The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suzuki, T.
Right arrow Articles by Honda, Z.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suzuki, T.
Right arrow Articles by Honda, Z.-i.
The Journal of Immunology, 1998, 161: 3694-3701.
Copyright © 1998 by The American Association of Immunologists

Essential Roles of Lyn in Fibronectin-Mediated Filamentous Actin Assembly and Cell Motility in Mast Cells1

Takeshi Suzuki*, Shunsuke Shoji*, Kazuhiko Yamamoto*, Shigeyuki Nada{dagger}, Masato Okada{dagger}, Tadashi Yamamoto{ddagger} and Zen-ichiro Honda2,*

* Department of Internal Medicine and Physical Therapy, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan; {dagger} Division of Protein Metabolism, Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka, Japan; and {ddagger} Department of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the requirement for c-Src in extracellular matrix (ECM)-mediated fibroblast motility has been well established, the roles of hemopoietic Src family protein tyrosine kinases in leukocyte migration have not been fully elucidated. To address the issue, we analyzed fibronectin (Fn)-mediated adhesion signaling in rat basophilic leukemia (RBL) 2H3 cells overexpressing 1) Csk, 2) a membrane-anchored, gain-of-function Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(-)). Parent RBL2H3 cells, expressing autoactivated c-kit, readily adhered to Fn-coated surface, developed typical leukocyte adhesion machinery (podosome), and migrated toward Fn without cytokine priming, thus provided a simple experimental system to analyze Fn-mediated outside-in signaling. While overexpression of Csk or the Csk mutants did not significantly affect cell adhesion to the Fn surface or {alpha}5 integrin recruitment to the attachment sites, Csk suppressed and mCsk almost abolished Fn-mediated tyrosine phosphorylation of paxillin, filamentous actin assembly to podosomes, and cell migration, but mCsk(-) did not. Coexpression of LynA devoid of C-terminal negative regulatory tyrosine in mCsk cells successfully restored Fn-mediated podosome formation and cell migration. Coexpression of c-Src lacking the C-terminal tyrosine reconstructed podosomes, but could not restore the cell migration regardless of its expression level. Collectively, these observations provide evidence that Src family protein tyrosine kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated cytoskeletal changes leading to cell locomotion in RBL2H3 cells, and they suggest that Lyn and c-Src are differentially involved in cell motility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interactions of leukocyte surface integrins with extracellular matrices (ECMs)3 or with their counter-receptors on endothelial cells are critical steps for inflammatory responses (1, 2, 3, 4). The integrin engagement induces leukocyte adhesion and locomotion (5, 6, 7, 8), augments the release of specific granules and mediators (5, 9, 10, 11), and up-regulates the expression of proinflammatory cytokines (12). A characteristic event following integrin engagement is the assembly of multimolecular adhesion machinery at the cytoplasmic surface of ECM-integrin attachment sites. The most thoroughly characterized example of the adhesion machinery is the focal adhesion (FA) in fibroblasts. FA serves as an attachment site for the long actin filaments and is newly formed in the dynamic cell protrusions (13, 14). FA is composed of integrins, multiple cytoskeletal, and adaptor proteins as well as several signaling molecules, such as protein kinases and small molecular GTPases (14, 15, 16). These characteristics have implicated FA as a key signaling complex in structural integrity and also in cell locomotion.

One mechanism underlying the molecular assembly is tyrosine phosphorylation of cytoskeletal and adaptor proteins, which provides sites for the recruitment of Src homology (SH) 2 domains (16, 17). Evidence has been accumulated for the pivotal roles of c-Src in the phosphorylation events in fibroblasts. In the basal state, c-Src activity is suppressed by the phosphorylation of its C-terminal negative regulatory tyrosine (18), and the reaction is catalyzed by C-terminal Src kinase (Csk) (19, 20, 21). When cells adhere to fibronectin (Fn)-coated surface, c-Src is activated (22, 23) and is redistributed from cytosol to FAs (24). c-Src and FA kinase colocalized at FA seem to co-ordinately phosphorylate cytoskeletal and adaptor proteins, such as paxillin and p130cas, thus amplifying the molecular assembly (15, 17, 25, 26, 27, 28, 29). In addition, c-Srcnull fibroblasts exhibit defective spreading and migration on ECMs, indicating the facilitating roles of c-Src in integrin-mediated fibroblast locomotion (22, 23, 30, 31).

Compared with the established roles of c-Src in the adhesion events in fibroblasts, the functional significance of hemopoietic Src family kinases in the signaling has not been fully explored. Recent studies have revealed that Lyn and Fgr are activated and translocated to cytoskeletal fraction (32) and are colocalized with Syk upon neutrophil adhesion over fibrinogen-coated surface (33). By using Fgr and Hck double-knockout mice, Lowell et al. have successfully shown that these hemopoietic Src kinases are involved in neutrophil spreading and O2- release through various integrins, and that their roles are overlapping (34). However, it has not been fully elucidated whether Src family kinases are required for ECM-mediated assembly of leukocyte adhesion machinery (podosome) (4) and cell motility or whether hemopoietic Src family kinase could transmit signals leading to these events. In the current study we have examined these issues through the analysis of rat basophilic leukemia (RBL) 2H3 mast cell lines overexpressing Csk, a gain-of-function, membrane-anchored Csk mutant (mCsk) (35, 36), and a kinase-defective mCsk (36). We show herein evidence suggesting that Src family kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated podosome formation and cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

All the culture media and Geneticin were purchased from Life Technologies Oriental (Osaka, Japan). FCS was obtained from Equitec (Ingram, TX). Bovine plasma Fn was purchased from Biomedical Technologies (Stoughton, MA). Type I collagen was obtained from Nitta Gelatin (Osaka Japan). Fatty acid-free BSA and Latex beads were purchased from Sigma (St. Louis, MO). Rhodamine-phalloidin was obtained from Molecular Probes (Eugene, OR). Protein G-Sepharose was obtained from Pharmacia-LKB (Uppsala, Sweden).

Antibodies

Anti-integrin {alpha}5 polyclonal Ab, AB1928P, was obtained from Chemicon (Temecula, CA). Anti-paxillin mAb was purchased from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine mAb, 4G10, was obtained from ICN (Costa Mesa, CA). Anti-c-Myc mAb (9E10) was purchased from Boehringer Mannheim (Indianapolis, IN). FITC-conjugated goat anti-rabbit IgG (heavy + light chains) Ab and FITC-conjugated rabbit anti-mouse IgG (heavy + light chains) Ab were purchased from MBL (Nagoya, Japan). Anti-Csk polyclonal Ab was prepared as previously described (19, 20).

