
antiphosphotyrosine and anti--catenin antibodies. B,
confluent CHO cell cultures grown on coverslips were challenged exactly
as in A and then fixed in the same isotonic or hypertonic
medium supplemented with 4% paraformaldehyde. After extensive washing,
the cells were permeabilized, blocked, and stained with an
anti--catenin primary and a fluorescein isothiocyanate-labeled
anti-Goat IgG secondary antibody. C, cells were treated with
isotonic or hypertonic medium for 30 min in the presence or absence of
PP2. N-cadherin was immunoprecipitated from the lysates, and the
immunocomplexes were analyzed for the presence of N-cadherin
(upper panel) and -catenin (lower panel) using
the corresponding specific antibody. WB, Western blotting;
PY, phosphotyrosine; IP,
immunoprecipitation.
|
|
To further substantiate the effect of osmotic shock on -catenin
distribution and to assess the potential role of Fyn in this process,
we performed experiments using WT and Fyn/ embryonic fibroblasts
(Fig. 8). Under resting conditions, these
fibroblasts show little cytosolic labeling, and -catenin distributes
predominantly in sharp, continuous lines along the periphery of the
neighboring polygonal cells. The morphology and catenin distribution of
these cells is quite epitheloid-like, suggesting that they represent an
early phase of the epithelial-mesenchymal transition. Although under
isotonic conditions -catenin distribution was similar in WT and
Fyn/ cells, their response to hypertonicity differed. In WT cells,
a 10-min hypertonic exposure induced major changes; the peripheral
staining became much weaker and discontinuous, whereas diffuse, often
punctate, cytosolic labeling occurred. After 30 min, the cell contacts
were hardly visible (Fig. 8, upper panels). In contrast, in
Fyn/ cells, hypertonic challenge for 10 min resulted in only a
minor widening of the peripheral staining, and most cells remained
labeled in a sharp polygonal pattern. After 30 min, the peripheral
labeling became weaker and less distinct, and more cytosolic staining
was present. Nevertheless, the cell borders were still clearly
distinguishable. (Fig. 8, lower panels). Treatment of
Fyn/ cells with PP2 still further reduced but did not prevent the
dissociation of -catenin. Similar observations were made when
-catenin distribution was followed (not shown).
View larger version (94K):
[in this window]
[in a new window]
|
Fig. 8.
Comparison of the hypertonicity-induced
changes in -catenin distribution in WT and
Fyn/fibroblasts. WT and Fyn/ cells were grown to
confluence on glass coverslips. The cells were then exposed to isotonic
or hypertonic solution for 10 or 30 min, as indicated. Immunostaining
for -catenin was performed exactly as in Fig. 7B.
|
|
Taken together, these findings suggest that hyperosmolarity induces
redistribution of -catenin in CHO cells and embryonic fibroblasts.
Tyrosine phosphorylation accelerates and augments this process, and Fyn
kinase is an important contributor to the phenomenon. However, the
involvement of other tyrosine kinase-dependent (PP2-sensitive) and -independent (PP2-insensitive) mechanisms also seem
to play an important role in the process.
| |
DISCUSSION |
The primary goal of this study was to explore osmotically
responsive signaling pathways that may serve as potential links between
changes in cell size and the organization of the cytoskeleton. Our
major findings show that 1) FER kinase is a volume-sensitive enzyme
that is tyrosine phosphorylated and activated upon cell shrinkage; 2)
the Src family member Fyn kinase is an important upstream mediator of
the osmotic FER response; 3) hypertonic stress induces the tyrosine
phosphorylation of key substrates of Fyn and FER, including elements of
the cortical skeleton (cortactin), and constituents of the cell-cell
contact apparatus (- and -catenin and p120Cas); and
4) cell shrinkage triggers the redistribution of -catenin, and this
reaction appears to be related to the increased tyrosine phosphorylation of the molecule.
Potential Mechanism of Osmotic FER Activation and Its Role in
Hypertonic Signaling--
To our knowledge, this is the first report
implicating a Src family kinase, specifically Fyn, in the activation of
FER. This conclusion is based on our findings that PP2, a selective Src family inhibitor, abolishes osmotic FER phosphorylation, and the shrinkage-induced FER response is substantially mitigated in
Fyn-deficient cells but remains intact in Src-deficient cells. Little
is known about the upstream activators of FER. Epidermal growth factor and platelet-derived growth factor were reported to induce its phosphorylation and association with the respective receptors (15).
These findings suggest that receptor tyrosine kinases, directly or
indirectly, are involved in FER activation. It is noteworthy that
hyperosmolarity was shown to elicit clustering of growth factor
receptors, an effect that might result in partial receptor activation
even in the absence of the corresponding ligands (35). Importantly,
these receptors can also bind Fyn and phosphorylate it on a residue
(Tyr28) unique to this Src kinase (36). Thus, it is
conceivable that hypertonicity, through the ensuing growth factor
receptor aggregation, recruits Fyn and FER, and the former kinase
potentiates the phosphorylation of the latter. The residual,
Fyn-independent phosphorylation of FER might be due to
autophosphorylation or could be catalyzed by the receptor itself. In
any case, the Fyn-related phosphorylation seems to be the predominant
mechanism in the osmotic FER stimulation.
