Cell Volume-dependent Phosphorylation of Proteins of the Cortical Cytoskeleton and Cell-Cell Contact Sites

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 β-catenin, α-catenin, and p120Cas and caused the dissociation of β-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.

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)(2)(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)(8)(9)(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)(16)(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, p120 Cas (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)(22)(23)(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.
Media-Bicarbonate-free RPMI 1640 was buffered with 25 mM Hepes to pH 7.4 (osmolarity 290 Ϯ 5 mosM). The isotonic sodium medium (Iso-Na) consisted of 140 mM NaCl, 3 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 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 MgCl 2 , 1 mM CaCl 2 , 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 NaHCO 3 and supplemented with 10% fetal calf serum, and 1% antibiotic suspension (penicillin and streptomycin, Sigma), under humidified atmosphere of air/CO 2 (19:1) at 37°C. Wild Type (WT), FynϪ/Ϫ, SrcϪ/Ϫ 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 treat-ments 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 FuGene TM 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 HCO 3 -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 Na 3 VO 4 and 20 l/ml protease inhibitor mixture. To obtain cell suspension from cultured cells, confluent monolayers were treated with Ca 2ϩ and Mg 2ϩ -free Iso-Na medium supplemented with 5 mM Na 2 EDTA (5 ml/10-cm dish). After 10 min, the detached cells (Ͼ90%) 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 ϫ g for 10 min, precleared for 1 h using 40 l 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 l of protein G-Sepharose, and the beads were washed four times with lysis buffer containing 1 mM Na 3 VO 4 . 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 [Val 5 ]-angiotensin II as substrate (15). Cell lysates obtained from iso-or hypertonically treated WT and FynϪ/Ϫ cells and containing equal amount of protein (500 -1000 g) were subjected to immunoprecipitation using the anti-FER2 antibody. The precipitates were washed with kinase buffer (50 mM Tris-HCl and 10 mM MnCl 2 , pH 7.7) supplemented with 0.25 mM Na 3 VO 4 . The beads were then incubated for 10 min at 30°C in 25 l of kinase buffer containing 5 mM [Val 5 ]-angiotensin II, 5 M ATP (potassium salt), and 10 Ci of [␥-32 P]ATP/sample. The reaction was terminated by the addition of 35 l of ice-cold stop solution (20% trichloroacetic acid and 5 mM ATP). 35 l 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 g 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 l of assay buffer containing 10 mM MgCl 2 , 50 mM Tris-HCl (pH 7.4), 40 M ATP, and 1.2 Ci of [␥-32 P]ATP. 15 min later the reaction was terminated by 30 l of 2ϫ Laemmli buffer, and the samples were boiled and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.
Immunfluorescence Microscopy-Confluent cultures grown on cover-slips 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 phosphatebuffered 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 pH i were monitored fluorometrically using the indicator dye BCECF, as described before (29). Confluent cultures of WT and FynϪ/Ϫ cells grown on glass coverslips were loaded with 1 M 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 Ϯ 5 nm/440 Ϯ 5 nm), single emission (530 Ϯ 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 pH i 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 Na 2 EDTA 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 Ϯ 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.
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 Ϸ70% 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.
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 activa- 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 M 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. tion 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 FynϪ/Ϫ cells, the shrinkage-induced FER phosphorylation was strongly reduced compared with the wild type. In contrast, in SrcϪ/Ϫ 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 FynϪ/Ϫ 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 FynϪ/Ϫ 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 ϩ /H ϩ exchanger (29), were also altered in the FynϪ/Ϫ cells. Shrinkage induced strong and essentially similar activation of ERK-2 in WT and FynϪ/Ϫ cells. In accordance with this, similar ERK phosphorylation was observed in both cells, when whole cell lysates were probed with a phosphospecific ERK antibody (Fig. 3A). Fig. 3B shows that hyperos-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 (FynϪ/Ϫ) fibroblasts, as in Fig. 1. FER activity was determined by an in vitro kinase assay using angiotensin II as substrate (see "Experimental Procedures"; n ϭ 4 for each condition; p Ͻ 0.01 for isotonic medium versus hypertonic medium in the WT). B, FER immunoprecipitates prepared from the same cell types and from Src-deficient (SrcϪ/Ϫ) fibroblasts were probed with antiphosphotyrosine (anti-PY) and then with anti-FER. WB, Western blotting.

