Adenovirus-mediated Overexpression of C-terminal Src Kinase
(Csk) in Type I Astrocytes Interferes with Cell Spreading and
Attachment to Fibronectin
CORRELATION WITH TYROSINE PHOSPHORYLATIONS OF PAXILLIN AND
FAK*
Yoshiharu
Takayama
§,
Sakae
Tanaka¶,
Katsuya
Nagai§, and
Masato
Okada§
From the § Division of Protein Metabolism, Institute for
Protein Research, Osaka University, Suita, Osaka 565-0871 and
¶ Department of Orthopedic Surgery, Faculty of Medicine, The
University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
 |
ABSTRACT |
To examine the role of C-terminal Src kinase
(Csk), a negative regulatory kinase of Src family tyrosine kinases, in
the cell adhesion mechanism of the nervous system, wild-type Csk (Csk), and a kinase-deficient mutant of Csk (Csk-
K) were overexpressed in
primary cultured type I astrocytes by infecting them with the recombinant adenovirus. Overexpression of Csk repressed the in vitro kinase activity of Src to as little as 10% that of control cells and interfered with cell spreading and cell attachment to fibronectin. Focal adhesion assembly and the organization of actin stress fibers were also disrupted in cells overexpressing Csk. On the
other hand, overexpression of Csk-
K induced tyrosine phosphorylation of cellular proteins, including the paxillin and focal adhesion kinase
(FAK) and enhanced to some extent the cytoskeletal organization and the
rate of cell spreading on fibronectin, indicating that Src or its
relatives was functionally activated in the cells. Paxillin was also
tyrosine-phosphorylated in Csk-overexpressing cells, indicating that it
can serve as a substrate of Csk. The phosphorylation state of paxillin
in cells overexpressing Csk was indistinguishable from that in cells
expressing Csk-
K in that both phosphorylated paxillins bound equally
to SH2 domain of Csk and were co-immunoprecipitated with Csk. In
contrast, tyrosine phosphorylation of FAK and its in vitro
autophosphorylation activity were increased only in cells expressing
Csk-
K. In Csk-expressing cells, the kinase activity of FAK was
substantially decreased to 20-30% that of control cells, even though
the expression level of FAK was rather increased. These findings
suggest that Csk regulates Src family tyrosine kinases that play
essential roles in the regulation of cell adhesion via a
FAK-dependent mechanism and that the tyrosine phosphorylation of paxillin alone may not be sufficient for the regulation of the cell adhesion mechanism in astrocytes.
 |
INTRODUCTION |
Protein-tyrosine phosphorylation is a crucial step in the signal
transduction cascades triggered by a variety of extra cellular stimuli
that modulate cellular functions. Receptor-type tyrosine kinases are
activated by ligand binding and directly transduce the extracellular
information into intracellular tyrosine phosphorylation events, whereas
nonreceptor tyrosine kinases function as signal transducers in concert
with receptor-like molecules that lack tyrosine kinase activities.
Src family tyrosine kinases are nonreceptor tyrosine kinases that are
functionally linked to some receptor-type tyrosine kinases (1, 2),
G-protein-coupled receptors (3), cytokine receptors (4), and cell
adhesion molecules/receptors (5, 6). Because of their functional
redundancy, the essential roles of Src family tyrosine kinases still
remain to be elucidated (7). Src family tyrosine kinases are
membrane-associated proteins having Src homology 2 (SH2)1 and SH3 domains, which
are important for protein-protein interaction. The activity of Src
family tyrosine kinases is regulated through intramolecular interaction
between the SH2 domain and the phosphorylated tyrosine at the
C-terminal regulatory site (Tyr-527) (8), and it is predicted that
displacement of this interaction by another molecule that can bind to
the Src SH2 or SH3 domain or dephosphorylation of Tyr-527 can activate
the kinase. Although the mechanism of activation coupled with receptor
stimulation is still unclear, the tyrosine kinase Csk (C-terminal Src
kinase) has been identified as a negative regulator of Src family
tyrosine kinases (9, 10). Csk acts specifically on the regulatory sites
of the family members, and targeted disruption of the csk
gene caused constitutive activation of Src family tyrosine kinases
accompanied by a defect in neurulation (11, 12). On the other hand,
overexpression of Csk in some cell lines induced repression of cellular
signaling mediated by Src family tyrosine kinases (13, 14). From these observations, it has been suggested that a balance between Src family
tyrosine kinases and Csk might define the basal function of Src family
tyrosine kinases.
It is also known that some members of the Src family are targeted to
cell adhesion plaque during the process of cell adhesion (15, 16).