Preparation of expression plasmids and stable expression in RBL2H3 cells

RBL2H3 cells were maintained in DMEM supplemented with 10% FCS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin at 37°C in humidified 5% CO2 as previously described (36). RBL2H3 cell lines overexpressing cDNAs for rat Csk, a kinase-defective Csk (Csk(-)) in which Lys222 is replaced with Arg, a mCsk possessing myristylation signal sequence of rat c-Src (c-Src tag: (M)GSNKSKPKDASQRRR), and its kinase-defective form (mCsk(-)) were described previously (36).

To create mutated rat c-Src and human LynA whose corresponding C-terminal amino acids (amino acids 527–536 for rat c-Src and amino acids 505–512 for human Lyn A) possessing the negative regulatory tyrosine were replaced with a myc epitope tag (TSVDEQKLISEEDLN), the SpeI site was artificially introduced at the junctional sites of the cDNAs using QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. The C-terminal short nucleotide sequences were cut out with SpeI, and oligonucleotides coding for the myc epitope tag flanked with SpeI and XbaI were ligated with the cDNAs at the C-termini. The resultant cDNAs, termed {Delta}Src and {Delta}Lyn, were subcloned into pBluescript SK- (Stratagene). Clones without misincorporation of the nucleotides were selected, subcloned into an expression vector, pCAGGS (provided by Dr. Miyazaki) (37), and stably introduced into mCsk-expressing cells by cotransfection with a puromycin-resistant vector. Puromycin-resistant clones were screened by immunoblotting with anti-myc epitope mAb (9E10) (38). Multiple independent cell lines expressing {Delta}Src or {Delta}Lyn were established and used for functional studies.

Cell adhesion experiments

Twenty-four- or 96-well flat-bottom polystyrene plates without chemical or mechanical coating (Iwaki, Chiba, Japan) were coated with various concentrations of Fn in PBS overnight at 4°C. Wells were washed once with PBS, incubated with 20 mg/ml BSA in PBS for 1 h at 37°C for blocking, and again washed twice with PBS and once with DMEM before experiments. Adherent RBL2H3 cells serum starved for 24 h were washed twice with PBS and detached from dishes by treatment with HBSS containing 0.05% trypsin and 0.53 mM EDTA (Life Technologies Oriental) at 37°C for 5 min. Cells were harvested with gentle pipetting with DMEM, and trypsin was immediately neutralized with 0.5 mg/ml soybean trypsin inhibitor. Cells were washed once with DMEM and kept at suspension in DMEM buffered with 10 mM HEPES-NaOH, pH 7.4, at 37°C for 10 min.

To examine cell adhesion to Fn-coated surface, 1 x 105 cells were added to the Fn-coated wells of a 96-well plate and incubated at 37°C for the indicated periods. At the end of the incubation, medium and unbound cells were removed by aspiration, and wells were washed twice with PBS. Adherent cells were fixed and stained with a 0.4% crystal violet/methanol (w/v) solution for 30 min, and absorbance at 595 nm was measured with a microplate reader (model 550, Bio-Rad, Hercules, CA) as previously described (39).

To obtain the cell lysate of Fn-stimulated cells, 1 x 106 cells were added to the Fn-coated wells of a 24-well plate and incubated as described above. At the end of the incubation periods, adherent cells were solubilized with Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM ß-glycerophosphate, and 1 mM PMSF), or with SDS lysis buffer (20 mM Tris-HCl (pH 7.4), 1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM ß-glycerophosphate, and 1 mM PMSF) as previously described (36). Insoluble materials were removed by centrifugation at 12,000 rpm for 10 min, and the supernatant was used as total cell lysate.

Immunoprecipitation

Cells were solubilized with Nonidet P-40 lysis buffer, and total cell lysates were prepared. The total cell lysate was incubated with Abs for 1 h at 4°C, and then with 15 µl of a suspension of protein G-Sepharose beads (50% slurry) for another 1 h at 4°C under continuous rotation. The beads were washed three times with 500 µl of Nonidet P-40 lysis buffer, and bound materials were eluted with 2% SDS sample buffer.

Immunoblotting

Immunoprecipitated proteins or the total cell lysate was subjected to 10% SDS-PAGE, and separated proteins were electrotransferred onto nitrocellulose membranes. Proteins were probed with first Abs and then reacted with horseradish peroxidase- or alkaline phosphatase-conjugated second Abs. Signals were detected by chemiluminescence (ECL Western blotting system, Amersham, Arlington Heights, IL) or by color development as previously described (36).

Cell staining with fluorescence-labeled reagents

Cells cultured on Fn- or type I collagen-coated Lab-Tek chamber slides (Nunc, Naperville, IL) overnight were serum starved for 3 h. The adherent cells were washed twice with PBS and fixed with PBS containing 4% formaldehyde and 5% sucrose for 20 min at 25°C. The fixed cells were rinsed twice with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min at 25°C, and again rinsed twice with PBS. Filamentous actin (F-actin) was stained with rhodamine-conjugated phalloidin for 30 min at 37°C. For immunostaining, cells were incubated with 20 mg/ml BSA in PBS for 30 min at 37°C for blocking and incubated with first Abs in the blocking solution for 1 h. After washing with an excess volume of 0.1% Triton X-100 in PBS, the first Abs were probed with FITC-conjugated second Abs. The samples were mounted, viewed, and photographed with an Olympus BX50 microscope (Olympus, New Hyde Park, NY) equipped with epifluorescent filters.

Loading of cells with Fn-coated microbeads

Cell loading with Fn-coated beads was conducted essentially as previously described (40). Small (mean diameter, 3 µm) or large (mean diameter, 11.9 µm) latex beads were coated with 50 µg/ml of Fn before the assay (40). Cells were cultured on type I collagen-coated Lab-Tek chamber slides (Nunc) overnight and serum starved for 3 h. Then, cells were loaded with the Fn-coated beads at 37°C for the indicated periods in DMEM, fixed, permeabilized, and stained with fluorescent-labeled reagents as described above. To quantitatively examine protein recruitment to the attachment sites, beads whose entire circumference was located within a cell were selected, and the number of the beads whose attachment sites were positively stained with the immunofluorescent reagents was counted.