Once activated, FER can relay and amplify the response by acting on its
substrates. Of these, we assessed whether the Fyn-dependent FER activation is involved in the hypertonic cortactin phosphorylation. Several findings are consistent with the participation of FER in this
phenomenon: 1) FER coprecipitated with cortactin, suggesting a direct
interaction between these proteins; 2) overexpression of FER induced
cortactin phosphorylation even under isotonic conditions, and this
reaction was strongly potentiated by hyperosmolarity; 3) the same
tyrosine residues (421, 466, and 482) were required for the
hypertonicity-induced responses that were predominantly targeted upon
FER overexpression; and 4) kinase-inactive FER reduced the osmotic
cortactin phosphorylation. Several possibilities may account for the
observation that the kinase-dead FER had only a partial inhibitory
effect on the hypertonic cotactin response. First, it has been reported
that, in various systems, neither kinase inactive FES (37) nor
kinase-dead FER (17) was able to exert potent dominant negative
effects. The likely reason is that these kinases act in an oligomeric
form (16, 17), and incorporation of one or more kinase-inactive
molecule into the complex does not interfere significantly with the
overall activity. Thus, very high levels of kinase-defective mutant may
be required for substantial suppression of FER activity. Moreover,
strong reduction in the colony stimulating factor-induced cortactin
phosphorylation by K591R FER was observed only in a cytosolic fraction
(15). This suggests that negative FER may preferentially affect certain cortactin populations. Finally, in addition to activating FER, Fyn
kinase may also be involved in the direct phosphorylation of cortactin.
The recognition of the osmotic sensitivity of FER may help interpret
earlier findings regarding hypertonic signaling. Specifically, osmotic
shock was reported to cause the activation of STATs (signal transducers and activators of
transcription) 1 and 3 (38, 39). The response was partially
mediated by Janus kinases (Jak1 and 2) and partially by an as yet
unidentified tyrosine kinase(s) (39). The Fyn/FER pathway may have a
role in both mechanisms. Regarding the Jak-dependent route,
Abe and Berk (40), using the same cell type as we did, have recently
reported that Fyn is an important upstream component in the oxidative
stress-induced Jak2 activation. Because Fyn is activated by osmotic
shock as well (6), it is very likely to be involved in the hypertonic Jak activation. Moreover, the FER-related c-Fes has been shown to
phosphorylate STAT3, in a Jak-independent manner (41). If FER can
catalyze a similar reaction, it may be the unidentified, Jak-independent kinase for the
STATs.2
Volume-dependent Phosphorylation of Elements of the
Adherens Junctions and the Cortical Cytoskeleton--
Perhaps the most
intriguing novel observation of this study is that hyperosmolarity
provokes the Fyn-dependent phosphorylation of cell-cell
contact proteins. Tyrosine phosphorylation has been long recognized as
one of the central mechanisms in the regulation of adherens junctions
(see Ref. 20 for a review), but neither its exact role nor the identity
of the responsible kinases has been entirely elucidated. Overexpression
of oncogenic Src proteins (21, 22, 25) or FER (18), as well as the
inhibition of tyrosine phosphatases (27, 28), provoke phosphorylation
and concomitant disassembly of the contacts. Growth factor-induced phosphorylation of -catenin has also been correlated with contact disruption (43), suggesting that this mechanism has physiological relevance, as well. On the other hand, Fyn was also implicated in the
normal assembly of the cell-cell junctions in keratinocytes (23). Novel
observations however strongly suggest that the catalytic activity of
Src kinases plays an important role in the dissociation of the contacts
in normal cells. Owens et al. (24) found that pharmacologic
or genetic interference with the basal activity of Src kinases
stabilized cell contacts and abrogated the dissociation of single cells
from epithelial sheets. The biochemical basis of such an effect has
also begun to emerge; Roura et al. (28) showed that Src
phosphorylates -catenin in vitro on Tyr86 and
Tyr654 and that the latter phosphorylation results in
decreased E-cadherin--catenin binding. The reaction did not alter
the -catenin--catenin association. Furthermore, because Src
phosphorylated Tyr654 with low efficiency, it was concluded
that in situ the reaction might be catalyzed by another
kinase, e.g. by FER (28). Our results are entirely
consistent with the above scenario; cell shrinkage activated Fyn and
FER and caused increased catenin phosphorylation and the dissociation
of -catenin from the contact sites. In addition, shrinkage appeared
to promote the uncoupling of N-cadherin from -catenin, whereas we
could not detect any change in the -catenin--catenin binding. The
involvement of tyrosine phosphorylation in -catenin redistribution
is evidenced by the mitigation of the process in Fyn/ or
PP2-treated cells. However, caution should be exercised when
interpreting these data. First, we have no formal evidence that the
membrane-associated -catenin pool was (also) phosphorylated. In
favor of this possibility, our preliminary observations show that FER
translocates to the membrane upon shrinkage (not shown). Another caveat
is that tyrosine phosphorylation does not seem to be an absolute
requirement. Rather it has a synergistic effect; although the process
is slower, dissociation still occurs also in Fyn/ and PP2-treated
cells. Tyrosine phosphorylation accelerates disassembly and may make it
irreversible, but other consequences of |
TOP
ABSTRACT
INTRODUCTION