Cells transformed by v-Src cause rearrangement of the actin
cytoskeleton structure accompanied by anchorage-independent growth
(17). In Src-transformed and Csk-deficient cells, tyrosine phosphorylation of some focal adhesion proteins, including FAK (focal
adhesion kinase) and paxillin, were elevated, implicating the role of
Src in the regulation of cell adhesion (7, 18). The cell adhesion
mechanism not only allows cells to adhere to one another but also
transduces signals involved in the regulation of cytoskeletal
organization and gene expression. In such a signaling cascade, the
nonreceptor tyrosine kinase FAK is regarded as a critical signaling
molecule, as deduced from observations of FAK-deficient and
FAK-overexpressing cells (19, 20). FAK is enriched in focal adhesion
through interaction with a cell adhesion receptor, integrin, and is
activated upon cell attachment on the extracellular matrix (ECM) (21,
22). The activation is accompanied by autophosphorylation of Tyr-397,
which creates an Src SH2 binding site (23). Src is subsequently
activated through this interaction, thereby phosphorylating FAK at
Tyr-925 to create another binding site for the signaling molecule
Grb2/Sos to drive the mitogen-activated protein kinase cascade (21, 24,
25). Furthermore, a recent study has indicated that tyrosine
phosphorylation of paxillin by Src may be a critical step in focal
adhesion assembly (26). These observations suggest that Src is an
important mediator of signals originating from cell adhesion through
FAK, although the molecular mechanism leading to the regulation of
cytoskeletal organization and gene expression has yet to be elucidated.
The nervous system is a highly complex structure composed of a variety
of cells, including neuronal and glial cells. During the development of
the nervous system, cell-cell and cell-ECM interaction is one of the
key processes in the highly organized proliferation of precursor cells,
differentiation, axon guidance, synapse formation, and cell death.
There is a line of evidence suggesting that Src family tyrosine kinases
are involved in the regulation of cell-cell and/or cell-ECM interaction
in the nervous system (27-29). Previously, we have shown that
suppression of Src family kinases by the expression of a
membrane-targeted Csk in neurally differentiated P19 cells induced
inhibition of cell-cell interaction mediated by the neural cell
adhesion molecule (6). To further elucidate the involvement of Csk in
the regulation of the cell adhesion mechanism mediated by the FAK/Src
pathway, we here employed primary cultured type I astrocytes and an
adenovirus-mediated gene transfer system to obtain the efficient
overexpression of Csk in primary cultures. Overexpression of the
wild-type Csk readily repressed the kinase activity of Src and
inhibited cell spreading and attachment to the ECM. Biochemical links
between the phenotype and the tyrosine phosphorylation of cell adhesion
proteins were also investigated.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
To obtain a primary culture of type I
astrocytes from mice (ICR strain), brain cortex regions were dissected
from embryos at embryonic day 18 (E18) and immediately soaked in
ice-cold isotonic buffer (137 mM NaCl, 0.17 mM
Na2HPO4, 0.22 mM
KH2PO4, 5.5 mM glucose, 59 mM sucrose, 5.4 mM KCl). After removing the
meninges, the tissues were shattered with scissors and incubated at
37 °C for 1 h in the presence of 0.1% dispase (Life
Technologies, Inc.) and 0.05% DNase I (Boehringer Mannheim). The
dispersed cells were further dissected by pipetting with a 10-ml
disposable pipette fitted with a 0.2-ml tip for use in a nonautomated
pipetter. The cell suspension was filtered through double sheets of
lens cleaning paper (Fuji Film) to remove undispersed fragments. After
removing dispase and DNaseI by centrifugation, cells were resuspended
in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10%
fetal bovine serum (Hyclone). Cell suspensions were plated in tissue
culture-grade dishes coated with 10 µg/ml fibronectin (Sigma) and
maintained at 37 °C and 5% CO2 in a humidified
atmosphere. Cells were used for experiments after passaging twice to
remove neuron-like cells.
Vector Construction--
The MluI fragment of Csk
cDNA was blunted, ligated with PvuI linker, then
inserted into the PacI site of the cosmid cassette of
pAx1CAwt, which carries the CAG promoter (a gift from Dr. I. Saitoh and
Dr. J. Miyazaki) (30, 31). A kinase-negative mutant of Csk (Csk-
K)
was generated by substituting Arg for Lys-222 by the Kunkel method. The
construction of the mutant was confirmed by direct sequencing. Csk-
K
was inserted into pAx1CAwt as described for Csk. The recombinant
adenovirus was obtained as described elsewhere (30). Briefly, the
cassette containing the Csk expression unit was co-transfected into
human kidney 293 cells together with an adenovirus genome DNA-terminal
protein complex, which was digested at several sites with
EcoT2321 or AseI/EcoRI. The targeted
recombinant adenovirus carrying the csk gene (Ax1CATcsk) or
its kinase-deficient mutant (Ax1CATcsk-
K) was generated by
overlapping recombination. After maintaining the 293 cells for 10-15
days, the virus clones were isolated.
Bacterial Fusion
Proteins--
Glutathione-S-transferase (GST) fusion
proteins containing rat cDNA fragments of Csk domains SH2
(nucleotides 71-299), SH3 (nucleotides 300-626), or SH2/3
(nucleotides 71-626) were synthesized as described previously(32).