Cell migration assay

Cell migration assay using a chemotaxis microchamber (41) was performed as described previously (42). The lower wells of a 48-well chemotaxis chamber (Neuroprobe, Pleasanton, CA) were filled with 100 µg/ml of Fn in DMEM or with DMEM alone (control), and a polycarbonate filter (5 µm pore size; Neuroprobe) was layered onto the wells. After assembly of the upper chamber, 50 µl of cell suspension (1 x 106/ml in DMEM) was added to the upper wells. The chambers were incubated for the indicated times at 37°C in humidified 5% CO2. After the incubations, the filter was removed, and cells that remained at the upper surface of the filter were scraped out. The filter was fixed and stained with Diff-Quik (International Reagents, Kobe, Japan), and cells that migrated through pores to the lower side of the filter were counted by light microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adhesion of RBL2H3 cells to Fn-coated surface is not affected by the expression of Csk, mCsk, or mCsk(-)

In preliminary experiments we have confirmed that RBL2H3 cells, expressing constitutively active c-kit, readily attach to and migrate on Fn-coated surface without cytokine priming (43, 44, 45). These characteristics provided a simple experimental system to explore the Fn-induced outside-in signal. To analyze the roles of Src family kinases in the signaling cascade, we used RBL2H3 cell lines overexpressing Csk, a mCsk (36, 46), and a kinase-defective mCsk functions as a dominant negative molecule (36). A representative immunoblotting of RBL2H3 cells expressing these molecules is shown in Figure 1Go.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. Expression of Csk and its mutants in RBL2H3 cells. Cells were lysed with Nonidet P-40 lysis buffer, standardized for protein concentration, and subjected to immunoblotting with polyclonal anti-Csk Ab (lane 1, wild-type RBL2H3; lane 2, Csk transfectant; lane 3, mCsk transfectant; lane 4, mCsk(-) transfectant). The migrated positions of Csk, mCsk, and mCsk(-) are indicated by arrows.

 
We first tested whether Csk and its mutants affect cell adhesion. As shown in Figure 2Go, expression of Csk, mCsk, or mCsk(-) did not alter cell adhesion to the dishes coated with various concentrations of Fn (0.46–10 µg/ml) at 60 min or cell adhesion to 10 µg/ml Fn-coated dishes during incubation periods of 10–80 min. These findings were reproducible in at least two clones for each Csk-based molecule. It was also noted that recruitment of {alpha}5 integrin subunit, a component of mast cell Fn receptor (very late Ag-5) (47, 48), to Fn attachment sites was not inhibited by Csk or its mutants (see Fig. 4Go, B and C). These observations indicate that the changes in Csk activity did not alter RBL2H3 cell adhesion to Fn-coated surface.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. Expression of Csk and its mutants did not alter cell adhesion to Fn-coated surface. A, Parent RBL2H3 cells and cells expressing Csk and its mutants were seeded onto plates coated with various concentrations of Fn. Cells were allowed to adhere for 60 min at 37°C, and the extent of the cell adhesion was examined by staining the adherent cells with crystal violet, as described in Materials and Methods. Data are representative of four separate experiments with almost identical results and indicate the mean of duplicate wells on the same plate. B, Cells were seeded onto plates coated with Fn at 10 µg/ml. After allowing cells to adhere for various time at 37°C, the extent of cell adhesion was quantitated as described in Materials and Methods. Data are expressed as the mean ± SD from five separate experiments performed in triplicate. A significant difference was not detected in each pair of the four clones (p > 0.05) during the incubation periods.

 


View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 4. Immunofluorescence studies of molecular assembly to Fn adhesion sites. A, Cells were cultured on Fn (30 µg/ml)-coated dishes for 24 h, and F-actin accumulated on the ventral surfaces was stained with rhodamine-conjugated phalloidin. a, Wild-type cells; b, Csk transfectant; c, mCsk transfectant; d, mCsk(-) transfectant. Scale bar = 10 µm. B, Cells were plated onto type I collagen (a, e, i, and m, wild-type cells; b, f, j, and n, Csk transfectant; c, g, k, and o, mCsk transfectant; d, h, l, and p, mCsk(-) transfectant) and incubated with small (mean diameter, 3 µm; a–l) or large (mean diameter, 11.9 µm; m–p) beads coated with 50 µg/ml Fn for 30 min. Localization of integrin {alpha}5 (a–d) and tyrosine-phosphorylated proteins (e–h) was detected by indirect immunofluorescent staining. Localization of F-actin was detected by rhodamine-labeled phalloidin (i–l and m–p). In the experiments using small beads (a–l), views were focused on the equator of the bead marked by a white arrowhead. Highlighted black arrowheads indicate other beads bound to the cells. Magnified views were shown in the insets. In the experiments in which large beads were used (m–p), views were focused around the basal attachment sites of the beads. Equators of the beads were drawn as a white circle. Scale bar = 10 µm. C, Beads positively stained for integrin {alpha}5, tyrosine-phosphorylated proteins, and F-actin were scored. The ordinate indicates the percentage of beads positive for immunofluorescence for each molecule. At least 50 beads were scored in each experiment, and data are expressed as the mean ± SD from three separate experiments. *, p < 0.01 compared with parent RBL2H3 cells (RBL-WT) by t test.

 
Csk inhibits adhesion-dependent protein tyrosine phosphorylation

We next explored whether Csk and its mutants affect Fn-mediated protein tyrosine phosphorylation. As shown in Figure 3GoA, adhesion of parent RBL2H3 cells to Fn-coated surface induced tyrosine phosphorylation of about 70 kDa (indicated by double asterisks in Figure 3Go) and about 120 kDa (indicated by single asterisk) proteins, findings consistent with earlier reports (9, 49). In Csk transfectants, these signals were reduced and delayed, and these inhibitory effects were more prominent in mCsk transfectants. Expression of mCsk(-) did not appreciably suppress the approximately 120-kDa signal; rather, it augmented the approximately 70-kDa signal. In addition, a constitutive tyrosine phosphorylation of the approximately 60-kDa protein(s) (indicated by # in Figure 3Go) was observed in mCsk(-) cells. This protein(s) was not identified in the current study, but it might correspond to a v-Src substrate, p62dok (50, 51).



