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*
Department of Internal Medicine and Physical Therapy, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan;
Division of Protein Metabolism, Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka, Japan; and
Department of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 
Src and 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 Src or 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 1
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, 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
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. 4, B and C). These
observations indicate that the changes in Csk activity did not alter
RBL2H3 cell adhesion to Fn-coated surface.
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We next explored whether Csk and its mutants affect Fn-mediated
protein tyrosine phosphorylation. As shown in Figure 3A, adhesion of parent RBL2H3
cells to Fn-coated surface induced tyrosine phosphorylation of about 70
kDa (indicated by double asterisks in Figure 3) 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 3) 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).
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B, 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 4Aa, 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. 4, A, b and c), while it was clearly
observed in mCsk(-)-expressing cells (Fig. 4Ad).
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 4B, a–d, integrin 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. 4B,e–l). In many cases, F-actin accumulation exhibited dot-like
protrusions around the beads (Fig. 4B, i and
l). The localized F-actin assembly was more clearly
visualized in the experiments using large beads (mean diameter, 11.9
µm; Fig. 4B, 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 4C. 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 5, 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.
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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. 6Ai). These mutants (termed
Lyn and 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 Lyn and Src were
obtained. A representative immunoblotting with the
anti-c-myc tag Ab of vector controls and cells
expressing Src or Lyn is shown in Figure 6Aii.
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Src- or
Lyn-expressing mCsk cells, but not in vector controls (Fig. 6B). The number of positively stained beads was scored, and
the data are summarized in Figure 6C. Lyn restored the
impaired recruitment of tyrosine-phosphorylated proteins and F-actin by
mCsk, whereas Src augmented it.
We next tested whether Src or Lyn could rescue the defective cell
migration toward Fn. As shown in Figure 6D, Fn-mediated
migration in the vector control cells was kept at low levels, as
expected. Coexpression of Lyn restored the impaired migration of
mCsk cells, or, rather, enhanced it, whereas Src expression did not
regardless of their expression levels. Apparently, Src expression
further inhibited migration to lower levels than those in vector
controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A), 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
5 integrin in RBL2H3 cells. Firstly, Csk and mCsk did
not inhibit the recruitment of 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 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. 4, 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
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 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. 4A), 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 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 (Lyn
and 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 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, Src restored the F-actin assembly, but could
not rescue the impaired cell migration regardless of its expression
levels. Apparently, 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
Src and 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/Lyn lo.
cells migrated significantly more than mCsk/Lyn hi. cells (Fig. 6D). The inverse correlation between the expression levels
of 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 Src restored podosome
formation more markedly than Lyn did (Fig. 6C), and that
Src inhibited cell motility (Fig. 6D), 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.
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Acknowledgments |
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Footnotes |
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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:
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.
Received for publication March 19, 1998. Accepted for publication May 27, 1998.
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P. Piccardoni, R. Sideri, S. Manarini, A. Piccoli, N. Martelli, G. de Gaetano, C. Cerletti, and V. Evangelista Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin Blood, July 1, 2001; 98(1): 108 - 116. [Abstract] [Full Text] [PDF]
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A. Oda, H. D. Ochs, L. A. Lasky, S. Spencer, K. Ozaki, M. Fujihara, M. Handa, K. Ikebuchi, and H. Ikeda CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk Blood, May 1, 2001; 97(9): 2633 - 2639. [Abstract] [Full Text] [PDF]
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 T. Yuasa, M. Ono, T. Watanabe, and T. Takai Lyn Is Essential for Fc{gamma} Receptor III-Mediated Systemic Anaphylaxis but Not for the Arthus Reaction J. Exp. Med., March 5, 2001; 193(5): 563 - 572. [Abstract] [Full Text] [PDF]
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 Z. Korade-Mirnics and S. J. Corey Src kinase-mediated signaling in leukocytes J. Leukoc. Biol., November 1, 2000; 68(5): 603 - 613. [Abstract] [Full Text]
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T. Suzuki, H. Kono, N. Hirose, M. Okada, T. Yamamoto, K. Yamamoto, and Z.-i. Honda Differential Involvement of Src Family Kinases in Fc{gamma} Receptor-Mediated Phagocytosis J. Immunol., July 1, 2000; 165(1): 473 - 482. [Abstract] [Full Text] [PDF]
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 Z.-i. Honda, T. Suzuki, H. Kono, M. Okada, T. Yamamoto, C. Ra, Y. Morita, and K. Yamamoto Sequential Requirements of the N-Terminal Palmitoylation Site and SH2 Domain of Src Family Kinases in the Initiation and Progression of Fcvarepsilon RI Signaling Mol. Cell. Biol., March 1, 2000; 20(5): 1759 - 1771. [Abstract] [Full Text]
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P. Lock, F. Casagranda, and A. R. Dunn Independent SH2-binding Sites Mediate Interaction of Dok-related Protein with RasGTPase-activating Protein and Nck J. Biol. Chem., August 6, 1999; 274(32): 22775 - 22784. [Abstract] [Full Text] [PDF]
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 A. V. Grishin, O. Azhipa, I. Semenov, and S. J. Corey Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis PNAS, August 28, 2001; 98(18): 10172 - 10177. [Abstract] [Full Text] [PDF]
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L. Geng, S. Pfister, S.-K. Kraeft, and C. E. Rudd Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: Differential involvement of the FYB SH3 domain PNAS, September 25, 2001; 98(20): 11527 - 11532. [Abstract] [Full Text] [PDF]
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120 kDa (*) and 