Cell Adhesion Assay--
Astrocytes expressing
-galactosidase, wild-type Csk, or Csk-
K were detached from
culture dishes by treatment with trypsin (0.25% trypsin in
phosphate-buffered saline (PBS), pH 7.4) for 10 min at 37 °C. The
detached cells were suspended in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, and portions of the cells were
plated at a concentration of 2.5 × 104
cells/cm2 on tissue culture-grade dishes or plates coated
with 10 µg/ml fibronectin. At the indicated intervals, the cultures
were washed twice with PBS to remove unattached cells. Attached cells
were fixed with 3.5% formaldehyde in PBS for 10 min and washed twice with PBS. The number of attached cells was estimated by staining with
0.1% crystal violet in PBS. After washing twice with PBS, the dye was
eluted with 500 µl of 10% acetic acid, and the absorbance at 600 nm
was measured. The background value was obtained from empty plates
coated with fibronectin.
Antibodies--
Mouse monoclonal antibodies against
phosphotyrosine (clones 4G10 and PY20, Transduction Laboratories),
recombinant horseradish peroxidase-conjugated anti-phosphotyrosine
antibody (RC-20, Transduction Laboratories), anti-focal adhesion kinase
(FAK) monoclonal antibody (clone 77, Transduction Laboratories),
anti-v-Src monoclonal antibody (clone Mab 327, Calbiochem),
anti-paxillin antibody (clone 177, Transduction Laboratories), anti-Csk
monoclonal antibody (clone 52, Transduction Laboratories),
anti-vinculin monoclonal antibody (clone V284, Cymbus Bioscience), and
anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (clone
G-A-5, Boehringer Mannhein) were employed. For Western blotting,
horseradish peroxidase-conjugated anti-mouse IgG (Zymed
Laboratories Inc.) and horseradish peroxidase-conjugated anti-rabbit IgG (Zymed Laboratories Inc.) were used as
secondary antibodies.
Immunofluorescence Staining--
Primary culture type I
astrocytes were plated onto glass coverslips coated with collagen. The
cells were allowed to grow for 5 days after infection with the
adenovirus. They were then fixed in 3.7% formaldehyde in PBS for 10 min, followed by treatment with 0.1% Triton X-100 in PBS for 10 min.
After this, the cells were incubated with anti-paxillin,
anti-phosphotyrosine (PY20), GFAP antibodies and further incubated with
fluorescein isothiocyanate- or Texas red-conjugated anti-mouse
antibodies. For F-actin staining, rhodamine-conjugated phalloidin was
used instead of the antibodies.
X-Gal Staining--
Cells fixed with 3.7% formaldehyde were
washed twice in PBS and then incubated with X-gal staining solution (50 mM K3Fe(CN)6, 50 mM
K4Fe(CN)6, 20 mM MgCl2,
0.01% sodium deoxycholate, and 0.2% X-gal
(5-bromo-4-chloro-3-indolyl-
-galactoside) in PBS) for 3 h at
37 °C.
Western Blotting--
Proteins separated by SDS-PAGE were
transferred onto a nitrocellulose membrane (Schleicher & Schuell).
Subsequently, the membrane was treated with blocking reagent
(Tris-buffered saline containing 0.1% Tween 20 (Tween-TBS)) for 2 h at room temperature. The blocked membrane was probed with primary
antibodies and further incubated with a secondary antibody conjugated
with horseradish peroxidase. The immunoreactivity was visualized with
an enhanced chemiluminescence system (DuPont).
Immunoprecipitation--
Cells were rinsed twice with ice-cold
PBS and then lysed with 400 µl of ice-cold radioimmune precipitation
buffer (10 mM Tris-HCl (pH7.4), 0.15 M NaCl,
1% Nonidet P-40, 1 mM EDTA, 0.1% SDS, 0.5% sodium
deoxycholate, 5 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 10 µg/ml aprotinin) or TNE buffer (20 mM Tris-HCl (pH 7.4),
0.15 M NaCl, 1% Nonidet P-40, 1 mM EDTA, 5 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). The cell
lysates were incubated with 20 µl of 10% pansorbin cells
(Calbiochem) for 30 min at 4 °C, after which the pansorbin cells
were removed by centrifugation to clear the lysate. The precleared
lysate was incubated with the respective antibody for 1 h at
4 °C. Subsequently, 0.1 µg of anti-mouse IgG was added, and the
complexes were further incubated for 15 min at 4 °C. The
immunocomplexes were recovered from the lysates by incubation with 20 µl of 10% pansorbin cells for 30 min at 4 °C, followed by
sequential washing with a cushion of 500 µl of radioimmune
precipitation buffer containing 1 M sucrose, twice with
radioimmune precipitation buffer buffer, and twice further with TNE
buffer. The immunocomplexes obtained were resuspended in 20 µl of TNE buffer.
In Vitro Kinase Assay--
The kinase activity of Src and FAK
were determined by quantifying the amount of radiolabeled phosphate
incorporated into acid-treated enolase or polyglutamate tyrosine
(poly-EY), respectively. Src and FAK were immunoprecipitated from the
cell lysate as described above. An aliquot of immunocomplex was mixed
with 18 µl of an ice-cold reaction mixture (10 mM PIPES,
pH 7.0, 10 mM MnCl2, 2.5 µg of rabbit mouse
enolase (Sigma), and 5 µM ATP), and the kinase reaction
was initiated by the addition of 5 µCi of [
-32P]ATP.