View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 3. Temporal profiles of Fn-mediated protein tyrosine phosphorylation. A, Parent RBL2H3 cells and cells expressing Csk or Csk mutants were kept in suspension for 10 min and allowed to adhere to Fn (30 µg/ml)-coated wells for 10 to 80 min. Adherent cells were lysed in 1% SDS buffer, standardized for protein concentration, and subjected to immunoblotting with anti-phosphotyrosine mAb, 4G10. The migration positions of molecular mass markers (kilodaltons) are shown at the left. Major adhesion-dependent tyrosine phosphorylation signals (~120 kDa (*) and ~70 kDa (**) signals), and a constitutive signal in mCsk(-) cells (#) are indicated by arrows at the right. B, Cells were stimulated as described above. Adherent cells were lysed with 1% Nonidet P-40 lysis buffer, standardized for protein concentration, and immunoprecipitated with 4G10 as described in Materials and Methods. Immunoprecipitated proteins were subjected to immunoblotting with anti-paxillin mAb. The migrated positions of paxillin and Ig heavy chain are indicated by an arrowhead and an arrow, respectively.

 
An adaptor protein, paxillin, has been assumed to be a c-Src substrate (28, 29) and has been presumed to play a crucial role in FA assembly (52). As shown in Figure 3GoB, tyrosine phosphorylation of paxillin was inhibited by Csk and was more profoundly inhibited by mCsk. The mCsk(-) did not exert a negative effect; instead, it augmented the basal phosphorylation level. These findings were reproducible at least two clones for each Csk-based molecule.

Csk inhibits podosome formation and cell locomotion stimulated by Fn

We next explored whether the altered Csk activity affects Fn-mediated formation of adhesion machinery and cell locomotion. As shown in Figure 4GoAa, RBL2H3 cells developed characteristic dot-like F-actin assembly (podosome) at Fn attachment sites. Staining with anti-vinculin Ab revealed colocalization of the F-actin bundles with vinculin (not shown). In Csk- and mCsk-expressing cells, F-actin assembly to podosomes was markedly reduced, even when cells were fully spread (Fig. 4Go, A, b and c), while it was clearly observed in mCsk(-)-expressing cells (Fig. 4GoAd).

To further evaluate the effects of Csk activity on Fn-induced protein assembly, cells were loaded with Fn-coated microbeads for 30 min, and proteins recruited to the attachment sites were studied according to previously reported procedures (40, 53). As shown in Figure 4GoB, a–d, integrin {alpha}5 subunit, a component of mast cell Fn receptor (very late Ag-5) (47, 48), was almost equally recruited to the attachment sites in wild-type cells and in the transfectants. However, the recruitments of tyrosine-phosphorylated proteins and F-actin were affected by Csk and its mutants; in wild-type cells and in mCsk(-)-expressing cells, the attachment sites were positively stained with anti-phosphotyrosine Ab (PY) and with rhodamine-conjugated phalloidin (F-actin), whereas these signals were profoundly diminished in Csk- and mCsk-expressing cells (Fig. 4GoB,e–l). In many cases, F-actin accumulation exhibited dot-like protrusions around the beads (Fig. 4GoB, i and l). The localized F-actin assembly was more clearly visualized in the experiments using large beads (mean diameter, 11.9 µm; Fig. 4GoB, m and p). The dot-like F-actin bundles were developed in wild-type cells and in mCsk(-) cells, while they were hardly detectable in Csk- or mCsk-expressing cells. The number of the beads positively stained with the fluorescent reagents was scored, and the data are summarized in Figure 4GoC. Csk and mCsk inhibited the molecular assembly to the attachment sites in an stepwise manner, whereas mCsk(-) did not.

We next compared Fn-mediated cell migration by the Boyden chamber assay method using Fn as a substratum (54). As shown in Figure 5Go, RBL2H3 cells time-dependently migrated to Fn. Csk inhibited and mCsk inhibited even more the cell migration. The mCsk(-) slightly suppressed the cell migration, but its extent was reproducibly smaller than that in Csk- or mCsk-expressing cells.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. Cell migration toward Fn. Fn-mediated migration of parent RBL2H3 cells and cells expressing Csk, mCsk, or mCsk(-) was studied by Boyden chamber assay using Fn (100 µg/ml) as a substratum. Cells that migrated through the pores (diameter, 5 µm) to the lower side of the filters were counted by light microscopy under high power field (x400). Cells were counted in 10 fields/well. The data represent the mean ± SE (wild-type, n = 9; others, n = 3). The data shown are from a single experiment that is representative of four independent experiments that were conducted with almost identical results. *, p < 0.01 compared with parent RBL2H3 cells (RBL-WT) by t test.

 
Differential effects of Lyn and c-Src devoid of C-terminal negative regulatory tyrosine on mCsk-suppressed podosome formation and cell migration

The stepwise inhibitory effects of Csk and mCsk on Fn-mediated podosome formation and cell migration strongly suggested that activation (or structural change to an "open" conformation (22, 23)) of Src family protein tyrosine kinases is required for these functions. To further evaluate the hypothesis, we tested whether Src family kinases lacking C-terminal negative regulatory tyrosine (a substrate for Csk (19)) could restore the impaired functions in mCsk cells. To this end, we created mutated Lyn and c-Src, two major Src family kinases expressed in RBL2H3 cells (55) whose corresponding C-terminal amino acid sequences (amino acids 505–512 for LynA and amino acids 527–536 for c-Src) were replaced with a c-myc epitope tag sequence (Fig. 6GoAi). These mutants (termed {Delta}Lyn and {Delta}Src, respectively) or a puromycin-resistant vector alone were stably introduced into the mCsk-expressing cells, and clones with high (hi.) and low (lo.) expression levels of {Delta}Lyn and {Delta}Src were obtained. A representative immunoblotting with the anti-c-myc tag Ab of vector controls and cells expressing {Delta}Src or {Delta}Lyn is shown in Figure 6GoAii.