FIG. 3. Fyn-deficient cells remain viable and osmotically responsive.
A, WT and FynϪ/Ϫ 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 ϩ /H ϩ exchanger, cells were first pretreated for 1 min with 5 M HOE 694, a selective blocker of NHE-1, and then challenged with the hypertonic medium supplemented with the same concentration of the inhibitor. molarity 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 FynϪ/Ϫ 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.
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 hemagglutinintagged 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 FERtransfected 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, ac- companied 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).
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 Ϯ 5.9%, n ϭ 5) in hypertonic cortactin phosphorylation. After similar normalization, the expression of kinase-active FER resulted in a 22.8 Ϯ 5.5-fold (n ϭ 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 Tyr 421 , Tyr 466 , and Tyr 482 . 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 p120 Cas (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 (Ϸ90 and Ϸ105 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,FIG. 5. Hypertonicity induces phosphorylation of cell-cell contact proteins ␤-catenin, ␣-catenin, and p120 Cas . CHO cells were preincubated under isotonic conditions and then further exposed to isotonic (I) or hypertonic (H) medium (Iso-Na ϩ 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 p120 Cas (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. 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 p120 Cas 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.
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 Ca 2ϩ 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 FynϪ/Ϫ cells.
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)(26)(27)(28). However, in keratinocytes, tyrosine phosphorylation of ␣-catenin was correlated with the assembly of the junctions (23).

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 M). ␤-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.
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.
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 FynϪ/Ϫ embryonic fibroblasts (Fig. 8). Under resting conditions, these fibroblasts show little cytosolic labeling, and ␤-catenin distrib-utes 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 epithelialmesenchymal transition. Although under isotonic conditions ␤-catenin distribution was similar in WT and FynϪ/Ϫ 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 FynϪ/Ϫ 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 FynϪ/Ϫ 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).
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 p120 Cas ); 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 (Tyr 28 ) 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 FIG. 8. Comparison of the hypertonicity-induced changes in ␤-catenin distribution in WT and Fyn؊/؊fibroblasts. WT and FynϪ/Ϫ 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. 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 factorinduced 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 FERrelated 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-in-duced 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 Tyr 86 and Tyr 654 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 Tyr 654 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 FynϪ/Ϫ 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 FynϪ/Ϫ and PP2-treated cells. Tyrosine phosphorylation accelerates disassembly and may make it irreversible, but other consequences of osmotic shock (e.g. alteration in Ser/Thr phosphorylation (44), activation of small G proteins (45), actin redistribution (46,47), and increase in contractility (48)) might also be essential for the phenomenon. Our preliminary data suggest that hypertonicity induces PP2insensitive serine dephosphorylation of ␤-catenin. 3 This mechanism may also contribute to the observed hypertonic effects, because vascular endothelial growth factor, an agent known to cause decreased cell adhesion, has been shown to cause serine dephosphorylation of catenins (49).
Besides being involved in cell-cell adhesion, ␤-catenin can also function as a transcription factor (50). In the cytosol it can bind to the T cell-factor/lymphoid enhancer factor, and the complex shuttles to the nucleus where it regulates gene expression. Future studies should address the intriguing possibility of whether hypertonicity can induce the expression of genes known to be regulated by ␤-catenin (e.g. ZO-1 (50) a binding partner for both cortactin (51) and ␣-catenin (52)).
Although the physiological role of cortactin tyrosine phosphorylation is not well understood, this condition is usually associated with decreased actin cross-linking (10) and increased cell motility (11,53). Importantly, elevated ␤-catenin phosphorylation was also correlated with increased cell migration (54). It is tempting to speculate that mechanical stimuli, perhaps partially via the Fyn/FER pathway, may contribute to the regulation and coordination of cell motility and cell-cell attachment. Chemical stimuli can switch the cells from an immobile and attached conformation to a mobile and free state. 2 While this work was under revision, a manuscript by Priel-Halachmi et al. (42) was published on the website of the Journal of Biological Chemistry reporting that FER is indeed capable of inducing phosphorylation in STAT3. 3 A. Kapus, unpublished observation.
At the same time, the ensuing changes in cell shape or size may act as an important regulatory feedback that can modify the phosphorylation and organization of the cytoskeletal proteins. The overall contact remodeling may be due to the combined presence of and the interaction between chemical and mechanical stimuli, such as tyrosine phosphorylation and altered tension. It is noteworthy that increased contractility was associated with cortactin phosphorylation (55) and ␤-catenin reorganization (56,57). Moreover, hyperosmolarity was shown to induce myosin light chain phosphorylation (48), suggesting that it may directly affect contractility. Thus, the observed hypertonic responses may be manifestations of regulatory reactions that normally occur in a less intense manner during physiological shape changes. In addition, they may serve as protective measures against osmotic shock, e.g. by uncoupling the shrinking cells and releasing the traction or by reinforcing the cortical skeleton (58). Taken together, the Fyn/FER pathway is one of the signaling routes that may translate alterations in cell volume or shape into changes in cytoskeletal organization and gene transcription.