After incubation for 10 min at 30 °C, an equal volume of 2×
SDS-PAGE sample buffer was added, and the samples were boiled for 3 min
to terminate the reaction. The phosphorylated proteins or substrates
were resolved by SDS-PAGE and visualized by autoradiography or
quantified by a BAS2000 image analyzer (Fuji Film).
 |
RESULTS |
Overexpression of Csk Represses the Src Activity--
To analyze
the role of Csk under near in vivo conditions, wild-type Csk
(Csk) and the kinase-negative Csk mutant (Csk-
K) were transiently
overexpressed in the primary cultured type I astrocytes. Recombinant
adenovirus vectors were used to obtain an efficient introduction of
ectopic genes into the primary cultures. Type I astrocytes were
infected by Ax1CAT-lacZ (10 multiplicity of infection
(m.o.i.)), Ax1CATcsk (1.25-10 m.o.i.), or Ax1CATcsk-
K (10 m.o.i.),
and the expression of Csk protein was determined by Western blotting 5 days after infection. Although the expression of Csk in the control
cells and cells infected by Ax1CAT-lacZ was at quite low
levels, Csk protein was overexpressed in cells infected by Ax1CATcsk or
Ax1CATcsk-
K in an m.o.i.-dependent manner (Fig.
1A). The efficiency of the
infection was confirmed by X-gal staining of cells infected by
Ax1CAT-lacZ, which showed that almost 100% efficiency was
achieved (Fig. 2A). Depending
upon the dose of Csk protein, the in vitro kinase activity
of Src was repressed in cells overexpressing Csk to as little as 10%
that of the control cells. However, activation of the in
vitro kinase activity of Src could not be detected in cells
expressing Csk-
K (Fig. 1B).

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Fig. 1.
Dose-dependent effect of Csk
overexpression on specific activity of Src. A, the
lacZ gene ( -galactosidase ( -Gal),
lane 2), wild-type Csk (Csk, lanes 3-6), or
kinase-negative mutant of Csk (Csk- K, lane 7) were
introduced into primary cultured astrocytes by the recombinant
adenovirus. In the case of Csk, the m.o.i. was varied in the range from
1.25 to 10. Expressions of Csk and Src were detected by Western
blotting. To estimate the kinase activity of Src, the immunoprecipitate
with anti-Src antibody was incubated with enolase and
[ -32P]ATP. The radioactivities incorporated into Src,
and enolase was visualized by autoradiography. B, the total
activity of Src was estimated by counting the radioactivity
incorporated into enolase. The relative specific activity of Src was
estimated by dividing the total activity by the amount of Src in the
immunoprecipitates.
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Fig. 2.
Inhibition of cell spreading on collagen by
overexpression of Csk. A, to determine the efficiency
of infection with the recombinant adenovirus, primary culture
astrocytes were infected with 10 m.o.i. of the recombinant
adenovirus carrying the lacZ gene (Ax-CATlacZ).
Forty-eight h after infection, the cells were fixed with formaldehyde
and incubated with X-gal-staining solution. B-E, cells were
plated onto dishes coated with collagen. After 2 days, astrocytes were
infected with 10 m.o.i. of the recombinant adenovirus carrying the
lacZ gene (Ax-CATlacZ (C)), Csk
(Ax-CATCsk (D)), or Csk- K (Ax-CAT-Csk- K
(E)). Cells were allowed to grow for 5 days in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum in a 5%
CO2 atmosphere. The cell morphologies of
adenovirus-infected cells were compared with those of the control cells
(B) by means of phase-contrast microscopy.
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Overexpression of Csk Interferes with Cell Spreading--
The
effect of Csk overexpression on the morphology of astrocytes was
observed under phase-contrast microscopy. The control astrocytes
exhibited widely spread, polygonal shapes with obvious cytoplasmic
edges, although some developing cells were rounded (Fig.
2B). Cells expressing
-galactosidase did not show any
morphological change (Fig. 2C). In contrast, overexpression
of Csk induced a dramatic change in the cell morphology; the majority
of the cells had spread very poorly and remained rounded with only a
few attachment sites (Fig. 2D). The morphology of cells
expressing Csk-
K appeared to be almost the same as that of the
control cells, although some had spread more vigorously than the
control cells (Fig. 2E).
Overexpression of Csk Interferes with Cell Attachment to
Fibronectin--
To compare the abilities of cells to attach to the
ECM, cell adhesion assays were performed. The control cells and those
expressing Csk-
K began to attach to fibronectin-coated dishes within
20 min after plating, and about 60% of them were attached after around 60 min. In contrast, the Csk-expressing cells did not begin to attach
to the dishes until 60 min after plating (Fig.