View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 6. Differential effects of {Delta}Src and {Delta}Lyn on the mCsk-suppressed podosome formation and cell migration. A, i, Schematic representation of c-Src, {Delta}Src, and {Delta}Lyn. In {Delta}Src and {Delta}Lyn, C-terminal corresponding sequences of c-Src (amino acids 527–536) and LynA (amino acids 505–512) were removed and replaced with c-myc epitope tag 14 amino acids. A, ii, {Delta}Src or {Delta}Lyn was transfected into mCsk cells with a puromycin-resistant vector, and high (hi.) and low (lo.) expressors were obtained. Vector controls (mCsk/puro; lanes 1 and 2), {Delta}Src (mCsk/{Delta}Src; lanes 3 and 4), and {Delta}Lyn (mCsk/{Delta}Lyn; lanes 5 and 6) expressors were lysed with SDS sample buffer, standardized for cell number, and subjected to immunoblotting with anti-c-myc epitope tag mAb, 9E10, or with anti-Csk Ab. The migrated positions of {Delta}Src, {Delta}Lyn, Csk, and mCsk are indicated by arrows. Expression levels of mCsk were similar among the clones. B, A vector control (mCsk/puro) (a and d), mCsk/{Delta}Src hi. cells (b and e), and mCsk/{Delta}Lyn hi. cells (c and f) were loaded with Fn-coated (50 µg/ml) beads (a–c: mean diameter, 3 µm; d–f: mean diameter, 11.9 µm), and the accumulations of phosphotyrosine (a–c) and F-actin (d–f) were examined. Scale bar = 10 µm. C, Beads positively stained for phosphotyrosine or for F-actin were scored as described in Figure 4Go. The ordinate indicates the percentage of beads positive for the fluorescent stainings. At least 50 beads were scored in each experiment. Data are expressed as the mean ± SD from three separate experiments. *, p < 0.01 compared with wild-type RBL2H3 cells (RBL-WT) by t test. D, Wild-type RBL2H3 cells, mCsk/puro cells, mCsk/{Delta}Src, and mCsk/{Delta}Lyn cells were assayed for cell migration toward Fn as described in Figure 5Go. The number of wild-type RBL2H3 cells that migrated to the lower surface was defined as 100%. The ordinate indicates the mean ± SD from three separate experiments performed in triplicate. *, p < 0.01 compared with parent RBL2H3 cells (RBL-WT) by t test.

 
When these cells were loaded with Fn-coated beads, tyrosine-phosphorylated proteins (PY) and dot-like F-actin bundles were clearly observed at the attachment sites in {Delta}Src- or {Delta}Lyn-expressing mCsk cells, but not in vector controls (Fig. 6GoB). The number of positively stained beads was scored, and the data are summarized in Figure 6GoC. {Delta}Lyn restored the impaired recruitment of tyrosine-phosphorylated proteins and F-actin by mCsk, whereas {Delta}Src augmented it.

We next tested whether {Delta}Src or {Delta}Lyn could rescue the defective cell migration toward Fn. As shown in Figure 6GoD, Fn-mediated migration in the vector control cells was kept at low levels, as expected. Coexpression of {Delta}Lyn restored the impaired migration of mCsk cells, or, rather, enhanced it, whereas {Delta}Src expression did not regardless of their expression levels. Apparently, {Delta}Src expression further inhibited migration to lower levels than those in vector controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As an approach to study the roles of Src family kinases in integrin-induced leukocyte motility, Fn-mediated outside-in signaling was investigated in RBL2H3 cells overexpressing Csk, mCsk, or mCsk(-). In the previous study, it was confirmed that basal and high affinity IgE receptor-stimulated Lyn activities are stepwise suppressed in Csk- and mCsk-overexpressing cells (36). RBL2H3 cells readily adhere to Fn-coated surface, develop F-actin assembly to podosomes (see Fig. 4GoA), and migrate toward Fn without cytokine priming (inside-out signaling). These characteristics of RBL2H3 cells have provided a suitable experimental system to focus upon outside-in signaling. Our data indicate that overexpression of Csk or the mutated Csks do not significantly affect the interaction of Fn with {alpha}5 integrin in RBL2H3 cells. Firstly, Csk and mCsk did not inhibit the recruitment of {alpha}5 integrin to the attachment sites with Fn-coated beads. Similar observations have been reported in human foreskin fibroblasts, where treatment of the cells with herbimycin A, a relatively Src-selective tyrosine kinase inhibitor, does not suppress {alpha}5 integrin assembly to the Fn-coated bead surface (40). Secondly, cell adhesion to the Fn-coated dish was not significantly affected by the overexpression of the Csk-based molecules. It was also noted that cell spreading over Fn or type I collagen was not appreciably influenced by Csk or by mCsk (Fig. 4Go, A and B, and data not shown). As for the roles of Src family kinases in the adhesion properties of hemopoietic cells, Lowell et al. have demonstrated that HcknullFgrnull murine neutrophils stimulated with TNF are defective in ß2 integrin-mediated tight adhesion and cell spreading, while these functions are preserved when the cells are primed with phorbol ester (34, 56). These findings suggest that requirement of Src family kinases for these functions are dependent on the signaling pathways through which integrins are activated, and that Src family kinases are not critically involved in protein kinase C-stimulated cell adhesion on ECMs. Although the mechanisms of the integrin activation in RBL2H3 cells should be further clarified, autoactivation of c-kit in the cells (43), that potentially leads to the protein kinase C pathway via phospholipase C{gamma} and phosphatidyl inositol 3-kinase (4, 44, 45) is a feasible candidate that could explain the constitutive adhesion of RBL2H3 cells and also the lack of inhibitory effects of Csk on cell adhesion.

Although the initial interaction of {alpha}5 integrin with Fn was not affected by Csk or mCsk overexpression, postintegrin signaling was significantly inhibited by the augmented Csk activity. Firstly, it was found that Fn-mediated tyrosine phosphorylation of cellular proteins, including paxillin, was stepwise inhibited by Csk and mCsk, but not by mCsk(-). Secondly, the assay using Fn-coated microbeads quantitatively showed that Csk and mCsk strongly suppressed the recruitment of tyrosine-phosphorylated proteins and short F-actin bundles to podosomes, but mCsk(-) did not. In addition, the assembly of the short F-actin bundle was hardly detectable at the ventral surface of Csk- and mCsk-expressing cells even under fully spread conditions (Fig. 4GoA), thereby indicating that cell spreading and podosome formation could be separable events. Podosome was originally described in v-Src-transformed tumor cells (57) and has been assumed to be a key signaling machinery to facilitate the motility of hemopoietic cells (4). The Csk-mediated inhibition of podosome formation might suggest that development of podosomes in hemopoietic cells is also regulated by the elevated basal activity of Src family kinases in these cell types (36, 58). Thirdly, we showed that Csk inhibited and mCsk almost abolished Fn-dependent cell migration, findings consistent with the essential roles of podosomes for cell migration. The mCsk(-) only partially reduced the migration. Collectively, Csk and mCsk inhibited the series of events in a stepwise manner that was dependent on the catalytic activity. Although the substrate for Csk is not strictly confined to Src family kinases (59), the correlation between the Csk activity and the extent of inhibition strongly suggest that Src family kinases are required for the signaling cascade. One exception in the correlation was that the cell locomotion was slightly suppressed by mCsk(-), albeit the inhibition was reproducibly smaller than that by Csk or mCsk. The reason for the apparent discrepancy is not clear, but a possible explanation might be that the augmented intrinsic c-Src activity by the dominant negative effects of mCsk(-) (36) hampered the cell migration (see below).