3A), and the majority still
remained unattached even after 6 h, when attachment of the control
cells and those expressing Csk-
K were almost completed (data not
shown). The morphologies of cells 40 min after plating are compared in
Fig. 3, B-D. It can be seen that cells expressing Csk-
K
spread more rapidly than control cells (Fig. 3D), whereas none of cells expressing Csk had become attached at this point (Fig.
3C).

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Fig. 3.
Interference with cell attachment to
fibronectin by overexpression of Csk. A, astrocytes
expressing -galactosidase (Control), Csk, or Csk- K
were collected from tissue culture dishes by treatment with trypsin.
Portions of the suspended cells (1 × 105 cells) were
plated onto 12-hole cell culture plates (growth area, 4 cm2) coated with 10 µg/ml fibronectin. After incubation
for the indicated periods, the numbers of cells attached to the
fibronectin were estimated as described under "Experimental
Procedures." The data shown were obtained from three independent
experiments. The error bars represent the standard deviation
(n = 4). B-D, phase contrast images of
control cells (B), cells expressing Csk (C), and
cells expressing Csk- K (D), taken 40 min after
plating.
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Focal Adhesion Structure and Organization of F-actin Cytoskeleton
in Csk-overexpressing Astrocytes--
To examine the molecular basis
of the phenomena observed in Csk-expressing astrocytes, the
organization of the F-actin cytoskeleton and the distribution of the
focal adhesion proteins were analyzed. Cells expressing
-galactosidase, Csk, or Csk-
K were all stained by an antibody
against GFAP, confirming that these cells maintained their astrocyte
characteristics (Fig. 4,
A-C). Staining with rhodamine-conjugated phalloidin
indicated that the total amount of F-actin in the Csk-expressing cells
was comparable with that in the other cell types. However, the F-actin
in the Csk-expressing cells displayed a disordered structure and was
condensed at the peripheral regions of the cells (Fig. 4E).
In some cells expressing Csk-
K, the organization of F-actin was more
evident than in the control cells (Fig. 4, D and
F). The organization of focal adhesion was examined by
staining with anti-paxillin and anti-phosphotyrosine antibodies (Fig.
4, G-L). Patchy staining around the cell peripheries, which
is characteristic of focal adhesion, was observed in the control cells
and in those expressing Csk-
K, whereas the focal adhesion staining
was dramatically reduced in the Csk-expressing cells, indicating that
the formation or maintenance of a focal adhesion structure was
disturbed by Csk expression.

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Fig. 4.
Effect of Csk overexpression on assembly of
focal adhesion and organization of the actin cytoskeleton Five
days after infection with the recombinant adenovirus, primary cultured
type I astrocytes expressing -galactosidase (A,
D, G, and J), Csk (B,
E, H, and K), or Csk- K
(C, F, I, and L) were
stained with anti-GFAP antibody (A-C), rhodamine-conjugated
phalloidin (D-F), anti-paxillin monoclonal antibody
(G-I), or anti-phosphotyrosine antibody
(J-L).
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Tyrosine Phosphorylation of Paxillin and FAK in Astrocytes
Expressing Csk and Csk-
K--
The effects of Csk and Csk-
K
expression on the tyrosine phosphorylation of cellular proteins were
investigated by Western blotting with anti-phosphotyrosine antibody
(Fig. 5A). In the control and
-galactosidase-expressing cells, the tyrosine phosphorylation levels
were relatively low (lanes 1 and
2). Overexpression of Csk induced the tyrosine
phosphorylation of several proteins in a dose-dependent
manner, with prominent phosphorylation being obtained for 70- and
100-kDa proteins (lanes 3-6). Overexpression of Csk-
K
also induced the tyrosine phosphorylation of some proteins, including a
70-kDa protein, although the phosphorylation of a 125-kDa protein was
induced in place of a 100-kDa protein (lane 7).

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Fig. 5.
Tyrosine phosphorylation of paxillin and FAK
in astrocytes expressing Csk and Csk- K. A, tyrosine
phosphorylation patterns in total cell lysates (15 µg of protein) of
the control (lane1), and -galactosidase
( -Gal)-expressing (lane 2), Csk-expressing
(lanes 3-6), and Csk- K-expressing (lane 7)
cells. In the case of Csk-expressing cells, m.o.i. was varied between
1.25 and 10. Tyrosine-phosphorylated proteins were detected by Western
blotting with anti-phosphotyrosine antibody (4G10). B,
expression levels of vinculin, FAK, and paxillin in total lysates (15 µg of protein) of the control (lane 1) and
-galactosidase-expressing (lane 2), independent cultures
of Csk-expressing (lanes 3 and 4), and
independent cultures of Csk- K-expressing (lanes 5 and
6) cells. The expression level of each protein was detected
by Western blotting. C and D, tyrosine
phosphorylation (PY) of paxillin (C) and FAK
(D) in control (lane 1),
-galactosidase-expressing (lane 2), Csk-expressing
(lane 3), and Csk- K-expressing cells (lane 4)
cells. The cell lysates (500 µg of protein) were subjected to
immunoprecipitation (IP) with anti-paxillin (C)
or anti-FAK (D) antibodies. Tyrosine-phosphorylated paxillin
and FAK in the immunocomplexes were, respectively, detected by Western
blotting with recombinant anti-phosphotyrosine antibody (RC20,
upper panel) or anti-paxillin and anti-FAK antibodies
(lower panel).