Through the analysis of mCsk-expressing RBL2H3 cells, it was found that these cells provided a condition where the initial interaction of Fn with {alpha}5 integrin was preserved, whereas Fn-mediated podosome formation and cell locomotion were almost abolished. As an approach to further evaluate the roles of Lyn and c-Src, two major Src family kinases expressed in RBL2H3 cells (55), we introduced epitope-tagged, constitutively activated forms of Lyn and c-Src ({Delta}Lyn and {Delta}Src) into mCsk-expressing cells. It has been suggested that the augmented basal catalytic activity of such C-terminal tyrosine-unphosphorylatable kinases is not sufficient to initiate organized cellular functions, including B cell receptor-mediated calcium mobilization (58) or TCR-mediated IL-2 production (46), and that participation of these molecules in receptor-originated signaling machinery is still required to transmit these remote signals. Therefore, this approach could provide information about whether once activated kinases could be involved in a given signaling pathway. We found that {Delta}Lyn successfully restored mCsk-suppressed F-actin assembly to podosomes and also cell migration toward Fn. These findings strongly suggest that Lyn activation could transmit sufficient signal to Fn-mediated cytoskeletal changes leading to cell migration in RBL2H3 cells. On the contrary, {Delta}Src restored the F-actin assembly, but could not rescue the impaired cell migration regardless of its expression levels. Apparently, {Delta}Src further suppressed it. Therefore, continuous activation of c-Src seems to be sufficient for F-actin aggregation, but inhibitory to cell motility. Obviously, the constitutive activation of the mutated c-Src could not fully reproduce the highly regulated innate signaling pathway. Thus, these negative results do not mean that receptor-activated c-Src transmits minimal or negative signal to the cell migration. However, the opposite effects of the two autoactive kinases still raise the possibility that Lyn and c-Src differentially participate in cell motility. Considering the overlapping roles of {Delta}Src and {Delta}Lyn in the podosome assembly, continuous activation of Lyn and c-Src might oppositely influence the turnover of the adhesion machinery (13, 54). It should be noted that mCsk/{Delta}Lyn lo. cells migrated significantly more than mCsk/{Delta}Lyn hi. cells (Fig. 6GoD). The inverse correlation between the expression levels of {Delta}Lyn and the migration levels suggests that excessive activation of Lyn is inhibitory to cell migration. Although Lyn activity is required for podosome formation and cell migration, excessive activation of Lyn might stabilize the adhesion machinery that leads to decreased cell mobility. Considering that {Delta}Src restored podosome formation more markedly than {Delta}Lyn did (Fig. 6GoC), and that {Delta}Src inhibited cell motility (Fig. 6GoD), it might be argued that activation of c-Src more potently fixes the adhesion machinery than Lyn does. Further study is required to understand the precise mode of actions of Lyn and c-Src in the adhesion signaling and to elucidate the structural determinants responsible for the differential effects of these kinases.

In summary, this study has provided evidence that Src family kinases are required, and activation of Lyn could transmit signal for Fn-mediated cytoskeletal changes leading to RBL2H3 cell motility. Considering that hemopoietic cells express multiple Src family kinases in a lineage-specific manner (60), these family members might possess different functions in ECM-mediated signaling cascade that leads to cell migration. In addition, in view of the highly redundant roles of Src family kinases in a number of biologic functions (27, 34, 61), the experimental design in which mCsk-expressing cells are used as a functionally Src family kinase-deficient background could complement the knowledge obtained from gene knockout studies.


   Acknowledgments
 
We thank Dr. J. Miyazaki for the generous gifts of pCAGGS and pCXN2 vectors, and H. Ota-Ichijo, and M. Saka for excellent technical assistance.


   Footnotes
 
1 This work was supported by grants-in-aid from the ministry of Education, Science, Sports, and Culture and by grants from Astra Research Foundation and Manabe Research Foundation. Back

2 Address correspondence and reprint requests to Dr. Zen-ichiro Honda, Department of Internal Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail address: Back

3 Abbreviations used in this paper: ECM, extracellular matrix; FA, focal adhesion; SH, Src homology; Csk, C-terminal Src kinase; Fn, fibronectin; RBL, rat basophilic leukemia; mCsk, a membrane-anchored C-terminal Src kinase; F-actin, filamentous actin. Back