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To identify the tyrosine-phosphorylated proteins, several candidate
proteins involved in cell adhesion were examined with regard to their
expression and tyrosine phosphorylation levels. The expressions of the
focal adhesion proteins vinculin, FAK, and paxillin was first confirmed
by Western blotting (Fig. 5B). The expression of vinculin
was not affected by the expression of Csk or Csk-
K. The total amount
of paxillin was not affected, but mobility shifts on SDS-PAGE, probably
because of the phosphorylation, were observed with cells expressing Csk
and Csk-
K. A slight increase in the expression of FAK was observed
only in the case of Csk-expressing cells. Overall, however, there were
no obvious changes in the expression of focal adhesion proteins in
either cell type.
Tyrosine phosphorylation of the proteins was detected by
immunoprecipitation followed by Western blotting. As shown in Fig. 5C, tyrosine phosphorylation of paxillin (70 kDa) was
dramatically elevated in cells expressing Csk or Csk-
K. FAK (125 kDa) was found to be heavily tyrosine-phosphorylated only in cells
expressing Csk-
K (Fig. 5D). The tyrosine phosphorylation
of other proteins having a similar Mr to that of
FAK, such as p130cas or PYK2 (CAK
), was not detected (data
not shown). Thus, it is suggested that the 70-kDa protein
phosphorylated in both Csk- and Csk-
K-expressing cells was paxillin,
whereas the 125-kDa protein in cells expressing Csk-
K was FAK. The
phosphorylated 100-kDa protein found in Csk-expressing cells was not identified.
Phosphorylation of Paxillin by Src and Csk in Vitro--
Paxillin
is known to be a good substrate for Src, Csk, and FAK (33). To further
confirm the identification of the 70-kDa protein as paxillin and
determine the kinase responsible for paxillin phosphorylation in the
cells, Src, Csk, and Csk-
K were co-immunoprecipitated with paxillin,
and the immunocomplexes were subjected to in vitro kinase
assay (Fig. 6). Paxillin was
phosphorylated by Src or Csk but not by Csk-
K, suggesting that
paxillin phosphorylation was mediated by Csk itself in Csk-expressing
cells. In cells expressing Csk-
K, the paxillin was phosphorylated by
a kinase(s) activated by the expression of Csk-
K, possibly Src
and/or FAK.

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Fig. 6.
In vitro phosphorylation of paxillin by Src
and Csk. In vitro phosphorylation of immunoprecipitated
paxillin (lane 1), Src (lane 2), Src + paxillin
(lane 3), Csk (lane 4), Csk + paxillin
(lane 5), Csk- K (lane 6), and Csk- K + paxillin (lane 7). Src and paxillin were immunoprecipitated
from cell lysates (100 and 500 µg of proteins, respectively) prepared
with radioimmune precipitation buffer from control astrocytes. Csk and
Csk- K were immunoprecipitated from cell lysates (200 µg of
protein) prepared with TNE buffer from Csk- and Csk- K-expressing
cells, respectively. In the cases of lanes 3, 5,
and 7, the two proteins were simultaneously
immunoprecipitated from the same cell lysates. The immunocomplexes
obtained were then subjected to in vitro kinase assay in the
absence of ectopic substrate. The phosphorylated proteins were resolved
by SDS-PAGE and detected by autoradiography.
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It has been reported that Src and Csk phosphorylate the identical
paxillin residues in vitro (33) To compare the properties of
the tyrosine-phosphorylated paxillins in cells expressing Csk and
Csk-
K, the abilities of the two species of paxillin to interact with
Csk in vitro were investigated. As shown in Fig.
7A, the tyrosine-phosphorylated paxillins in cells expressing Csk and Csk-
K
were both co-precipitated with GST fusion proteins containing the SH2
domain of Csk. There were no differences in the amount of paxillin
precipitated, the migration pattern on SDS-PAGE, or the tyrosine
phosphorylation level between the two species of paxillin. In addition,
they were equally co-immunoprecipitated with Csk from the cell lysates
of cells expressing Csk or Csk-
K (Fig. 7B). These
observations suggest that the phosphorylation states of the two species
of paxillin were functionally identical.

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Fig. 7.
Interaction of tyrosine-phosphorylated
paxillin with SH2 domain of Csk. A, 10 µg of GST fusion
proteins (GST (lanes 1 and 5), GST-CskSH2
(lanes 2 and 6), GST-CskSH3
(lanes 3 and 7), GST-CskSH2/SH3
(lanes 4 and 8)) were incubated with cell lysates
(500 µg of protein) prepared with TNE buffer from Csk-expressing
(lanes 1-4) or Csk- K-expressing (lanes 5-8)
cells for 12 h at 4 °C. The GST fusion proteins were then
collected by incubation with glutathione-Sepharose 4B beads (Amersham
Pharmacia Biotech) for 1 h. Immunoreactivities against
anti-phosphotyrosine antibody ( -PY, upper
panel) and anti-paxillin antibody (lower panel) in the
collected beads were detected by Western blotting. B, TNE
cell lysates (500 µg of protein) prepared from control astrocytes
(lane 1), -galactosidase ( -gal)-expressing
cells (lane 2), Csk-expressing cells (lane 3), or
Csk- K-expressing cells (lane 4) were subjected to
immunoprecipitation with anti-Csk antibody. The immunoreactivities in
the immunocomplex was detected as described in A. The
arrows indicate the position of paxillin on SDS-PAGE.
|
|
Kinase Activity of FAK in Astrocytes Expressing Csk or
Csk-
K--
Tyrosine phosphorylation and the kinase activity of FAK
have been reported to increase upon cell attachment to fibronectin (22). In addition, FAK-deficient cells displayed poor spreading and a
rounded cell morphology, indicative of the critical role of FAK in the
regulation of cell adhesion (19). As Csk-overexpressing cells displayed
the phenotype similar to that of FAK-deficient cells, we estimated the
kinase activity of FAK in cells expressing Csk or Csk-
K. The
autophosphorylation activity and the activity for an ectopic substrate
(poly-EY) in immunoprecipitated FAK were determined by in
vitro kinase assays (Fig. 8). The
amount of FAK in immunoprecipitates were evaluated by Western blotting
(Fig. 8A, upper panel). The relative activity of
them were estimated by dividing the total activity by the amounts of
FAK in immunoprecipitates (Fig. 8B). In Csk-expressing
cells, both the relative autophosphorylation and substrate
phosphorylation activities of FAK were decreased to 20-30% those of
control cells. In cells expressing Csk-
K, however, relative
autophosphorylation activity was elevated 1.5-fold over that in control
cells, which is consistent with the observation that FAK tyrosine
phosphorylation was elevated in astrocytes expressing Csk-
K.

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Fig. 8.
Specific activity of FAK in cells expressing
Csk and Csk- K. FAK was immunoprecipitated (IP) from
TNE cell lysates (500 µg of protein) prepared from control astrocytes
(Mock, lane 1), -galactosidase
( -Gal)-expressing cells (lane 2),
Csk-expressing cells (lane 3), or Csk- K-expressing cells
(lane 4). About one-tenth of the immunoprecipitates was used
in the in vitro kinase assay to determine the total activity
of FAK as described under "Experimental Procedures," except that 5 µg of polyglutamate-tyrosine (poly-EY) was added to the reaction
mixture as a substrate. The radioactivity incorporated into poly-EY was
quantified by a BAS2000 image analyzer (Fuji Film). To estimate the
autophosphorylation activity of FAK, the immunoprecipitates were
subject to in vitro kinase assay in the absence of poly-EY.
The radioactivity incorporated in FAK was visualized by autoradiography
(A, lower panel) and quantified by a BAS2000
image analyzer as well. The amounts of FAK in the immunoprecipitates
were estimated by Western blotting with anti-FAK monoclonal antibody
(A, upper panel). The relative activity of FAK
(autophosphorylation and poly-EY phosphorylation) was estimated by
dividing the total activity of FAK by the amounts of kinase in the
immunoprecipitates (B).
|
|
 |
DISCUSSION |
We have shown that adenovirus-mediated overexpression of Csk in
type I astrocytes inhibited cell spreading and attachment to the ECM,
and these effects were accompanied by defects in the cell adhesions and
cytoskeletal organization. By contrast, overexpression of Csk-
K
enhanced the cell adhesion capability to some extent. These findings
suggested that overexpressed Csk may target a critical point(s) in the
regulation of cell adhesion machinery. In cells expressing Csk, the
in vitro Src activity was repressed in a manner dependent on
the amount of Csk protein expressed, showing that the function of the
Src was successfully attenuated by Csk. Unexpectedly, overexpression of
Csk-
K did not have a dominant-negative effect on in vitro
Src activity. However, the tyrosine phosphorylation of some cellular
proteins, including the Src substrates paxillin and FAK, was increased
in cells expressing Csk-
K. It has been reported that overexpression
of Csk in CHO cells enhanced insulin-stimulated dephosphorylation of
FAK, whereas overexpression of Csk-
K inhibited the dephosphorylation
of FAK and paxillin but somewhat enhanced the basal tyrosine
phosphorylation of FAK (34). These findings together with our
observations suggest that Src or its relatives are functionally
activated in cells overexpressing Csk-
K. Although we do not have
direct evidence, it is likely that the turnover rate of phosphorylation
at the C-terminal regulatory site of Src is accelerated through
inhibition of Csk by the overexpression of Csk-
K and that the
transiently activated Src may be involved in the accumulation of
tyrosine-phosphorylated proteins. In cells overexpressing Csk, tyrosine
phosphorylation of several proteins, including paxillin and an as-yet
unidentified 100-kDa protein, was increased, suggesting that Csk, when
substantially overexpressed, can act on proteins other than Src family
tyrosine kinases, although the physiological meanings of this has yet
to be determined.