Received for publication March 19, 1998. Accepted for publication May 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berton, G., S. R. Yan, L. Fumagalli, C. A. Lowell. 1996. Neutrophil activation by adhesion: mechanisms and pathophysiological implications. Int. J. Clin. Lab. Res. 26:160.[Medline]
  2. Hamawy, M. M., S. E. Mergenhagen, R. P. Siraganian. 1995. Protein tyrosine phosphorylation as a mechanism of signalling in mast cells and basophils. Cell. Signalling 7:535.[Medline]
  3. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
  4. Wei, J., L. M. Shaw, A. M. Mercurio. 1997. Integrin signaling in leukocytes: lessons from the {alpha}6ß1 integrin. J. Leukocyte Biol. 61:397.[Abstract]
  5. Fuortes, M., W. W. Jin, C. Nathan. 1993. Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor. J. Cell Biol. 120:777.[Abstract/Free Full Text]
  6. Kinashi, T., T. A. Springer. 1994. Steel factor and c-kit regulate cell-matrix adhesion. Blood 83:1033.[Abstract/Free Full Text]
  7. Mercurio, A. M., L. M. Shaw. 1988. Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum. J. Cell Biol. 107:1873.[Abstract/Free Full Text]
  8. Shaw, L. M., J. M. Messier, A. M. Mercurio. 1990. The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the {alpha}6ß1 integrin. J. Cell Biol. 110:2167.[Abstract/Free Full Text]
  9. Hamawy, M. M., S. E. Mergenhagen, R. P. Siraganian. 1993. Cell adherence to fibronectin and the aggregation of the high affinity immunoglobulin E receptor synergistically regulate tyrosine phosphorylation of 105–115-kDa proteins. J. Biol. Chem. 268:5227.[Abstract/Free Full Text]
  10. Nathan, C. F.. 1987. Neutrophil activation on biological surfaces: massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J. Clin. Invest. 80:1550.
  11. Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, S. D. Wright. 1989. Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins. J. Cell Biol. 109:1341.[Abstract/Free Full Text]
  12. Lin, T. H., C. Rosales, K. Mondal, J. B. Bolen, S. Haskill, R. L. Juliano. 1995. Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells: a possible signaling role for the Syk tyrosine kinase. J. Biol. Chem. 270:16189.[Abstract/Free Full Text]
  13. Lauffenburger, D. A., A. F. Horwitz. 1996. Cell migration: a physically integrated molecular process. Cell 84:359.[Medline]
  14. Gumbiner, B. M.. 1996. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345.[Medline]
  15. Jockusch, B. M., P. Bubeck, K. Giehl, M. Kroemker, J. Moschner, M. Rothkegel, M. Rudiger, K. Schluter, G. Stanke, J. Winkler. 1995. The molecular architecture of focal adhesions. Annu. Rev. Cell. Dev. Biol. 11:379.[Medline]
  16. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11.[Medline]
  17. Schaller, M. D., J. T. Parsons. 1994. Focal adhesion kinase and associated proteins. Curr. Opin. Cell. Biol. 6:705.[Medline]
  18. Jove, R., H. Hanafusa. 1987. Cell transformation by the viral src oncogene. Annu. Rev. Cell. Biol. 3:31.
  19. Nada, S., M. Okada, A. MacAuley, J. A. Cooper, H. Nakagawa. 1991. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature 351:69.[Medline]
  20. Nada, S., T. Yagi, H. Takeda, T. Tokunaga, H. Nakagawa, Y. Ikawa, M. Okada, S. Aizawa. 1993. Constitutive activation of Src family kinases in mouse embryos that lack Csk. Cell 73:1125.[Medline]
  21. Imamoto, A., P. Soriano. 1993. Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice. Cell 73:1117.[Medline]
  22. Kaplan, K. B., K. B. Bibbins, J. R. Swedlow, M. Arnaud, D. O. Morgan, H. E. Varmus. 1994. Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527. EMBO J. 13:4745.[Medline]
  23. Kaplan, K. B., J. R. Swedlow, D. O. Morgan, H. E. Varmus. 1995. c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev. 9:1505.[Abstract/Free Full Text]
  24. Howell, B. W., J. A. Cooper. 1994. Csk suppression of Src involves movement of Csk to sites of Src activity. Mol. Cell. Biol. 14:5402.[Abstract/Free Full Text]
  25. Bockholt, S. M., K. Burridge. 1995. An examination of focal adhesion formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-, and yes- mice. Cell Adhes. Commun. 3:91.[Medline]
  26. Vuori, K., H. Hirai, S. Aizawa, E. Ruoslahti. 1996. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol. Cell. Biol. 16:2606.[Abstract]
  27. Thomas, S. M., P. Soriano, A. Imamoto. 1995. Specific and redundant roles of Src and Fyn in organizing the cytoskeleton. Nature 376:267.[Medline]
  28. Turner, C. E., Jr J. Glenney, K. Burridge. 1990. Paxillin: a new vinculin-binding protein present in focal adhesions. J. Cell. Biol. 111:1059.[Abstract/Free Full Text]
  29. Sabe, H., A. Hata, M. Okada, H. Nakagawa, H. Hanafusa. 1994. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src. Proc. Natl. Acad. Sci. USA 91:3984.[Abstract/Free Full Text]
  30. Schwartz, M. A.. 1993. Signaling by integrins: implications for tumorigenesis. Cancer Res. 53:1503.[Free Full Text]
  31. Hall, C. L., L. A. Lange, D. A. Prober, S. Zhang, E. A. Turley. 1996. pp60c-src is required for cell locomotion regulated by the hyaluronan receptor RHAMM. Oncogene 13:2213.[Medline]
  32. Yan, S. R., L. Fumagalli, G. Berton. 1996. Activation of SRC family kinases in human neutrophils: evidence that p58C-FGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity. FEBS Lett. 380:198.[Medline]
  33. Yan, S. R., M. Huang, G. Berton. 1997. Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen. J. Immunol. 158:1902.[Abstract]
  34. Lowell, C. A., L. Fumagalli, G. Berton. 1996. Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions. J. Cell Biol. 133:895.[Abstract/Free Full Text]
  35. Chow, L. M., A. Veillette. 1995. The Src and Csk families of tyrosine protein kinases in hemopoietic cells. Semin. Immunol. 7:207.[Medline]
  36. Honda, Z., T. Suzuki, N. Hirose, A. Makoto, T. Shimizu, S. Nada, M. Okada, C. Ra, Y. Morita, K. Ito. 1997. Roles of C-terminal Src kinase in the initiation and the termination of the high affinity IgE receptor-mediated signaling. J. Biol. Chem. 272:25753.[Abstract/Free Full Text]
  37. Miyazaki, J., S. Takaki, K. Araki, F. Tashiro, A. Tominaga, K. Takatsu, K. Yamamura. 1989. Expression vector system based on the chicken ß-actin promoter directs efficient production of interleukin-5. Gene 79:269.[Medline]
  38. Evan, G. I., G. K. Lewis, G. Ramsay, J. M. Bishop. 1985. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5:3610.[Abstract/Free Full Text]