There is accumulating evidence that FAK and Src are essential signaling
molecules in the regulation of the cell adhesion mechanism (19-23). We
have demonstrated here that the tyrosine kinase activity of FAK was
decreased in cells overexpressing Csk and that the expression of
Csk-
K induced elevation of the autophosphorylation activity and
tyrosine phosphorylation level of FAK. The phenotype of Csk-expressing
cells was also consistent with that of cells obtained from
FAK-deficient mice (19), suggesting that loss of FAK function may
account for some aspects of the phenotype of Csk-expressing cells. It
is well known that FAK activation induced by cell attachment recruits
Src to activate it, thereby inducing the phosphorylation of Src
substrates, including FAK itself and paxillin (23). This
phosphorylation recruits Grb2-Sos and Paxillin-Crk-C3G adapter protein
complexes into the focal adhesion and ultimately activates the Ras and
mitogen-activated protein kinase signaling pathway (21). The
Ras-mitogen-activated protein kinase pathway is involved in the
regulation of cell adhesion by modulating the organization of the
cytoskeletal structure. Because Src was targeted in Csk-expressing
cells, the pathway downstream of Src might be shut down in the cells,
resulting in defects in cytoskeletal organization. Thus, it is more
likely that loss of FAK activation in Csk-expressing cells is a
secondary result of loss of cell adhesion elicited by defects in the
cytoskeletal organization mediated by Src.
We observed that the expression level of FAK was slightly increased in
cells expressing Csk, in which the kinase activity of FAK was
repressed. This suggests that there is an up-regulation system in FAK
expression regulated by the total activity of FAK itself. A similar
phenomenon was observed in the cells obtained from csk
knockout mice, in which the expression of Src was down-regulated by the
constitutive activation of Src because of the lack of Csk (18). Further
studies on the molecular mechanism leading to the feedback regulation
of gene expression might provide a new clue to understanding the roles
of tyrosine kinases.
Recently, it has been shown that overexpression of the C-terminal
portion of FAK inhibits tyrosine phosphorylation of FAK and paxillin
and, in addition, delays cell spreading and focal adhesion assembly
(26) and that the inhibitory effects of the C-terminal FAK can be
rescued by coexpression of FAK or Src but not by a FAK mutant that
fails to bind Src and paxillin (26). Because tyrosine phosphorylation
of paxillin is always observed when overcoming the inhibition of cell
spreading by the C-terminal FAK, it is suggested that tyrosine
phosphorylation of paxillin mediated by Src is a critical step in focal
adhesion assembly. In this study, the tyrosine phosphorylation of
paxillin was induced by overexpressing active Csk in a manner
independent of FAK and/or Src. Paxillin in Csk-expressing cells was
tyrosine-phosphorylated at an equivalent level to that in cells
expressing Csk-
K, and the two species of phosphorylated paxillins
were functionally identical in terms of specific interaction with the
SH2 domain of Csk. In addition, it has been shown that Src, Csk, and
FAK phosphorylate identical sites of paxillin in vitro (33).
However, cell morphology and the ability of cells to spread and attach to the ECM were selectively abrogated in cells expressing Csk. Thus it
is suggested from the findings of this study that although tyrosine
phosphorylation of paxillin may be required, it is not sufficient to
regulate the cell adhesion mechanism of astrocytes. Identification of
another molecule(s) downstream of Src may be necessary to elucidate the
pathway leading to the regulation of cytoskeletal organization.
This work has further highlighted the importance of Src in the
regulation of the cell adhesion mechanism by using Csk, a regulator kinase of Src. A crystallographic analysis of the repressed form of
c-Src indicated that displacement of the intracellular binding between
the SH2 domain and the C-terminal phosphotyrosine (Tyr-527) by another
protein can activate Src without dephosphorylation of Tyr-527 (35).
Indeed, it is shown that autophosphorylated FAK can act as an activator
through such a mechanism. However, overexpression of Csk readily
modulated the kinase activity of Src in the cells, suggesting that a
tyrosine phosphatase(s) directed to Tyr-527 is also important during
the process of cell adhesion. To identify such a phosphatase, our
system for obtaining efficient overexpression of Csk may be useful.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Izumu Saitoh and Dr.
Jyunichi Miyazaki (Institute for Medical Science, University of Tokyo)
for giving us the pAdex1CAwt cosmid cassette.
 |
FOOTNOTES |
*
This research was supported by a Grant-in-aid for Scientific
Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: +81-6-6879-8632;
Fax: +81-6-6879-8633; E-mail: ytaka{at}protein.osaka-u.ac.jp.
The abbreviations used are:
SH, Src homology; FAK, focal adhesion kinase; m.o.i., multiplicity of infection; GST, glutathione-S-transferase; poly-EY, polyglutamate-tyrosine; ECM, extracellular matrix; PBS, phosphate-buffered saline; GFAP, glial
fibrillary acidic protein; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid;
-gal,
-galactosidase.
 |
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