  39. Morita, H., H. Yasumitsu, H. Harada, M. Umeda, T. Takaoka. 1988. Secretion of a novel cell-adhesive protein distinct from fibronectin by mouse L.P3 cells growing in protein- and lipid-free synthetic medium. Cell Struct. Funct. 13:481.[Medline]
  40. Miyamoto, S., S. K. Akiyama, K. M. Yamada. 1995. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267:883.[Abstract/Free Full Text]
  41. Falk, W., Jr R. H. Goodwin, E. J. Leonard. 1980. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J. Immunol. Methods 33:239.[Medline]
  42. Klemke, R. L., M. Yebra, E. M. Bayna, D. A. Cheresh. 1994. Receptor tyrosine kinase signaling required for integrin {alpha}vß5-directed cell motility but not adhesion on vitronectin. J. Cell Biol. 127:859.[Abstract/Free Full Text]
  43. Tsujimura, T., T. Furitsu, M. Morimoto, Y. Kanayama, S. Nomura, Y. Matsuzawa, Y. Kitamura, Y. Kanakura. 1995. Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3. Int. Arch. Allergy Immunol. 106:377.[Medline]
  44. Kinashi, T., J. A. Escobedo, L. T. Williams, K. Takatsu, T. A. Springer. 1995. Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C{gamma}1 pathways. Blood 86:2086.[Abstract/Free Full Text]
  45. Kinashi, T., T. A. Springer. 1995. Regulation of cell-matrix adhesion by receptor tyrosine kinases. Leuk. Lymphoma 18:203.[Medline]
  46. Chow, L. M., M. Fournel, D. Davidson, A. Veillette. 1993. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk. Nature 365:156.[Medline]
  47. Fehlner-Gardiner, C. C., S. Uniyal, C. G. von Ballestrem, B. M. Chan. 1996. Differential utilization of VLA-4 ({alpha}4ß1) and -5 ({alpha}5ß1) integrins during the development of mouse bone marrow-derived mast cells. Differentiation 60:317.[Medline]
  48. Yasuda, M., Y. Hasunuma, H. Adachi, C. Sekine, T. Sakanishi, H. Hashimoto, C. Ra, H. Yagita, K. Okumura. 1995. Expression and function of fibronectin binding integrins on rat mast cells. Int. Immunol. 7:251.[Abstract/Free Full Text]
  49. Hamawy, M. M., C. Oliver, S. E. Mergenhagen, R. P. Siraganian. 1992. Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion. J. Immunol. 149:615.[Abstract]
  50. Yamanashi, Y., D. Baltimore. 1997. Identification of the abl- and ras-GAP-associated 62 kDa protein as a docking protein, Dok. Cell 88:205.[Medline]
  51. Carpino, N., D. Wisniewski, A. Strife, D. Marshak, R. Kobayashi, B. Stillman, B. Clarkson. 1997. p62dok: a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells. Cell 88:197.[Medline]
  52. Tachibana, K., T. Sato, N. D’Avirro, C. Morimoto. 1995. Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK. J. Exp. Med. 182:1089.[Abstract/Free Full Text]
  53. Grinnell, F., B. Geiger. 1986. Interaction of fibronectin-coated beads with attached and spread fibroblasts: binding, phagocytosis, and cytoskeletal reorganization. Exp. Cell Res. 162:449.[Medline]
  54. Illc, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, T. Yamamoto, S. Aizawa. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377:539.[Medline]
  55. Eiseman, E., J. B. Bolen. 1992. Engagement of the high-affinity IgE receptor activates Src protein-related tyrosine kinases. Nature 355:78.[Medline]
  56. Nishizuka, Y.. 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607.[Abstract/Free Full Text]
  57. Tarone, G., D. Cirillo, F. G. Giancotti, P. M. Comoglio, P. C. Marchisio. 1985. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp. Cell Res. 159:141.[Medline]
  58. Hata, A., H. Sabe, T. Kurosaki, M. Takata, H. Hanafusa. 1994. Functional analysis of Csk in signal transduction through the B-cell antigen receptor. Mol. Cell. Biol. 14:7306.[Abstract/Free Full Text]
  59. Autero, M., J. Saharinen, T. Pessa-Morikawa, M. Soula-Rothhut, C. Oetken, M. Gassmann, M. Bergman, K. Alitalo, P. Burn, C. G. Gahmberg, T. Mustelin. 1994. Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase. Mol. Cell. Biol. 14:1308.[Abstract/Free Full Text]
  60. Bolen, J. B., R. B. Rowley, C. Spana, A. Y. Tsygankov. 1992. The Src family of tyrosine protein kinases in hemopoietic signal transduction. FASEB J. 6:3403.[Abstract]
  61. Crowley, M. T., P. S. Costello, C. J. Fitzer-Attas, M. Turner, F. Meng, C. Lowell, V. L. J. Tybulewicz, A. D. DeFranco. 1997. A critical role for Syk in signal transduction and phagocytosis mediated by Fc{gamma} receptors on macrophages. J. Exp. Med. 186:1027.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Kannan, A. Audet, H. Huang, L.-j. Chen, and M. Wu
Cholesterol-Rich Membrane Rafts and Lyn Are Involved in Phagocytosis during Pseudomonas aeruginosa Infection
J. Immunol., February 15, 2008; 180(4): 2396 - 2408.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
J M W Gee, V E Shaw, S E Hiscox, R A McClelland, N K Rushmere, and R I Nicholson
Deciphering antihormone-induced compensatory mechanisms in breast cancer and their therapeutic implications
Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S77 - S88.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Kitaura, T. Kinoshita, M. Matsumoto, S. Chung, Y. Kawakami, M. Leitges, D. Wu, C. A. Lowell, and T. Kawakami
IgE- and IgE+Ag-mediated mast cell migration in an autocrine/paracrine fashion
Blood, April 15, 2005; 105(8): 3222 - 3229.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
E. Yokoo, Y. Yatomi, T. Takafuta, M. Osada, Y. Okamoto, and Y. Ozaki
Sphingosine 1-Phosphate Inhibits Migration of RBL-2H3 Cells via S1P2: Cross-Talk between Platelets and Mast Cells
J. Biochem., June 1, 2004; 135(6): 673 - 681.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
T. Oka, M. Hori, A. Tanaka, H. Matsuda, H. Karaki, and H. Ozaki
IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells
Am J Physiol Cell Physiol, February 1, 2004; 286(2): C256 - C263.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
D. Pradip, X. Peng, and D. L. Durden
Rac2 Specificity in Macrophage Integrin Signaling: POTENTIAL ROLE FOR Syk KINASE
J. Biol. Chem., October 24, 2003; 278(43): 41661 - 41669.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Kono, T. Suzuki, K. Yamamoto, M. Okada, T. Yamamoto, and Z.-i. Honda
Spatial Raft Coalescence Represents an Initial Step in Fc{gamma}R Signaling
J. Immunol., July 1, 2002; 169(1): 193 - 203.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Ueda, M. Mizuki, H. Ikeda, T. Tsujimura, I. Matsumura, K. Nakano, H. Daino, Z.-i. Honda, J. Sonoyama, H. Shibayama, et al.
Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration
Blood, May 1, 2002; 99(9): 3342 - 3349.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USA